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structure and function of the digestive t r a c t of the grasscarp cover photograph: Pharyngealteethofthegrasscarp. Usually,awelldevelopedpharyngealmasticatoryapparatusispresentinstomachlessfish.Thefoodisfragmentedbetweentheventralteeth(photograph) andadorsalmasticatoryplate.Scanningelectron.micrographx45. 0000 Promotor: dr. L.P.M. Timmermans, hoogleraar in de algemene dierkunde Co-Promotor: dr. J.W.M. Osse , hoogleraar in de algemene dierkunde /uiuoYzo? k HENRI W.J. STROBAND STRUCTURE AND FUNCTION OF THE DIGESTIVE TRACT OF THE GRASSCARP Proefschrift t e r v e r k r i j g i n g van de graad van doctor in de landbouwwetenschappen, op gezag van de rector magnificus, dr.H.C. van der Plas, hoogleraarindeorganische scheikunde, in het openbaarteverdedigen opwoensdag 10september 1980 des namiddags tehalfdrieindeaula vandeLandbouwhogeschool teWageningen »i CONTENTS/INHOUD VOORWOORD 6 GENERAL INTRODUCTION 9 A. Kaderstelling 9 B. Structure and function of the digestive t r a c t of f i s h e s . . . 1 1 C. Objectives of the experiments 24 GENERAL DISCUSSION AND SUMMARY 26 Conclusions 32 REFERENCES 34 SAMENVATTING (Zieookbijlage) 38 PUBLICATIONS: A. Stroband , H.W.J. Growth and d i e t dependant s t r u c t u r a l adaptations of the digestive t r a c t i n j u v e n i l e grasscarp ( Ctenopharyngodon idella, Val.) J . Fish B i o l . 11, 167-174 (1977) B. ^Ê Stroband , H.W.J. & F.M.H. Debets. The u l t r a s t r u c t u r e and renewal of the i n t e s t i n a l epithelium of the j u v e n i l e grasscarp, Ctenopharyngodon idella (Val.) Cell Tiss. Res. 187, 181-200 (1978) C. £ Stroband , H.W.J. , H. v . d . Meer & L.P.M. Timmermans. Regional functional d i f f e r e n t i a t i o n i n the gut of the grasscarp, Ctenopharyngodon idella (Val.) Histochemistry 64, 235-249 (1979) D. Stroband , H.W.J. & F.H. van der Veen. The l o c a l i z a t i o n f STELLINGEN 1. Er z i j n geen argumenten voor de veronderstelling dat b i j juveniele en adulte maagloze vissen de e i w i t v e r t e r i n g in het darmlurnen minder e f f e k t i e f zou z i j n dan b i j maaghoudende soorten. Dit proefschrift. 2. De opvatting van Trevisan, dat vooral het caudale deel van de graskarperdarm betrokken is b i j de resorptie van nutriënten, is o n j u i s t en komt voort u i t het trekken van voorbarige konklusies u i t alleen morfologische informatie. Trevisan, P. : Anat. Anz. 145, 237-248 (1979). 3. Het voorstel van Green & P h i l l i p s , graanmutanten te kweken met verminderde feedbackinhibitie van aspartaatkinase door lysine en threonine, om zo het gehalte aan één of meer aminozuren u i t de aspartaat-familie te vergroten, verdient s t e l l i g waardering maar l i j k t geen praktische bijdrage t o t de oplossing van het wereldvoedselvraagstuk te kunnen leveren. Green, C E . &P h i l l i p s , R.L. : Crop Science 14, 827-829 (1974). 4. De circadische f l u k t u a t i e s in gevoeligheid van diverse weefsels voor bepaalde hormonen suggereert dat hormoonreceptoren een korte levensduur hebben. Meier, A.H. ; John, T.M. &Joseph, M.M. : Comp. Biochem. P h y s i o l . 40A, 459-465 (1971). Meier, A.H. ; Trobec, T.N. ; Joseph, M.M. &John, T.M. : P r o c . Soc. Exp. B i o l . and Med. 37, 408-415 (1971). 5. De i s o l a t i e van zowel één g l y c o p r o t e i n e r i j k - als één glycoproteinearm gonaidotroop hormoon u i t hypofyses van beenvissen ondersteunt de opvatting dat "glob u l a i r e " en " v e s i c u l a i r e " P.A.S.- positieve gonadotrope cellen t o t hetzelfde (celtype behoren, maar s l u i t n i e t u i t dat er een tweede gonadotroop celtype, mog e l i j k niet P.A.S.- p o s i t i e f , aanwezig i s . Ng, T.B. &I d l e r , D.R. : Gen. Comp. Endocrinol. 38, 410-420 (1979). Peute, J. ; Goos, H.J.Th. ; de Bruin, M.G.A. &van Oordt, P.G.W.J. : Ann. B i o l . Anim. Bioch. Biophys. 13, 905-910 (1978). Ueda, H. : Bull. Fac. F i s h . Hokkaido Univ. 31, 1-15 (1980). 6. Het zou van groot belang voor de wereldvrede kunnen z i j n , onderzoek te verrichten naar de oorzaken van het verschijnsel dat achteruitgang van de levensstandaard veelal l e i d t t o t toename van onredelijk vijand-denken en stijgende wapenproduktie, en in d i t verband onder andere aandacht te schenken aan de mogel i j k e rol van het " M i l i t a i r Industrieë'el Komplex" . 7. De boycot van de Olympische Spelen i n Moskou, waarvoor de verschillende redenen op zichzelf terecht werden aangevoerd, doet vermoeden dat w i j , westerl i n g e n , het restant van de boterberg die aan de Sovjetunie is verkocht op ons hoofd hebben gesmeerd in plaats van ook onszelf boter op het brood te geven. 8. De "vertrossing" van de omroepen l e i d t er toe dat de a f l e i d i n g die wordt gebracht de bevolking a f l e i d t van de meest wezenlijke problemen van onze samenleving. 9. Het f e i t , dat de vervoersorganisaties bezwaar maken tegen het gebruik van "hun" belastinggelden ten behoeve van de aanleg van fietspaden betekent dat de wegvervoerders te weinig oog hebben voor de fietsende medemens en onderstreept derhalve de noodzaak van genoemde wielerpaden. 10.De "brede maatschappelijke discussie" over kernenergie z a l , wanneer het kabinet zich n i e t onthoudt van voorbarige uitspraken, vergelijkbaar b l i j k e n met een golfstroom : oeverloos en met voorspelbare bestemming. Proefschrift vanH.W.J.Stroband Structure and function of thedigestive tract of thegrasscarp. Wageningen, 10september 1980. of protein absorption during the transport of food i n the i n t e s t i n e of the grasscarp, Ctenopharyngodon idella (Val.) Submitted to J . Fish Biol Stroband, H.W.J. & Annet G. Kroon. The development of the stomach i n Clarias lazera and the absorption of protein macromolecules. Submitted to Cell Tiss.Res URRICULUM VITAE IJLAGE : Stroband , H.W.J., J.H.W.M. 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GENERALINTRODUCTION A:KADERSTELLING Hetinditproefschrift beschreven onderzoek werd gestartin1973 binnen de toenmalige afdeling DierkundevandeLandbouwhogeschool waarvandeleiding berustte bij prof. dr.J.W.M.Osseenmevr. dr.L.P.M.Timmermans.In die periode werdeenaantal jonge biologen aangetrokken inverband metde in 1970 gestarte studierichting Biologie aandeLandbouwhogeschool.Het isdanookniet verwonderlijk dat het onderzoek vanmeetafaan beïnvloed werd door drie faktoren: 1.Het moest toegankelijk zijn voor doctoraal studenten,niet alleeninde biologie maar ookinbijvoorbeeld voedingen(vee)teeltwetenschappen. Daarom diendehetvoldoende breedteworden opgezet. 2.Het diendetepassen binnenhet kadervanhet onderzoek vandeLandbouwhogeschool,datwil zeggendat"landbouwkundige" aspekten o.i. gewenst warenenhet onderzoek daarom nietaltezeer zuiverwetenschappelijkvan karakter moest zijn. 3.Develeoptezettenprédoctorale onderwijs elementen eisten relatief veel tijd op,indeeerste jaren zelfs praktisch 100%.zodat het onderzoek een trage start ondervond. De derde faktor leiddeertoe datergedurendeeenlange periode konworden nagedacht overdeinvulling vandeonderzoekstaak vandeafdeling,de eerste twee faktoren bepaalden medederichting waarin gedacht werd. Bijenkelevandebetrokken medewerkers bestond ervaring inhet onderzoek metvissen.Dewetenschap dat visseneenbelangrijke voedselbron (kunnen gaan) vormeninvele landen naasthet feit dat fundamenteel onderzoek aandeze lagere gewervelde dierenaandeLandbouwhogeschool ontbrak hebbenertoegeleid datvanmeetafaan vissenalsproefdieren het meestinaanmerking lekente komen. Tenslotte werd gekozen voor o.a. onderzoek naardevoedselopnameen-verwerking bij vissen,waarbij morfologisch onderzoek t.a.v.devoedsel verwerving werd uitgevoerd binnen desectie Functionele Morfologie terwijl binnende sectie Histologie/Ontwikkelingsbiologie onderzoek werd verricht naar bouwen funktievanhetdarmkanaal. Dit laatste onderzoek bestond uiteen tweetal projecten, waarin r e s p e c t i e v e l i j k vorm en functie van het darmepitheel en aspecten van endocriene r e g u l a t i e , met name structuur en funktie van hormoon producerende cellen,de aandacht hadden (Rombout). Van de diverse takken van onderzoek die zich l a t e r (met name na de s p l i t sing van de afdeling Dierkunde in een voorlopige vakgroep Experimentele Diermorfologie en Celbiologie en een voorlopige vakgroep Dierkunde) binnen de vakgroep E.D.C, ontwikkeld hebben is er één zeer d u i d e l i j k aan het darmonderzoek gerelateerd, namelijk het onderzoek naar de ontwikkeling van het immuun-apparaat b i j vissen (v. Muiswinkel). De r e l a t i e met d i t binnen de sectie Celbiologie bewerkte projekt l i g t vooral in de w a a r s c h i j n l i j k n i e t onaanzienlijke rol die afweercellen in het darmepitheel spelen b i j het voorkomen van i n f e k t i e s via het (maagloze) spijsverteringskanaal van de door ons onderzochte proefdieren. 10 GENERAL INTRODUCTION B :FORMANDFUNCTION OFTHEDIGESTIVE TRACT INTELEOSTS. 1. Morphology a. Mouth cavity, pharynx and esophagus. The digestive system of teleosts is not basically d i f f e r e n t from that of other vertebrates including mammals. In a number of respects, however, i t s structure is a s i m p l i f i e d one. Digestive and absorptive functions seem to be carried out by a lesser d i v e r s i f i e d morphological system. Mouth cavity and pharynx in aquatic vertebrates are f o r continuous resp i r a t o r y and discontinuous feeding functions, more so than in t e r r e s t r i a l vertebrates. G i l l s and associated structures occupy nearly the whole pharynx ( f i g . 3). Taste buds in the pharyngeal epithelium are numerous in many f i s h species (Curry, 1939; Mc Vay & Kaan, 1940; G i r g i s , 1952; Kapoor et a l . , 1975a; Sinha, 1976 a ; Sinha & Moitra, 1975 a ; f i g . 1, 2 ) . No salivary glands have been found (Fahrenholz, 1937). The position of the mouth, the presence or absence of teeth on the jaws, vomer, palatines and pharyngeal bones, the morphology of the g i l l rakers, and other characteristics are closely related to the feeding habits. Reviews on this subject have been presented by Suyehiro (1942) and Kapoor et a l . (1975 b ). The esophagus is usually lined with a squamous epithelium, j u s t as the pharynx ( f i g . 2). I t s surface shows concentric microridges, j u s t as e p i t h e l i al c e l l s in the skin of teleosts (Merrilees, 1974; Reutter et a l . , 1974). In the epithelium, mucous c e l l s are abundant and tastebuds may be present (Chitray, 1965; Sinha, 1976 b ; Sinha & Moitra, 1975 a ; Verigina, 1976; Moitra & Ray, 1977). Esophageal m u l t i c e l l u l a r glands are not common (Kapoor, 1975 ). b. Stomach. In about85%ofallteleostspecies theesophagus leads intothestomach. The other 15%ofthebony fishesdonothaveastomach (Jacobshagen, 1937); the esophagus enters theintestine directly.Thesame applies tothelarval stagesofmost speciesoffish (Balon, 1975),when they take exogenous food although their stomach hasnotyetdeveloped. 11 ^'•^,"•''.'. 12 The walls of the stomach and the i n t e s t i n e consist of s i m i l a r layers of tissue as found in higher vertebrates ,but the i n t e s t i n a l mucosa lacks a muscularis mucosae ( C i u l l o , 1975; Korovina, 1976). The stomach usually shows two d i s t i n c t sections: a corpus part with a l i n i n g of mucous-producing c e l l s with underlying gastric glands, and a p y l o r i c part without gastric glands (Mohsin, 1962; Kapoor et a l . , 1975 ; Moitra & Ray, 1977). The glands are formed by only one type of c e l l ; no d i s t i n c t i o n can be made between pepsin producing c e l l s and oxyntic c e l l s . I t has been shown that corpus gland cells produce pepsin as well as hydroc h l o r i c acid in bony fishes (Blake, 1936; Barrington, 1957; Bucke, 1970; Verma & Tyagi, 1974; Moitra & Ray, 1977; Noaillac Depeyre & Gas, 1978). The same applies to a l l nonmammalian Vertebrates (Smit, 1968). e. Pyloric aaeaae Pyloric caecae are found in many teleosts with a stomach. The histology is not d i f f e r e n t from that of the i n t e s t i n e and this suggests that t h e i r primary role is to enlarge the intestinal area (Moitra & Ray, 1977). i. Intestine. In stomachless f i s h and in f i s h larvae, the f i r s t part of the i n t e s t i n e is a widened tube, called i n t e s t i n a l bulb. I t is assumed to have a storage function (Babkin & Bowie, 1928; Mc Vay & Kaan, 1940; Berry & Low, 1970; / e r i g i n a , 1978 ). In the bigmouth b u f f a l o , Verigina (1976) found an i n t e s t i lal bulb with a very thick p a r t l y s t r i a t e d muscularis. This may be related to ;he mechanical processing of food, and might be seen as an adaptation to the loorly developed pharyngeal teeth and the absence of a pharyngeal plate in ;his species. The b i l e and pancreatic ducts, generally located closely t o g e t h e r , j o i n he i n t e s t i n a l bulb at a short distance from the entrance of the esophagus Rogick, 1931; Curry, 1939; G i r g i s , 1952; Noaillac-Depeyre & Gas, 1976; e r i g i n a , 1978 b ). ig. ].Scanning electronmicrograph of the rostral part of thebuccal cavity of a6-monthsold grasscarp (x 105).L = dorsal lip; V =valve prohibiting outflow ofwater during the expiratory phase of respiration. R=roof of thebuccal cavitywith concentrations of tastebuds (arrows), also present in the areadirectly behind the lip. 13 fig. 2.Scanning electronmicrograph ofpart of the roof of thepharynx. Note thepresence of concentric microridges onepithelial cells, possibly facilitating gas exchange of the cells and/or holdingmucous at the cell surface(Reutter et al., 1974). In the left lower corner,a tastebud (x 5250). 14 Morphologically,no clear regional differentiation was madeinthe intestineofteleostswithastomach.Some species havearectum,set apartfrom the restofgutbysomekindofvalveorfolds (Jacobshagen,1937;Ciullo, 1975). e. Histology of the intestinal mucosa Themucosaofthe teleostintestine showsamoreorless complex pattern of folds (fig.4 ) .Villi haveneverbeen observed,multicellular glandsand cryptsofLieberkühn,presentwithin thewallofpartsofthe mammalian intestine,areabsentinteleosts,exceptinthefamily Gadidae (Jacobshagen, 1937; Klust,1940;Bishop&Odense, 1966). Theepitheliumisasimple columnaroneandcontains threeepithelial cell types.Absorptiveenterocytesbearmicrovilli (fig.6)andciliahavebeen foundinafewspecies (Barrington,1957;Iwai, 1967 a 'b ;Bucke,1970;Verigina,1978 a ). Furthermore theepithelium contains mucous goblet cells (fig.3,4)andenteroendocrine cells. Betweenthethree epithelial cell types,migrating cellsmaybepresent (Rogick, 1931;Girgis,1952;Bullock,1963;Hale,1965;Smit, 1968;Bucke, 1970; Krementz&Chapman,1974;Weinberg, 1975;Davinaetal. 1980), These areprobably lymphocytes,macrophagesandgranular leucocytes. The presenceofsocalled "pear-shaped cells"or"rodletcells"intheepithelium lasbeen reported formany teleostspecies.Someauthors areofthe opinion thatthese cellsareinfactprotozoan parasites (Rhabdospora thelohani) b bannister, 1966; Iwai,1968). Al Hussaini (1949 , 1964)mentionedthe lossibilityofrodlet cells being developing mucous cells,but thisisnot :orrect (Hirji&Courtney, 1979). Catton (1957)suggested thatthe rodlet :ellsaregranulocytic leucocytes,but concentrationsinbloodcelliroducingorgans were never found.Many recent authors considertherodlet :ellsasunicellular glands (Leino, 1974;Grünberg&Hager, 1978;Matteyet 1., 1979). Cytology of absorptive cells. The general morphologyofthe absorptive enterocytesofbony fishes howsastriking resemblancewith thatofhigher vertebrates.Similarorgaellesarefound.Minor differencesarethelackofcomplex interdigitations 15 fig. 3.Lateral part of thepharynx floor,with twobranchial arches,gi filaments (F)and gill lamellae (L).On thebranchial arches the gill rakers (R) (x 100).Inset: Tastebuds (arrows)on the gill rakers (x 700). 16 of the l a t e r a l plasma membranes of adjacent c e l l s , and the presence of lamellar infoldings of the plasma membrane in the basal part of the c e l l s in teleosts (Yamamoto, 1966). These infoldings appear to be s i m i l a r to those described f o r the "basal l a b y r i n t h " of proximal tubule c e l l s of the kidney in mammals. A function in osmoregulation has been suggested (Yamamoto, 1966; Noaillac-Depeyre & Gas, 1973 b ). In stomachless f i s h three segments can be distinguished, on the strength of the morphology of the absorptive c e l l s , and thi smorphology is directly related to the absorption of food. Therefore, some information about the digestive enzymes and absorption in teleosts should be discussed before dealing with the regional d i f f e r e n t i a t i o n . 2. Physiology a) Digestive enzymes Theenzymesintheintestinal lumenofbony fishes are essentially similartothose foundinmammals.Intheory theyareproducedinthe pancreas, the gastric mucosa orthe intestinal mucosa (including pyloric caecae). Productionbythe intestineisdoubtful (Kenyon, 1925;Jany, 1976), although Kapooretal.,(1975)areoftheopinion that themain protein-,carbolydrate-andfatdigesting enzymesare also producedbythe pyloric caecae îndintestinal mucosa.Itismore likely,however,thatenzymemolecules, derived from the pancreas,havethetendencytoaccumulate inthe glycocalix )ftheenterocytes (Fänge&Grove, 1979). Thismayalso explainthepresence )fproteinases,lipaseandamylaseinextracts from carp intestine (Al ïussaini, 1949 ).Onlyanenterokinaseandprobablyanamino peptidaseare )roducedbythemucosaofthe fishgut(Creach, 1963;Ishida, 1936;Bondi 1Spandorf, 1954). In fishwithastomachthecorpus glands produce hydrochloric acidand >epsinogen.When activated theenzyme showsaoptimumpHofabout 2.5, /hichiscommoninvertebrates.Since more thanonepHoptimum has often been oundinacid protease activity (Alliotetal., 1974;Creach, 1963),asecond iroteolyticenzymeislikelytobepresent,probably cathepsin withan iptimumpHof3-3.5. Thishasthe samequantitative proteolytic activityas lepsininpikeandtrout (Buchs, 1954). Therearenoimportant differencesintheproductionofenzymesbythe 17 18 pancreas for f i s h with or without a stomach. In both cases the pH in the i n t e s t i n e is neutral to s l i g h t l y alkaline (Shcherbina & Kazlauskene, 1971; Creach, 1963; A l l i o t et a l . , 1974). Consequently there is no peptic a c t i v i t y in the digestive t r a c t of stomachless f i s h (Kenyon, 1925; Babkin & Bowie, 1928; Smit, 1968; Ishida, 1936; Kawai & Ikeda, 1972; Jany, 1976). The p r o t e o l y t i c enzymes produced by the pancreas are t r y p s i n , chymotrypsin and carboxy peptidases (Creach, 1963; A l l i o t et a l . , 1974; Fänge & Grove, 1979). Among the other enzymes in the i n t e s t i n a l lumen of f i s h are the carbohydrases. Amylase, maltase, glycogenase, sucrase and saccharase a c t i v i t i e s have been found in some stomachless teleosts by Ishida (1936), Sarbahi (1951), and Kawai & Ikeda (1971), invertase by Ishida (1936) and Dhaliwal (1975). Cellulase could not be detected in the teleost i n t e s t i n e ( I s h i d a , 1936; Migita & Hashimoto, 1949). The pancreatic j u i c e and the i n t e s t i n a l lumen of most of the studied teleosts contain also lipase (Babkin & Bowie, 1928; Agrawal et a l . , 1975; Goei, 1974; Sastry, 1974 a ' b ; Kapoor et a l . , 1975b; Falge & Shpannkhof,1976). Patton et al.1975, suggested the presence of another fat-hydrolysing enzyme in f i s h that may compete e f f e c t i v e l y with lipase as a major f a t digesting enzyme. Apart from endogenous enzymes, exogenous substances might be of importance f o r the digestion of food. The possible role of microorganisms in digestion has been studied f o r carp, grasscarp and tench by Jankevicius and colleagues (Syvokiene et a l . , 1974; Syvokiene & Jankevicius, 1976, 1977; Lubyanskiene et a l . , 1977). Other studies were made by Paris et a l . (1977) and Sacquet et a l . (1979)for carp, grasscarp and t r o u t . The results show that microorganisms nay play a role in the fermentation of carbohydrates and the digestion of Droteins, but only the l a t t e r may be physiologically relevant in stomachless f i s h . According to Dabrowski & Glogowski (1977), a u t o l y t i c enzymes in food night play an important role in the digestion of proteins in f i s h larvae. Other enzymes, probably produced by i n t e s t i n a l c e l l s , and located in the nembranes of the microvillous border of the absorptive enterocytes might be of Eig.4. Scanning electron micrograph of somemucosal folds in the intestinal hulb.Note themany mucous goblet cells (arrows) (x 150). :ig. 5.As fig. 4,x 1800.B=bacteria. fig.6.As fig.4, x 8750. Note thepresence ofmany microvilli on the epithelial cells. 19 significance at least in mammals (Ugolev, 1971; Gossrau, 1975). Only a few studies on this subject have been made for t e l e o s t s . Of interest are the findings of Fänge &Grove (1979) in white grunt. A dipeptidase activity was noticed, especially in the epithelium of the anterior i n t e s t i n e . In mammals proteins are likely to be broken down to oligopeptides, and these are absorbed (Smyth, 1971; Crampton, 1972). The presence of a dipeptidase, especially in the anterior gut musoca is in accordance with Babkin &Bowie (1928), Hickling (1966), Alliot et a l . , (1974) and Cockson &Bourn (1973), who found maximum proteolytic activity in the anterior intestine of the studied fish species. Similar results were obtained for lipase and amylase a c t i v i t i e s (Hickling, 1966; Al Hussaini, 1949 ) but Cockson &Bourn (1973) found similar amylase activity in anterior and posterior intestine of Barbus paludinosus. b. Absorption of nutrients. The expectation that the localization of most digestive enzyme a c t i v i t i e s in the anterior part of the intestine might lead to a proximal to distal gradient in absorption of nutrients has not yet been confirmed. There are a number of studies, however, that suggest this to be true for many fish. Alkaline phosphatase activity is higher in the anterior than in the posterior part of the gut of several teleosts (Al Hussaini, 1949 ; Arvey, 1960; Sastry, 1975; Srivastava, 1966). Khalilov (1969) found an increase in the size of the Golgi apparatus and in alkaline phosphatase activity in the anterior intestine of tench after a fatty meal. The same was found after feeding starch. Broussy &Serfaty (1958), Sivadas (1964), Iwai (1968, 1969), Tanaka (1972), Gauthier& Landis (1972), and Noaillac-Depeyre &Gas (1974, 1976) by applying morphological techniques found that most of the lipid was absorbed in the anterior intestine of carp, goldfish, Tilapia, and in a number of fish larvae. This was confirmed by physiological experiments (Shcherbina, 1973, concerning l i p i d s , Shcherbina & Sorvatchev, 1969 and Shcherbina et a l . , 1976, for proteins; Farmanfarmaian et a l . , 1972, Sastry en Garg, 1976 and Shcherbina et a l . , 1977 for absorption of sugar). Sastry en Garg (1976),however, found absorption of lipids all over the intestine of Ophiocephalus and Heteropneustus by the application of histochemical techniques. 20 3. Regional differentiation of the intestine. In stomachless teleosts and in f i s h larvae two i n t e s t i n a l segments can be often distinguished: an a n t e r i o r segment with enterocytes loaded with l i p i d p a r t i c l e s a f t e r a f a t t y d i e t , and a posterior segment with many pinoc y t o t i c vesicles in the apical part of the c e l l s (Yamamoto, 1966; Iwai, 1969; Gauthier & Landis, 1972; Tanaka, 1971; Noaillac Depeyre & Gas, 1973 a , 1974, 1976). In some cases a t h i r d segment (rectum) has been described. The caudal segment contains many lamellar i n f o l d i n g s , which according to Noaillac-Depeyre & Gas (1973 ) might have a specialized function in osmoregulation. Orally administered horseradish peroxidase was absorbed by c e l l s of the second segment in adult stomachless f i s h (Gauthier & Landis, 1972; NoaillacDepeyre & Gas, 1973 ). This capacity to absorb protein macromolecules is also known f o r suckling mammals, in which the digestive system i s s t i l l not f u l l y developed (Clark, 1959; Leissring et a l . , 1962; Kraehenbuhl & Campiche 1969; Staley et a l . , 1972). Some authors suggested that the presence of a "second segment" i s to be correlated to the lack of a stomach and peptic digestion in order to account f o r the i n e f f i c i ë n t protein digestion in the gut lumen (Yamamoto, 1966; Gauthier & Landis, 1972; Noaillac-Depeyre & Gas, 1976). I t is of i n t e r e s t that Shcherbina & Sorvatchev (1969) found a protein absorption of 78 %in carp. In f i s h with a stomach, even higher percentages j f ingested protein may be absorbed (Kapoor et a l . , 1975 ). I t should be realized, however, that protein d i g e s t i b i l i t y can be correlated to the ;ame extend with the composition of the d i e t (Inaba et a l . , 1962, 1963). (itamikado & Morishita (1965) found a protein d i g e s t i b i l i t y of 10% i f t r o u t vere fed with soybeans. Aoe et a l . (1974) noticed that f o r carp hydrolysates ) f casein (amino acids and peptides) are far i n f e r i o r in n u t r i t i v e value to intact p r o t e i n . Some native proteins seem d i f f i c u l t to be digestid by carp [Jany, 1976). K Relations between food preference and digestive tract Many authors t r i e d to correlate the morphology and physiological characters t i e s of the digestive t r a c t to the feeding habits of certain species, teviews were presented by Suyehiro (1942) and Kapoor et a l . (1975 ). The lossible relationships seem to be very complex. This applies in p a r t i c u l a r 21 to the lack of a stomach i n a number of t e l e o s t s : stomachless f i s h are supposed to be descendant from f i s h with a stomach and many of them are believed to have adapted t h e i r d i e t s , as a r e s u l t of which there are herbivorous, omnivorous and carnivorous stomachless fishes (Rogick, 1931; Klust, 1940; G i r g i s , 1952; Kapoor et a l . , 1975 b ; Kafuku, 1977). Many ecological studies indicate an apparent preference f o r one or several types of food, but others point to the opportunistic feeding behaviour of f i s h in periods of food s c a r c i t y . Carnivorous, herbivorous, e t c . only indicate general tendencies in feeding h a b i t s , and not c h a r a c t e r i s t i c habits. Only the length of the i n t e s t i n e seems to be c l e a r l y associated with the feeding habits of a p a r t i c u l a r species. Some herbivorous and microphagous stomachless f i s h have a r e l a t i v e l y long gut: 7 x to 24 x standard body length (Rogick, 1931; G i r g i s , 1952; Das & Nath, 1965; Sinha & Moitra, 1975 ' ; Kafuku, 1977). The gut length of omnivorous species l i k e Cyprinus cavpio and Barbus aonahonius is 2-3 times the body length (Curry, 1939; Das & Nath, 1965). Carnivorous species have the shortest gut lengths, approximately 0,5 - 0,8 times the body length (Klust, 1940; Das & Nath, 1965). Khanna & Mehrotra(1971) found a r e l a t i v e l y short gut in 5 species of carnivorous teleosts. A large v a r i a b i l i t y may be found w i t h i n a given species, f o r example in the cyprinid Carasaius auratus (Vickers, 1962). This seems to be related to the kind of food administered during the ontogeny. The same is known from some cyprinodonts (Hykes & Moravek, 1933). Therefore, the v a r i a b i l i t y is evidently less pronounced in stenophags (Kapoor e t a l . , 1975 ). An additional problem i s that seasonal factors (quantity of food?) may a f f e c t the gut length (Ciborowska, unpublished results). In some studies a r e l a t i o n is described between the d i e t and the a c t i v i t y of several digestive enzymes. According to Sarbahi (1951) and Kitamikado & Tachino (1961), carbohydrases are more abundant in the stomachless goldfish than in the largemouth bass. Carbohydrases show the highest a c t i v i t i e s in herbivorous f i s h (Vonk, 1927; Al Hussaini, 1949 ; Agrawal e t a l . , 1975). Turpayev (1941) found t r y p t i c a c t i v i t y to be dominant i n a carnivorous cyprinid (Asp-ius) , and in the herbivorous Soavdinus the amylase a c t i v i t y was evident. Agrawal et a l . (1974) noticed a higher peptidase a c t i v i t y in omnivorous than in herbivorous species. Kawai & Ikeda (1972) found a r e l a t i o n 22 of the a c t i v i t y of amylase and protease in carp to the composition of the food. According to Sinha (1978), protease and lipase are the major digestive enzymes in Cirrhinus mrigala larvae, which are zooplankton feeders. In juve- niles (omnivorous) and adults (herbivorous) strong amylase and weak protease a c t i v i t i e s have been observed. Of special i n t e r e s t is the development of the digestive t r a c t and i t s functions during ontogeny, as most f i s h larvae are f i r s t carnivorous, feeding on zooplankton. The food regime of omnivorous and herbivorous species changes during ontogeny. This may be correlated with changes in the morphology and physiology of the digestive apparatus. 23 GENERAL INTRODUCTION C :Objectivesofour investigations. Fromthestartofthis studywehave realized that some fish change their feeding habits during development. Grasscarp larvaewere supposed tobe zooplankton feeders,justasmost fish larvae,whereasthe adults were called herbivorous (Gidumal, 1958;Aliyev&Bessmertnaya, 1968;Fisher, 1968). The grasscarp mightbeofspecial importancetoagriculture becauseit feedsonandthus controls water weeds (Cross,1969;Stott &.Robson,1970; Opuszynsky, 1972;Michewiczetal., 1972).Thespeciesmightbealso valuable for consumption,asithasahigh growth rate (Hickling,1960;Anderson, 1970). The selectionofgrasscarpforthe present study wasnotbased primarilyonthe absenceofastomach. The first objective wastofind morphological evidencefor theadaptationof the intestinetothe changing feeding habits during ontogeny.Forthis purpose the experiment described inpaperAhas been carried out.Theresultswerenot encouraging,butour attention was attracted tothedistinct regional differentiationofthe intestineinthis stomachless species. Because,justasinmammals,theintestinal epithelium incyprinidsisregularly renewed (Vickers,1962;Hyodo,1965;Gas&Noaillac-Depeyre, 1974),the speciesmightbeusedfor studying thedifferentiationofenterocytes withinth successive segments. PaperBdealsmainly withtherenewal ofthe gutepitheliu and themorphologyofabsorptiveanddifferentiating cells. Many interesting results have been obtained,butthe unexpected discoveryoffunctional cellsin the proliferative area indicated thattheintestinal epithelium isnotthemost useful tissuefor studyingthe differentiationofabsorptive cells. Itappeared thatjustasinother cyprinidsasegmentwiththe abilityof pinocytosisispresentinthe caudal partofthe gut,andthismightberelated tothelackofastomach. Firstithadtobeascertained whether active absorption takes placeintherostral intestinal segmentandabsorptionofmacromoleculesbypinocytosismayberestricted tothesecond partofthegut.Inadditi the absorptive activity along themucosal foldshadtobeinvestigated. Therefc the experiments described inpaperChave been carried out.Totestwhetherthe abilitytoabsorp protein macromoleculesisrelated tothelackofastomach,t locationofprotein absorption inthe grasscarp intestinehadtobedetermined (paperD ) . 24 The development of the i n t e s t i n e in a teleost (Clarias lazera) in which a stomach is developed at the end of the larval stage i s described in paper E. I t was investigated whether the development of the stomach might be related to the disappearance of pinocytosis in the second gut segment. 25 GENERAL DISCUSSION AND SUMMARY General morphology of the digestive tract Structurallyandfunctionally the digestive tractoffishes hasmuchin common with thatofother vertebrates,butinfishesthesystemissimplified. Salivary glands areabsent,justasesophageal glands.Approximately 15%ofthe species doesnothaveastomach.Theintestineisless complexduetothe absenceoffoldsofKerkringandreal villi. Furthermore,most fishes lackthecrypts of Lieberkühn,the glandsofBrunnerandamuscularismucosae,thereisno differentiation insmallandlarge intestine,andinmany speciesarectum cannotberecognized. For these reasons fishes appeartooffer several advantagesforstudying morphological andphysiological aspects,ofthedigestive system. Food,preference and the need for animal protein Another advantageisthatmany species change their food preference during their life.Anexampleisthechinese grasscarp, Ctenopharyngodon idella, the animal used forour experiments. PaperAdealsmainly with changesinthemorphologyoftheintestineinrelationtogrowthandchanging dietofthe young grasscarp. Animal food was foundtostimulate rapid growth,also after theanimals becom capableofingesting plantmaterial.The composition ofthefood protein,especiallyastotheessential aminoacids maybethemain factor determiningthe effectivenessofthe food.Many plantmaterialsareknowntocontain only small amountsofmethionineandlysineandmuch vegetable material inthefood might causeanimproper amino acid balance (Shcherbinaetal. 1964,quotedbyPhillips 1969)andconsequentlyalowgrowth rate.Grasscarp,keptinponds forweed control,mayformaseriousthreattoother speciesasapredatoraswell asa competitorforfood. Length and regional differentiation of the gut The digestionand absorptionofplant foodingrasscarp ismainly facilitated byanincreaseofthe relative lengthofthe intestine from + 0,7toabout twic the bodylength.Thelatterwas foundin6monthsoldspecimensandalsoinadult (paper D ) ,andislowforapresumably herbivorous species. 26 Inmany other fish species alsoachangeinfeeding habits takes placeduring evelopment.Thisisalways relatedtoanincreaseingutlength from+0,5x odylengthinthecarnivorous larvaetomuch highervaluesinomnivorousand erbivorous juvenilesandadults.Infig.7thegutlengthsofseveral Cyprinids regivenatanumberofstagesofdevelopment.Intheherbivorous Catla oatla hemost rapid increaseinrelativegutlengthwas found,whilethegrasscarp howsaslower increaseingutlength than themirror-carp, Cyprinus oarpio, presumably omnivorousspecies. 5-, ^ X I- LU z > —I UJ * / AO 10 BODY LENGTH (cm) 7. GraphshowingtherelativelengthofthegutofsomeCyprinidlarvaeat severalages.FromStroband,H.W.J.&Dabrowski,K.R.,in:"Nutrition desPoissons"C.N.E.R.N.A.Paris (inpress). O-commoncarp,»-grasscarp (Ctenopharyngodon idella) ,A- silvercarp (Hypophthalmichthys molitrix) ,A-bighead (Aristiohthys nob-ilia)(after Ciborowska,unpublished data),*- common carp (after Klust, 1940), •-grass carp (afterStroband,1977)jjCatla aatla (after Kafuku, 1977). 27 Themorphology oftheintestinal epithelium ofthestomachless grasscarpis described inpaperAand,inmoredetail,inpaperB.Thegutshowsasimilar regional differentiation asinother cyprinids (Yamamoto,1966;Gauthier& Landis,1972;Noaillac-Depeyre&Gas,1974,1976).Theanterior segment shows thecharacteristics oflipid absorption;inthe"second segmenfenterocytescon tainmany pinocytotic vesiclesandoneormore supranuclear vacuoles probably representing large secundary lysosomes.Athird caudal segment contains entere cyteswith thecharacteristics ofwateroriontransport. The length oftheintestine isalso affected bythediet during early life. Vegetable food causesaslight increase ingutlength,whichwasalso foundir other species.Ourresults indicate thattheanterior segmentisinvolved int growth,andthismight well berelated tothemain absorptive function ofthis partoftheintestine (paperD ) . The intestinal epithelium as a cell renewal system The regional differentiation andtherelatively lesscomplex structureof1 mucosa seem tomakethefish intestine auseful model forstudying thediffère tiationofepithelial cells.InpaperBthecell renewal systemofthegutepi theliumofthegrasscarp isdescribed with light-microscopic radioautography, 3 using Hthymidine.Thesystem appeared tobecomparable tothatinthemammalian small intestine;proliferation takes place inthebasal partsofthemucc sal folds infishes,andinthecryptsofLieberkühn inthesmall intestineo1 mammals.Therenewal time inthegrasscarp isrelatively long:10-15daysat 20°C.InpaperBtheultrastructureoftheintestinal epithelium isdescribee for starved andfedspecimens. Functional absorptive cells proved tobepresent intheproliferative area whereas undifferentiated cells could notbeidentified. This isamajordifferenceinrespecttomammals,inwhich undifferentiated proliferative cellsin the crypts only become functional aftermigration towards theintestinal villi (Rijke, 1977). A comparison ofradioautoqraphs andelectronmicrographsforlarvaeof Barbus aonchonius (Romboutetal.,1980)and Clavias lazera (paperE)shows thatfuni tional absorptive cellsarecapableofproliferation. Similar resultswerefoi inthelarvaeoftheamphibian Xenopus laevis (Marshall &Dixon, 1978).The difference ofthecell renewal system inmammals andfishes isalso reflected the presenceofalkaline phosphatase activity inthemicrovillous borderof enterocytes intheproliferative area ofthegrasscarp (paper C ) ,which also 28 ndicatesthecellstobecapableofabsorption. Intracellular factors,relatedtothestateofdifferentiation,and extraellular factors (chalones,hormones,stimuli from the food)are possiblyinolvedindeterminingtherateofproliferationofvertebrate cells. Inhibition f proliferationbyintracellular factors possibly develops onlywhen thecells ave reachedarelatively high stateofdifferentiationinfishes.Inthis onnectionitisinteresting thatinthecolonofmammals functional cellsand ndifferentiated proliferative cellsareintermingled (Lipkin, 1973). This imliesamajor effectofthe intracellular factorsinblocking cell divisionin nedifferentiated cells. he digest-ion moment and absorption of protein and the function of the second gut An important factoristheregional differentiationofthe grasscarp intestine, idespecially the presenceofa"second segment"with pinocytotic activity.This isalso found aftertheingestionofexogenous foodinother stomachless fishes idinfish larvae,which usually are initially stomachless (Tanaka,1969; »perE ) . PaperCdealswith the activityofalkaline phosphataseandtheuptakeof 'allyadministered horseradish peroxidaseintheintestineofgrasscarp larvae idjuveniles.Aproximo-distal gradientinalkaline phosphatase activitycan ;demonstrated.This suggests that themain active absorption takes placein ieanterior gut segment. Peroxidase was demonstrated histochemicallyinentero'tesofonly the second segment.This implies thatthe enzymemust have been »sorbedbypinocytosis.Similar results have been found inother Cyprinids loaillac-Depeyre&Gas,1973)andinClarias lazera larvae (paperE ) . The enterocytesinthesecond segmentofthe grasscarp have also theability »pinocytosisoflarge ferritin particles (paper D). Since pinocytosisofmacro»leculestakes place alsointheintestineofsuckling mammals before the stolenfunctionsarefully developed, itiswidely believed thatthepresenceof "second segment"instomachless fishesisrelatedtothe lackofpepticdiistion.Recently,however,a"second segment"was also found intheintestine r the perch,afish withastomach (Noaillac-Depeyre&Gas,1979): this segment, wever,isrelatively small (+11 %ofthegutlengthand+20%infish larvae d stomachless fishes). ShcherbinaandSorvatchev (1969)andShcherbinaetal. (1976),whousedthe ertmarker technique,discovered thatmost proteinisabsorbed intheanterior 29 60 %ofthe carp intestine,i.e.inthe anterior gut segment.Intheirexperiments Shcherbinaetal. pooledthegut contentsof6specimens.Therefore, the locationofprotein absorption inthe grasscarp intestine has been determined alsoinindividual test animals.Theresultsaredescribed inpaperD.Itwill beproved that proteins arealmost completely absorbed within the anterior 40-50 %ofthegut i.e.intherostral two thirdofthe first segment.This implies adequate digestionofproteininthegutlumen,sincenonoticeable pinocytosisof macromoleculeswas found intheanterior segment.Ourresults also indicate thatessential amino acids are preferentially absorbed inthe grasscarp,justasinmammals (Orten,1963;Ben-Ghedaliaetal., 1974).The rapid disappearanceoflysineandarginine from the chyme suggestsatryptic breakdownofproteins. In their paperonthe perch,Noaillac-Depeyre&Gas (1979)suggested that protein digestion inthegut lumenoffishmightbeless efficient thanin mammals. They suggest that completionofhydrolysis takes placebyintracellular enzymesinthesecond segment.Our results prove thistobeincorrect proteins are digestedandabsorbed adequately withinthe anterior gut segment, butanexception should possiblybemadefor fish larvae,inwhich food reache the distal partofthe intestine very shortly after feeding (Fänge&Grove, 1979). Under physiological circumstances pinocytosisofafewmacromolecules undoub ly takes place.Thismayberelevantinaqualitative sense.Itmaybecompara tothesituation inadultmammals,inwhich macromolecules areabsorbed insma quantities andmaybeantigenicorbiologically active (Walker&Isselbacher, 1974; Warshawetal., 1974). Similarity between the anterior gut segment in fish and the small in mammals intestine Astoitsabsorptive functions,the anterior intestinal segmentoffishesY muchincommon with the small intestineofmammals.Mammals also showaproxin distal gradientinalkaline phosphatase activity,andinboth classesofverte brates sugars,lipidsandprotein aremerely absorbed inthe anterior partof thegut(Belletal., 1972;Crampton,1972;Farmanfarmaianetal., 1972;Shche binaetal., 1973;1976;1977;BenGhedaliaetal., 1974;Noaillac-Depeyre& Gas, 1976;paper B ) .Themorphological characteristicsoflipid absorptionare similarinmammalsandfishes,andinboththetransportofsugarsandaminoacidsissodium-dependent (Cartieretal., 1979). 30 The development of the stomach in Glorias lazera The digestive systemoffish larvae resembles thatofstomachless teleosts inmany respects:a"secondgutsegment"mayberecognized immediately afterthe beginningofexogenous feeding.Thisisalso trueforfish species that develop a stomachattheendofthelarval period. In paperEthe resultsare described ofanother attempttoestablishapossible relation betweenthe presenceofa"second segment"andthelackofastomach.The developmentofthe stomachinclarias lazera isdescribed,andits ultrastructureiscompared tothatofadult specimens.Itisofinterest that the stomach corpus glands contain only one cell type,which apparently is involvedinproducing hydrochloric acidandpepsinogen. From theultrastructure 3 of the glands,the H-thymidine labeling index,andthepHindicator tests,it isconcluded thatafunctional stomach ispresent from approximately 12days after fertilization. However,inlarvaeaswell asinjuvenile Cl. lazera a "second segment"ofapproximately 20%ofgut length shows theabilitytopinocytosisofhorseradish peroxidase.Thesamemay applytoadult specimens,since they also possessa"second segment"of20%ofgut length,asproved with lightand electron-microscopic studies.Therefore,the conclusion seems justified, thatthe abilitytopinocytosisofmacromoleculesisnotpositively correlated tothelackofafunctional stomach.Our results also indicate thataregional differentiationofthe intestine,includingagutsegmentwith pinocytosis,is a general featureofthedigestive tractinteleosts. Function of the stomach in vertebrates Since proteins aredigested adequately intheintestineofstomachless fishes -although food passes throughthegutinnotmore than about7hinthegrasscarp (Hickling, 1966)-thequestion ariseswhether peptic digestion inanacid environment,asfound inother vertebrates,isinfactofminor importance. AccordingtoBertin (1958),most stomachless teleosts possess some kindofmasticatory apparatus,likethepharyngeal teethinCyprinids. Fishes without this apparatusaresupposedtoneedastomach withalowpHtofractionate the food. Botharetherefore believedtofacilitate feeding with relatively largeorganisms. Itiswidely believed thatthestomach evolved togetherwiththe jawsbutprimarilyasastorage organ.Acid productionmayhave followed,andacted infractioning large food elementsandpossiblyinprotectingthestored food against bacterial digestion.The productionofpepsinfor thedigestionofproteinsin 31 the acid environment might have been the last important step in the evolution of the stomach (Barrington, 1957;Romer,1962;Waterman et al., 1971). From phylogenetic studies it isgenerally believed that the absence of astomach in teleosts isa secundary feature (Barrington, 1942). CONCLUSIONS 1.The grasscarp isastomachless teleost.The intestine does not containmulticellular glands. 2.The relative length of the grasscarp intestine increases from 0,7 x body length inyoung larvae to 2xbody length inadults.The gut length is the onlymorphological characteristic to changewhen the grasscarp turns from carnivorous to herbivorous feeding. 3.After feeding thegrasscarp with vegetable food,the increase ingut length is higher than in fishes fed with animal food. 4.Animal food stimulates rapid growth in the grasscarp,also after they are able to ingest plantmaterial. Grasscarp may represent a serious threat to other species as a predator aswell as acompetitor for food. 5.The intestine of the grasscarp shows a similar regional differentiation as found inother Cyprinids,with aproximo-distal gradient inalkaline phosphatase activity. 6.The anterior gut segment is involved in the absorption of lipids and proteins (and probably also of sugars),which are absorbed inenterocytes after hydrolysis in the lumen.Also in regard to themorphological characteristics of lipid absorption,the epithelium shows resemblance with the epithelium in the small intestine ofmammals. 7. A"second gut segment",running from +60-85 %of gut length, is characterized by enterocytes with a high pinocytotic activity. These cells are capable of absorbing protein macromolecules like horseradish peroxidase and ferritin. 8. Protein digestion isefficient despite the absence of a stomach. Quantitatively relevant amounts of protein are not absorbed by the"second segment". 9. 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Ongeveer 15% der vissoorten, waaronder de karperachtigen, is maagloos. Wat de darm b e t r e f t v a l t vooral het ontbreken van plooien van Kerkring, v i l l i met crypten en meercellige klieren i n de darmwand op. Bovendien kan er geen onderscheid worden gemaakt i n dunneen dikke darm. Eén en ander heeft er toe geleid dat vissen een steeds belangr i j k e r plaats innemen i n het fundamenteel onderzoek naar vorm en f u n k t i e van elementen van het s p i j s v e r t e r i n g s s t e l s e l . De graskarper is als larve carnivoor en voedt zich met zoöplankton. Tijdens z i j n ontwikkeling, w a a r s c h i j n l i j k al gedurende de eerste maanden, verandert het dieet en aangenomen wordt dat de volwassen graskarper herbivoor i s . Deze verandering l i j k t deze vissoort bijzonder geschikt te maken voor onderzoek naar de r e l a t i e tussen de struktuur van de darm en het d i e e t . U i t a r t i k e l A b l i j k t echter dat graskarpers, ook lang nadat ze voor het eerst i n staat z i j n plantaardig voedsel te n u t t i g e n , het snelst groeien wanneer ze met d i e r l i j k materiaal worden gevoed. De enige morfologische verandering die werd waargenomen gedurende de periode waarin het dieet zich zou wijzigen was een toename van de relatieve darmlengte van 0,7 t o t 2 maal de lichaamslengte. Dit is tamelijk kort voor een vis die verondersteld wordt herbivoor te z i j n , en w i j s t als zodanig meer op een omnivore levenswijze. Dit kan betekenen dat graskarpers, wanneer ze massaal worden uitgezet ter bes t r i j d i n g van waterplanten, een bedreiging worden voor andere soorten, zowel als predator alsook als konkurrent wat het voedsel b e t r e f t . In p u b l i k a t i e A, en meer gedetailleerd i n a r t i k e l B, wordt de morfologie van het darm epitheel beschreven. Er z i j n 3 darmsegmenten te onderscheiden. Het meest rostral e segment, dat + 60% van de darm beslaat, bezit resorberende cellen die dezelfde morfologische kenmerken van vetopname vertonen die ook i n enterocyten i n de dunne darm van zoogdieren worden aangetroffen. In het middelste of tweede segment (+ 25% van de darmlengte) komen resorberende cellen voor die veel pinocytotische a k t i v i t e i t vertonen. Het caudale of 3e segment (+ 15%) speelt w a a r s c h i j n l i j k een rol b i j de osmoregulatie. 38 Wegens deze vormverschillen, die ook een regionale d i f f e r e n t i a t i e t . a . v . •esorptie van bepaalde voedingsstoffen l i j k e n i n te houden (zo kunnen e i w i t :en in het tweede segment worden gepinocyteerd), werd de graskarper een ge;chikt object geacht om de d i f f e r e n t i a t i e van verschillende typen resorptie :ellen te bestuderen. In a r t i k e l Bwordt de vernieuwing van het darm epitheel beschreven. Deze ili j k t r e l a t i e f traag te verlopen: 10 à 15 dagen b i j 20°C tegenover 2 à 3 lagen b i j de meeste zoogdieren. In de dunne darm van zoogdieren is de proifererende "pool" van cellen gelokaliseerd i n de crypten van Lieberkühn. e bestaat u i t ongedifferentieerde c e l l e n , die zich d i f f e r e n t i e r e n tijdens e migratie naar de v i l l u s , alwaar zich funktionele cellen bevinden. De esultaten b i j de graskarper wijken in belangrijke mate af van d i t beeld. aar crypten ontbreken, bevinden zich de prolifererende cellen aan de basis 3 an de darmplooien, zoals b l i j k t u i t de H-thymidine labeling van epitheel e l l e n , die alleen i n d i t gebied optreedt. Er worden hier echter alleen unktionele cellen aangetroffen. In p u b l i k a t i e Ewordt aangetoond dat ook i j Clarias lazera larven de p r o l i f e r a t i v e cel "pool" i n het darm epitheel i t funktionele cellen bestaat. Bovendien b l i j k e n b i j deze vissoort f u n k t i o ele cellen u i t het maag epitheel i n staat te z i j n zich te delen. Terwijl oogdiercellen al vroeg i n het d i f f e r e n t i a t i e proces, lang vóórdat ze unktioneel worden, hun delingsvermogen verliezen, l i j k t de blokkering an de mogelijkheid t o t p r o l i f e r a t i e b i j vissen pas i n een veel l a t e r staium van de d i f f e r e n t i a t i e op te treden. Eén en ander houdt in dat de grasarper zich minder goed leent voor het bestuderen van morfologische aspecten an het d i f f e r e n t i a t i e proces van darm epitheel c e l l e n . In a r t i k e l Cwordt aangetoond dat min of meer komplete protéine macroDleculen (mierikswortel peroxidase) i n het tweede darmsegment kunnen worden =pinocyteerd. Terwijl i n het eerste segment de m i c r o v i l l i der resorptie ï l l e n een hoge alkalische fosfatase a k t i v i t e i t vertonen, ontbreekt deze -ijwel geheel in het tweede segment. Dit w i j s t er op dat a k t i e f transport )or de apicale celmembraan vooral plaatsvindt i n het eerste segment. Omdat ook pasgeboren zoogdieren de mogelijkheid t o t pinocytose van macro)leculen bezitten - vóórdat a l l e maagfunkties t o t ontwikkeling gekomen z i j n wordt wel aangenomen dat deze mogelijkheid in verband staat met het onteken van een maag en vooral van peptische e i w i t v e r t e r i n g . Deze hypothese lakte het wenselijk de e i w i t v e r t e r i n g en opname nader te onderzoeken. ertoe werden de experimenten zoals beschreven in p u b l i k a t i e D uitgevoerd. 39 B i j de graskarper b l i j k t , dat de 75% van het voedsel e i w i t die worden opgenomen vrijwel volledig door de r o s t r a l e 40% van de darm worden geresorbeerd. Een k w a n t i t a t i e f belangrijke rol kan dus, wat de e i w i t opname b e t r e f t , n i e t aan het 2e segment worden toegeschreven. U i t de experimenten b l i j k t tevens dat er een preferentie bestaat voor essentiële aminozuren, zoals dat ook b i j zoogdieren het geval i s . De r e l a t i e f snelle resorptie van arginine en lysine w i j s t op het belang van vooral tryptische vertering b i j de graskarper. Een tweede manier om na te gaan of de aanwezigheid van een tweede segment met pinocytotische a k t i v i t e i t i n r e l a t i e staat t o t het ontbreken van een maag is het bestuderen van de ontwikkeling van het spijsverteringskanaal van een maaghoudende v i s . B i j vislarven is de maag nog n i e t aangelegd, en de darm vertoont dezelfde regionale d i f f e r e n t i a t i e als die van maagloze vissen. In a r t i k e l Ewordt de vorming van de maag beschreven b i j de tropische meerval Clarias lazeva. Een bijzonderheid ten aanzien van de struktuur van de maag van vissen i s , dat - i n tegenstelling t o t zoogdieren - slechts één type k l i e r c e l voorkomt waarin zowel pepsinogeen als HCl wordt geproduceerd. Het tweede segment b l i j k t na de vorming en het funktioneel worden van de maag te b l i j v e n bestaan. B i j larven én b i j adulte specimen van clarias lazeva beslaat het + 23% van de darmlengte. Er bestaat dus geen r e l a t i e tussen de mogelijkheid macromoleculen op te nemen en het ontbreken van een maag. U i t a r t i k e l D b l i j k t bovendien dat de afwezigheid van een maag n i e t of nauwelijks van invloed i s op de e f f i c i ë n t i e van de e i w i t v e r t e r i n g . De funktie van het 2e segment b l i j f t dan ook o n d u i d e l i j k . B i j v i s l a r v e n , waarbij het voedsel zéér snel het achterste deel van de darm b e r e i k t , kan het een k w a n t i t a t i e f belangrijke rol spelen b i j de eiwitopname, maar b i j oudere vissen heeft de pinocytose van macromoleculen vermoedelijk alleen kwalitatieve betekenis. Ook b i j zoogdieren worden kleine hoeveelheden e i w i t als macromoleculen opgenomen. Deze hebben geen betekenis voor de voeding, maar z i j n w e l l i c h t op één of andere wijze biologisch a k t i e f (bijvoorbeeld als anti geen). U i t het f e i t dat b i j de graskarper ondanks het ontbreken van een maag en een v r i j snelle passage van het voedsel door de darm (+ 7 uur)toch een zeer e f f i c i ë n t e e i w i t v e r t e r i n g plaatsvindt v a l t af te leiden dat de maag met betrekking t o t de e i w i t v e r t e r i n g slechts van secundaire betekenis i s , of althans dat de productie van pepsine en/of zuur geen voorwaarde vormt voor een adequate vertering van voedseleiwitten. 40 Reprinted from J. Fish Biol. (1977) 11,167-174 Growthanddiet dependant structural adaptations ofthe digestivetract injuvenile grasscarp {Ctenopharyngodon idella,Val.) H. W. J. STROBAND Agricultural University, Department of Experimental Animal Morphology and Cell Biology, Hollandseweg 13, Wageningen The Netherlands {Accepted 8 June 1976) The morphology of the intestinal epithelium is described and compared with published data. In the gut of this stomachless fish, three zones can be distinguished based on light and electron microscopic observations of absorptive cells: a rostral zone with numerous fat droplets, a second zone with large P.A.S.-positive supranuclear vacuoles and pinocytoticvesicles,andathirdzonewithoutthesecharacteristics. Inthesezonesdeepinvaginations of the cell membrane is a feature of the basal part of the cells. The presence of pinocytotic vesicles in the second zone isconsidered evidence of the uptake of undigested protein-like substances. Juvenile grass carp from 15to 335 days, fed on either animal or vegetable food, were measured. The growth rates indicate that animal food stimulates rapid growth. The data also show a vegetable diet causes a slight increase in relative gut length, mainly in the length of thefirst zone. I. INTRODUCTION TheadultWhiteAmur or grasscarp(Ctenopharyngodon idellaVal.)eatslargequantitiesofaquaticplantsand asaresultiseffective inweedcontrol (Lin, 1935;Penzes& Tolg, 1966;Fischer, 1968;Cross, 1969; Stott &Robson, 1970). Thefish also has a high growth rate (Hickling, 1960; Anderson, 1970) which increases its economic significance, and consequently the grasscarp has beenthe subject ofmany investigations. Several authors have described the histology of the alimentary tract in Cyprinids (Rogick, 1931;Curry, 1939;Klust, 1940;McVay &Kaan, 1940;AlHussaini,1949a, b; Girgis, 1952). It appears to be difficult to relate histological and morphological adaptations to the types of food. According to Nikolsky (1956, cited by Hickling, 1966)theyounggrasscarp iscarnivorous until reachinga length of 2-5cm;whenits feedinghabitschangerapidly,anditreachestheherbivorousstageatalengthof3cm. InseveralCyprinids,fat andproteinappear tobeabsorbed indifferent partsofthe gut(Yamamoto, 1966;Iwai, 1968,1969;Gauthier &Landis, 1972; NoaillacDepeyre & Gas, 1973). The location, relative length and histological features of these parts werethe subject of our investigation. II. MATERIALS AND METHODS Anumberofgrasscarpfry(15 daysafter hatching)wereprovidedbythe'Organisatieter Verbetering van deBinnenvisserij', Holland in February 1975. Onarrivalthefisheswere 167 168 H. W. J. STROBAND dividedintothreegroups,allwerefed onTubificidae(theirownbodyweight aday). Group2 received additional plant material (young darnel). From the28th day after hatching(when thedarnel wasconsumedfor thefirsttime)thefishingroup2receivedtubifex and anexcessiveamount ofdarnelandthethirdgroupwasfedanexcessiveamount ofdarnelonly. After 50days the darnel wasreplaced by grass pellets and the daily amounts of tubifex werereduced to 100ga day per 30animals. Eachgroup of 30animals waskept in a 2001 tank at 23°C±1° C,withlightfor 14haday. Standardlengthandweightwereregularly measured. Forhistologicalexamination,theanimalsweresacrificed at 15,27,48 days,3and 7months, each time 2-4h after feeding. The wholefish(up to 2 months old; the ventral body wall removed) orsmallpiecesof tissue(fish 2months or older)werefixedin Flemmingsosmium tetroxidefixative(Romeis, 1968). Thetissueswereembedded inparaffin waxandcut at 1-4 urn. Azonaldifferentiation ofthegutwasmadehistologically. Todeterminethelengthofthe subdivisions,thenumberofhistologicalsectionsofeachzonewascounted. Infishlargerthan 50mm,thelengthofthetotalalimentarytractwasmeasuredbeforefixation,andpartsofthe gutwereexaminedhistologicallytolocatethetransitionofeachzone. For electron microscopical examination, the tissues were prefixed in a mixture of 1% osmium-tetroxideand2%glutaraldehydebuffered with0 1 M sodiumcacolylatebuffer pH7-2 for 15 min,and postfixed for45mininthesamemixturetowhichl%potassium bichromate was added. Fixation was carried out at 0°C. The tissues were embedded in epoxy resin. Ultrathin sectionswerecut on aLKBHuxleyor LKBIII Ultramicrotome and photographs weremadeonaPhilipsE.M.300electronmicroscope. TABLEI. Growth rate of grasscarp fed on animal or vegetable food Standardlength(mm) r Age(days) 15 28 48 57 70 110 206 335 Group1 (animal food) — — 43±4-7(8)* 49±7(3) 61±5-6(2) 95±7(2) 113±9-4(10) 120±8(10) Group2 (control animals) 16-3±1-4(6)* 25-7±2-8(9) 42-2±3-9(5) — 55± 7(2) 88± 5(2) — 110±13(10) Group 3 (plant food) — 31-9±3-2(8)* 45± 4(3) 56±5-4(4) 66-7±5-2(4) 76±8-l (10) 75± 6(10) *Thenumber offishexamined isindicated between brackets. III. GROWTH RATE A difference ingrowth ratewas observed betweengrasscarpfed ontubifex (group 1) and those fed on grass (group 3) (see Table I). Between the groups 1(animal food) and 2 (animal+plant food) no significant differences were found. Tubifex-fed fish grewabout0-8mm/day to an ageof 110days;thenthegrowthdiminished. A reduced growth rate for plant fed animals was noticed from the start of the experiment. The reduced growth of tubifex fed fish between 110 and 335 days may be partly due to crowding. The meanweight of specimensfrom the two groups is also different: at 30days this was 370mg, at 7 months the mean weight of plant fed animals was 7-3±2-6 g, and that oftubifex fed specimenswas24-7±7 g(for ten animalsper group). DIGESTIVE TRACT OF GRASS CARP 169 IV. MORPHOLOGY AND REGIONAL DIFFERENTIATION OF THE GUT In Cyprinids, the stomach is absent. The oesophagus opens directly into the intestine a few mm anterior to the entrance of the ductus choledochus. Thefirst limb or intestinal bulb (intestinal swelling) is wide. Its rostral part has complex branching mucosalfolds; at theend oftheintestinal swelling,thesefolds areat right angles to the long axis of the gut. From this area the diameter of the intestine decreasestowardstheanus. 15Days cp^ t o 27Days 48Days I- 5mm FIG. 1. A schematic drawing of the digestive tract of grass carp at three stages of development. Ventralview:cp,caudal pharynx;t,position ofpharyngeal teeth; o,oesophagus;ib,intestinal bulb; ip, intestine proper; a, anus. A and B,boundaries of the second zone of the intestine. Based onhistological features theintestinecanbedividedintothreezones(Fig.1). Table II showsthe relative length of the total alimentary canal, the intestine and its three zones in specimens of different ages and fed on different kinds of food. The relativelengthofthegutincreasesfrom approximately 1xstandard bodylengthin15 daysto 2xbody length in 210days. This increase was mainly due to growth of the firstzone. The relative length of the gutislargerin grass fed animals than in those fed on tubifex. The difference can be attributed to growth of thefirstzone of the intestine. 170 H. W. J. STROBAND TABLEII. Ratioofstandard bodylengthtolengthofthegutanditssubdivisions Intestine A Age(days) Totalcanal r Zonel Zone2 Zone3 0-4 0-4 0-6 0-6 0-3 0-3 0-3 0-3 005 01 0-2 0-2 10 11 1-4 1-4 Total 07 0-8 1-1 11 210(1) 210(1) 210(1) 210(1) 1-8 1-8 1-7 1-9 1-5 1-5 1-5 1-6 0-9 0-9 0-9 0-9 0-3 0-4 0-3 0-4 0-3 0-2 0-3 0-3 210(3) 210(3) 210(3) 210(3) 210(3) 2-4 2-2 20 1-9 20 20 1-9 1-7 1-6 1-7 1-3 1-2 10 0-9 0-9 0-4 0-4 0-3 0-3 15 27 48(1)* 48(3) 0-7 0-4 0-4 0-3 0-3 * (1)= group1fedonanimalfood. (3)= group3fedonplantfood. V. HISTOLOGY HISTOLOGY OF THE INTESTINAL EPITHELIUM The gut does not possess villiform projections, but the surface contains mucosal foldswhichgraduallydecreaseinheightfromtheanteriortotheposteriorpartsofthe intestine. The mucosa islinedwithacolumnar epithelium consistingoftwotypesof cells:absorptive cellswithmicrovilliand,lessfrequently, glandular cellsofthegoblet typewithP.A.S.positivecontents. The latter cellsare moreabundant inthe caudal part of the gut. Other celltypes found between the epithelial cellsinclude:lymphocytes (Plate I), cells with rod-like inclusions [probably Rhabdospora thelohani (Laguesse)], and a few acidophilic granular cells. At the base of the folds, mitotic stagescanbeobserved (PlateI). Specialattention waspaidto the appearance ofthe absorptive cellsthroughout the intestinal canal. These cells are high columnar with nuclei situated in the lower third, the nuclei have clear nucleoli, the cytoplasm is granularandhasa subapicalbasophilicband andabasalbasophilicarea. Theintestinewasdivided intothree distinct zonesonthebasisoftheappearanceof the absorptive cells. In the first zone (including the intestinal bulb and the rostral part of the intestine proper), many cells in the apical region of the mucosal folds showed a number of clear vacuoles between nucleus and the subapical basophilic cytoplasm(PlateI). Thevacuolesafter Bouinsfixationappearedemptybutshowedan osmiophilic substance afterfixationin Flemmingsfluid(Plate II). The nucleiin this zonebecomelargerandmoreroundedtowardstheapicalregionofthemucosalfolds. The cells contained small P.A.S. positive granules in the perinuclear cytoplasm. Thesecondzoneofthegutischaracterized bythepresence oflarge' supra-nuclear bodies' and vacuoleswith basophilic and P.A.S.positive contents (Plate III). Cells withvacuoleswere observed alloverthe mucosal folds. Thevolume ofthevacuoles PLATEI. Cross section through mucosal fold inthefirstzoneof intestine ofan animal of48days,fed on tubifex and plant material. Note the presence of clear vacuoles in absorptive cells at the apex of the fold. Mitotic figures can be seen at the base of the fold. A few goblet cells are present(arrows). Bouinfixative,Azan stain. DIGESTIVE TRACT OF GRASS CARP 171 increasedfrom rostraltocaudalinthesecondzone. Theapicalbasophilicareaofthe cellsisdistinct in this zone (Plate III). Finally, a third zoneisfound near theanus. Inthiszonetheabsorptivecellsdonotcontainvacuoles. This description is based on control and tubifex fed animals, between which no difference could bedetected. Theepithelium ofplantfedfishisnearlysimilarto that of animal fed specimens, the absorptive cells of the first zone of the gut do not, however,containvacuoles,andthecellsinallthreezonesareshorter. ULTRASTRUCTURAL FEATURES OF ABSORPTIVE CELLS* OF CONTROL ANIMALS The absorptive cells in the first zone of the intestine are typically columnar and contain well-developed microvilli (Plate IV). In the terminal web area beneath the microvillous border, cellular organelles are absent and the cytoplasmic matrix consists of a dense fibrillar meshwork. Between the terminal web and the nucleus (which is situated at one third of cell height) the cytoplasm is packed with vesicles varying in diameter. They are lessnumerous in the cytoplasm between nucleus and basal membrane. The vesicles contain small particles with a diameter of approximately 70nm (Plate IV). Similar particles were found in the numerous intercellular spaces. Mitochondria andpolyribosomes arepresent throughout thecell,the former aremorenumerousinthebasalregion (PlatesIV,V). In somecellsstrandsofrough endoplasmaticreticulum(RER)havebeendetected. Inthebasalpart ofthecellsand occasionally in apical positions lamellar structures are present, generally running paralleltothe longaxisofthecells(PlateV). Onlycellsattheapicesofthemucosal folds contain largevacuoleswith electron lucent contents (PlateIV). In the second zoneofthe gut the absorptive cellsshowinvaginations ofthe apical cell membrane into the terminal web area (Plate VI). One or more large supranuclear vacuoles, mostly surrounded by mitochondria, contain an electron lucent mass and numerous fibrillar structures (Plate VI). A distinct Golgi apparatus, a number of mitochondria and scattered strands of RER are present between these vacuoles and the nucleus. Many mitochondria, RER and lamellar structures are presentinthebasalcytoplasm. Thecellsofthethirdzoneoftheintestinearecharacterizedbytheirregulararrangement of the microvilli, which are short and few in number (Plate VII). The cells contain many mitochondria, especially in apical and basal position. A perinuclear Golgi apparatus is always present. Many vesicles and tubules of smooth endoplasmatic reticulum can be observed in the cytoplasm. Lamellar structures similar to thosedescribedforfirstzonecellsarealsopresentnearthebasalmembrane. VI. DISCUSSION Theobservation that thegrasscarpjuveniles growfaster onanimalthan vegetable food suggeststhefishesarenotherbivorousinthefirst7monthsafter birth. Thisisin accordancewith Fischer (1972)whostressed theneedfor animalproteinsfor normal growthofCtenopharyngodon idella. Thelengthof theintestinalcanalof 7month old specimensdoesnotindicate aherbivorouswayoflife sinceotherpresumed herbivorousCyprinidshaveanintestineofsixtimesthebodylength(Campostomasp.Rogick, *Details of ultrastructure are given as far as they underline the characteristics of the absorptive cells in the three zones, and to compare our results with experimental ultrastructural data in the literature. Adetailed description oftheintestinalultrastructure willbepublishedelsewhere. 172 H. W. J. STROBAND 1931)oreven15timesthebodylength(Labeohorie, Girgis,1952). Ourresultsdonot allowconclusionsastothetimeintervalinwhichthechangefrom acarnivoroustoa strictly herbivorouswayoflife takesplace. Olderjuveniles andevenadultgrasscarp have the samerelative gut length as our 7months oldfish(Inaba &Nomura, 1956; Berry &Low, 1970); consequently the suggestion of Inaba &Nomura (1956) that grass carp gradually acquires omnivorous habits after the first month and actually neverbecomesrealherbivorous seemsplausible. Hickling(1960)reported veryrapid growthofgrasscarponNapier-grass. Howeverthefishwerekeptinlargepondsand the stomach contents were not analysed, it cannot be excluded therefore that small amounts of animal food were swallowed with thegrass. McVay &Kaan (1940)werethefirstauthors to describe a regional differentiation ofthegutofaCyprinid. Accordingtotheseauthors,theanterior half ofthe goldfish Carassiusauratus,intestineischaracterizedbythepresenceofabsorptivecellswithout a single perinuclear vacuole, which was invariably found in cells of the posterior intestine. This issupported by our results in plant fed animals. In tubifex fed grass carp,however, osmiophilic droplets werefound in cellssituated inthe apical part of themucosalfoldsinthefirstzoneoftheintestine. Thissuggeststhat absorption offat takesplaceinthesecells. Gauthier &Landis(1972)found absorbed lipidsappearing firstinthe apicalcellsand later inmorelateralregionsoffolds inthe anterior gutof thegoldfish. Ourultrastructural examination indicatesthepresenceoflipid droplets, possiblychylomicrons,invesiclesofdifferent sizesand inintercellular spaces of only thefirstzoneoftheintestineofthegrasscarp. Thelargevacuolesfound attheapexof the mucosalfoldspossiblyrepresent stored lipids,sinceabsorption seemsmost active inthisarea. Theabsenceofpinocytoticinvaginationsinanterior intestine absorptive cellswasalso observed byYamamoto (1966)and Gauthier &Landis (1972)ingoldfish and byIwai(1969)injuvenilecarp. Therefore, fat isprobably hydrolysed inthe gutlumenand absorbed andresynthesized incellsofthefirstzoneofthegut. Thisis in accordance with Al Hussaini (1949è), Sarbahi (1951) and Hickling (1966), who statedthat lypolyticactivity islocated mainly inthe anterior intestine of stomachless teleosts. This mechanism of fat absorption closely resembles that of the intestinal triglycerideabsorption intherat(Cardelletal., 1967). In the grass carp, the large supranuclear vacuoles in the second zone of the gut containaP.A.S.-positive substance. ThisisconsistentwiththeresultsofGauthier & Landis (1972) and Noaillac-Depeyre & Gas (1973a), obtained in goldfish and carp respectively. The latter authors proved that digestion by amylase did not affect this affinity for P.A.S. Onthebasisofthisobservation and ofthestainingreaction ofthe substanceinthevacuoleswithtoluidineblue,theyconcludedthesubstanceisaneutral or slightly anionic glycoprotein. The same authors showed that proteins are absorbed asintact molecules in the epithelial cellsof the posterior intestine of goldfish and carp; in these cells they found indications of pinocytotic activity. Yamamoto (1966)concluded that pinocytosistakesplaceinabsorptive cellsoftheposterior half of the gut in the goldfish. Our results indicate that protein absorption in the grass carptakesplacebypinocytosisinthesecondzoneoftheintestine,asinothercyprinids. Studiesoftheactivitiesofseveraldigestiveenzymesmadeitclearthatpepticdigestion isabsentinthesestomachlessteleosts(Sarbahi, 1951;Ishida, 1936). Trypticactivity, however, was found in the posterior half of the intestine in grass carp (Hickling, 1965),goldfish (Sarbahi, 1951),carp,roach Rutilus rutilus and gudgeon Gobio gobio (Al Hussaini, 19496). The incomplete digestion of proteins by the absence of a DIGESTIVE TRACT OF GRASS CARP 173 stomach isprobably compensated by pinocytosis. A regional differentiation as in the gutofadultCyprinidswasobserved in someteleost larvae (Iwai, 1968a,b, 1969;Iwai& Tanaka, 1968a, b). Most authors do not distinguish different zones in what they call the posterior intestine of fishes. In the grass carp a caudal third zone can be clearly distinguished histologically from the second zone. The presence of relatively few short microvilli suggeststhat absorption isnot asimportant asinother zones ofthegut. The presence of many lamellar structures in the basal part of the absorptive cells is in accordance with the results of Noaillac-Depeyre & Gas (1973è) for the ' rectum ' of the goldfish. These authors suggested that this part of the gut is specialized in ion transport. Yamamoto (1966) did not pay special attention to the most posterior part of the intestine ofthe goldfish, but described lamellar structures inthe absorptive cellsof the whole intestine. If these structures are related to ion transport, our results indicate that in the grass carp the first and the third zone of the gut are best adapted to this function. Our results indicate the more rapid increase in relative length of the intestine of plant fed grass carp compared to that in tubifex fed specimens is merely a relative increase in length of the lipid absorbing zone of the gut. This zone may be also responsible for carbohydrate absorption, as a P.A.S. positive granulation was found in the absorptive cells, and amylase is found chiefly in the anterior intestine of a number of Cyprinids (Al Hussaini, 1949Ô,Sarbahi, 1951 ;Hickling, 1966). The cause of the enlargement of the gut and its physiological significance needs further investigation. The distribution of absorptive cells in the intestine and mucosal folds differs from that in mammals. The distinct regional differentiation in the carp intestine is considered useful for investigating the differentiation of epithelial cells. The cyprinid is a useful model for examining fat and protein absorption pathways, the kinetics of the epithelial cellsin different zones ofthe gut, and the influence of different kinds of food on the latter. Theauthorwishestothank Professor Dr J.W.M.Osseand Dr LucyP. M. Timmermans for theirstimulatinginterest,MrW.Valenfor preparingtheillustrationsand MrEleriforhis technical assistanceinelectron microscopy. References Al-Hussaini,A. H. (1949a). Onthefunctional morphology of the alimentary tract of some fish inrelationtodifferences intheirfeedinghabits. I:AnatomyandHistology. Q.J. microsc. Sei.90,109-139. Al Hussaini,A. H. (1949A). Onthefunctional morphology of the alimentary tract of some fishin relation to differences in their feeding habits. II: Cytology and Physiology. Q.J.microsc.Sei.90,323-354. Anderson, E. N. Jr. (1970). Traditional aqua culture in Hong Kong. J. Trop.Geogr. 30, 11-16. Berry,P.Y. &Low,M.P.(1970). Comparativestudiesonsomeaspectsofthemorphology and histology of Ctenopharyngodon idellus,Aristichthys nobilisand their hybrid (Cyprinidae). Copeia1970,4,708-726. Cardell,R.R.Jr.,Badenhausen,S.&Porter,K.R.(1967). Intestinaltriglyceride absorption intherat. / . CellBiol.34,123-155. Cross,D.G.(1969). Aquaticweedcontrolusinggrasscarp. J.Fish. Biol. 1,27-30. Curry,E.(1939). Thehistologyofthedigestivetubeofthecarp(Cyprinus carpio communis). J.Morph.65,53-78. 174 H. W. J. STROBAND Fischer, Z.(1968). Foodselection in grasscarp(Ctenopharyngodon idellaVal)underexperimentalconditions. PolskieArchwm.Hydrobiol.15,1-8. Fischer, Z. (1972). The elements of energy balance in grasscarp {Ctenopharyngodon idella Val). Part.Ill, Assimilability of proteins, carbohydrates and lipids byfishfed with plantandanimalfood. PolskieArchwm.Hydrobiol.19,83-95. Gauthier, G.F. &Landis,S.C.(1972). Therelationshipofultrastructural andcytochemical features to absorptive activityinthegoldfish intestine. Anat.Rec.172,675-701. Girgis, S.(1952). On the anatomy and histology of the alimentary tract of an herbivorous bottom-feeding cyprinoidfish,Labeohorie(Cuvier). J. Morph. 90,317-362. Hickling, C. F. (1960). Observations on the growthrate of the chinese grasscarp,Ctenopharyngodonidellus(Cuv.etVal). MalayAgric.J.43,49-53. Hickling,C.F.(1966). Onthefeeding processinthewhiteAmur, Ctenopharyngodon idella. J.Zool.140,408-419. Inaba,D.&Nomura,M.(1956). Onthedigestivesystemandfeedinghabitsofyoungchinese carpcollectedintheRiverTone. J.TokyoUniv.Fish.42,17-25. Ishida,J.(1936). Distribution ofthedigestiveenzymesinthedigestivesystemofstomachless fish. AnnotnesZool.Jap.15,263-284. Iwai, T. (1968a). Fine structure and absorption patterns of intestinal epithelial cells in rainbowtroutalevins. Z.Zellforsch.Mikrosk.Anat.91,366-379. Iwai, T. (19686). The comparative study of the digestive tract of teleost larvae V. Fat absorption in thegut epithelium of goldfish larvae. Jap.Soc.Sei.Fish. 34,973-978. Iwai, T. (1969). Fine structure of gut epithelial cells of larval and juvenile carp during absorption offat andprotein. Arch,histol.Jap.30,183-189. Iwai, T. & Tanaka, M. (1968a). The comparative study of the digestive tract of teleost larvae. III. Epithelialcellsintheposteriorgutofhalfbeaklarvae. Jap.Soc.Sei.Fish. 34,44-48. Iwai,T.&Tanaka,M.(1968b). Thecomparativestudyofthedigestivetractofteleostlarvae. IV. Absorption offat bythegut ofhalfbeaklarvae. Jap.Soc.Sei.Fish.34,871-875. Klust, G. (1940). Über Entwicklung, Bau und Funktion desDarmes beim Karpfen. (CyprinuscarpioL.). Int.Revueges.Hydrobiol.Hydrogr.40,88-173. Lin,S.Y. (1935)Life history of Waan Ue, Ctenopharyngodon idellus (Cuv. &Val.). Lignan Sei.J. 14,129-135. McVay,J.A.&Kaan,H.W.(1940). ThedigestivetractofCarassiusauratus.Biol.Bull.Mar. biol. Lab. Woods Hole.78,53-67. Noaillac-Depeyre,J. & Gas, N. (1973a). Absorption of protein macromolecules by the enterocytesofthecarp. (CyprinuscarpioL.). Z. Zellforsch. Mikrosk. Anat.146, 525541. Noaillac-Depeyre,J. &Gas,N. (1973b). Miseenévidenced'unezoneadaptée au transport des ions dans l'intestine de carpe commune (Cyprinus carpio L.). C.r.Hebd. Séanc. Acad. Sei.(Paris) 276,773-776. Penzes,B. &Tolg,I. (1966). Etude de la croissance et de l'alimentation de la ' grasscarp ' (Ctenopharyngodonidella)en Hongrie. Bull.fr. Piscic39,70-76. Rogick, M.D. (1931). Studieson thecomparative histology ofthedigestivetubeof certain teleost fishes. II. Aminnow (Campostoma anomalum). J. Morph.52,1-25. Romeis, B. (1968). Mikroskopische Technik. München: Oldenbourg Verlag. Wien. 16 Auflage. Sarbahi, D. S. (1951). Studies of the digestive tracts and the digestive enzymes of the goldfish, Carassius auratus (1.)and thelargemouth black bass,Micropterussalmoides (Lacépède). Biol. Bull. mar. biol. lab. WoodsHole,100,244-257. Stott, B. &Robson, T. O.(1970). Efficiency ofgrasscarp (Ctenopharyngodon idella Val.) in controllingsubmergedwaterweeds. Nature,Lond. 226,870. Yamamoto, T. (1966). An electron microscope study of the columnar epithelial cellin the intestineoffresh waterteleosts:goldfish (Carassiusauratus)andrainbowtrout(Salmo irideus).Z. Zellforsch. Mikrosk. Anat.72,66-87. Printed in Great Britain by Henry Ling Ltd., at the Dorset Press, Dorchester, Dorset Cell Tiss. Res. 187, 181-200 (1978) CellandTissue Research © by Springer-Verlag 1978 The Ultrastructure and Renewal of the Intestinal Epithelium of the Juvenile Grasscarp, Ctenopharyngodon idella (Val.) H.W.J. Stroband and F.M.H. Debets* Agricultural University, Department of Experimental Animal Morphology and Cell Biology, Zodiac, Wageningen, The Netherlands Summary.The intestinal absorptive epithelium of starved and fed fish has been studied electron microscopically.After feeding, cellsoftheproximal segment of theintestineshowmorphological characteristics oflipidabsorption. Absorptive cellsinthemiddle segment contain manypinocytoticvesiclesinboth fasted and fed specimens.Absorption of protein macromolecules issupposed to be one of themainfunctions ofthispartofthegut.Inthemostcaudalpartoftheintestine, absorptive cells carry relatively few and short microvilli. The proximal and distal segments show structural indications of a function in osmoregulation. The renewal of the epithelium has been studied with light microscopic autoradiography, using tritiated thymidine. The intestinal mucosal fold epithelium represents a cell renewal system. The cells proliferate at the base of the fold and migrate towards the apex in 10-15 days at 20°C. The functional absorptive cells proved to be generally present in the intestinal epithelium, including theproliferative area. Undifferentiated cellshavenot been identified. The results will be compared with data on absorption of lipid and protein macromolecules in teleostean and mammalian intestines and with descriptions of the cell renewal system in the mammalian intestine. Key words: Intestine - Teleost - Epithelium - Renewal - Ultrastructure. Introduction A differentiation of the intestinal epithelium was found in a number of Cyprinids (whichdonothaveastomach)(Yamamoto, 1966and Gauthier andLandis, 1972in the goldfish; Iwai, 1969inthecarp; Noaillac-Depeyre and Gas, 1976in the tench). Therostralpartofthegut,includingtheintestinalbulb,wasshowntoabsorblipids, Send offprint requests to: Dr. H.WJ. Stroband, Agricultural University, Department of Experimental Animal Morphology and Cell Biology, Zodiac, Prinses Marijkeweg 40, Wageningen, Niederlande * The authors wish to thank Prof. Dr. J.W.M. Osse and Dr. Lucy P.M. Timmermans for their stimulating interest, Mr. H.G. Eleri(T.F.D.L.) for his technical assistance in electron microscopy, and Mr. W.J.A. Valen for making the drawings and light microscopic photographs 0302-766X/78/0187/0181/$04.00 182 H.W.J. Stroband and F.M.H. Debets and the posterior part of the intestine absorbs protein by pinocytosis. NoaillacDepeyre and Gas(1973b) demonstrated that ion transport takes place in the most caudalpartofthegutofthegoldfish. Inarecentmorphological study,the grasscarp intestine was shown to consist of three segments (Stroband, 1977). In the goldfish (Vickers, 1962; Hyodo, 1965) and the carp (Gas and NoaillacDepeyre, 1974), the intestinal epithelium represents a cell renewal system. Cell proliferation takesplaceinthebasal part ofthemucosal folds, and thecellsmigrate to the top of the folds where they are probably discarded. The present study deals mainly with the renewal of the intestinal epithelium of thegrasscarp.Autoradiography wasappliedafter injecting 3 H-thymidine,to locate the supposedly present proliferative pool of cells and to trace the renewal of the epithelium. The ultrastructure ofabsorptivecellshasbeenstudied inthree areas of themucosalfold: thebase,thecentralpartandtheapex.Thepurposewasto obtain general information on the ultrastructure of absorptive cells and on the morphology of possibly differentiating cells,migrating from the proliferative pool totheapex ofthefolds. To identify functional absorptive cells,thedifferences have been studied between these cells in fed fish and in starved specimens. Materials and Methods Autoradiographic studies were made on 21 one year old juvenile grasscarp (Ctenopharyngodon idella, Val.) provided by O.V.B. Lelystad, Holland. These were injected intraperitoneally with methyl- 3 Hthymidine (1uCi/gram body-weight; spec, activity 18 Ci/mmol; Radiochemical Centre, Amersham, England). Before and during the experiment the fish were fed with Tubificidae. The water temperature was 20°C±1°C. Three animals were killed with ±0.3U/Oo MS222 (Sandoz, Basel, Switzerland) after 3h, 24h, 2days, 5, 10, 15and 21days.Tissues were fixed inBouin's fixative and embedded in paraffin. Sections of 3um were stained with haemalum and eosin. Radioautograms were made with slides preparedinKodak NT B2emulsion(Eastman-Kodak, Rochester,NewYork),diluted 1:1with distilled water. The slides were exposed for three weeks. Forelectronmicroscopicpurposes,7-9monthsoldgrasscarpwerestarvedfor 15days(2animals) or 40days(4animals).In addition, two specimens wereregularly fed. Three ofthe starvelings and the two regularly fed fishes were given tubifex approximately 3h before being killed. All these animals were anaesthetized in0.1°/ 00 MS222,and small fragments ofintestinal tissuewereexcised from the locations showninFig. 1.For electron microscopicalexamination, tissueswereprefixed for 15mininamixtureof 1% osmium tetroxide and 2 % glutaraldehyde buffered with 0.05M Na-cacodylate (pH 7.2) at 0"C. Postfixation was made in a similar mixture to which 1 %potassium bichromate was added. The tissues weresectionedonaLKBIIIultramicrotome.Thesectionswerecontrasted withuranylacetate, followed by lead citrate according to Reynolds (1963), and photographs were made on a Philips 300 electron microscope. Results 1. The Renewal of the Intestinal Epithelium 3h after injection of 3 H-thymidine, labelled epithelial cells can be noticed in the three segments of the gut. Most of these cells are found in the central segment (Table1), and they are concentrated in the basal third of the folds (Figs.2,4; Table1). Grasscarp Intestinal Epithelium 183 Table1.Percentageoflabelledcellsinagivenareaofthemucosalfoldsintwospecimens(IandII),3h after 3H-thymidine injection Area(in % of total fold length) 0%= baseof the fold 0-10 10-20 20-30 30-40 40-50 50-60 Percentage oflabelled cells in agiven area measured in 5-8 folds/segment proximal segment middle segment distal segment I II I II I II 3.3±0.5 1.1±0.4 1.3±0.7 6.5±1.0 2.9±1.6 8.2±1.8 4.6±1.1 2.5±0.8 2.0±1.0 0.9±0.4 0.7±0.4 10.2±2.6 2.3±1.7 3.3±0.9 3.7±1.1 0.7±0.5 1.7±1.3 3.9±1.1 1.5±0.4 0.6±0.3 - - - - - Figure4shows that labelled cellsmigrate totheapex ofthemucosal folds.The result after 10-15days are shown in Figure 3.Theheight inthefolds reached by labelled cells 48hafter 3 H-thymidineinjection andthelarge standard deviation in thiscolumn suggest aconsiderable individual variation intheproliferative activity of the intestinal epithelium. Three hours after injection labelling in the intestinal epithelium may be quite different from place to place; this indicated that proliferation varies in the folds with time. The well-labelled areas have been used for quantification. 2. The Ultrastructure of the Epithelium of theProximal Segment a) In FedFish.Most ofthecellsofthe(columnar) epithelium consist of absorptive cells.Goblet cellsarepresenttoo,andalso someendocrinecells(Fig.18;Rombout, 1977) and migrating cells (lymphocytes, granulocytes and macrophages). The apical part ofthe absorptive cellbears numerous microvilli(Fig.5)with an asymmetric cell membrane (Fig.8). The part of the cell adjacent to the lumen is covered with a thin fuzzy coating or glycocalyx. The cytoplasm of the microvilli containsanumber oflongitudinal fibrillar structures,penetratingtheterminalweb. The tips ofthe microvilli have a dense cap(Fig.5),named capitulum by Krementz and Chapman (1974). The lateral plasma membranes of adjacent cells run closely together from the microvillous border to thebasement membrane without complex interdigitations. Desmosomes are frequently encountered, most of them lying in the terminal web area whereajunctional complex ispresent(Fig.9).Inthebasal halfof thecells,the lateral plasma membranes form long lamellar infoldings parallel tothelong axisof the cells. These infoldings can be recognized by the regular distance between the membranes( ± 250Â) andbythepresence oflumen material with a relatively high electron density. Themembranes are relatively thick and asymmetrical, just asin the microvilli (Fig.6). The same type of infoldings originates in the basal plasma membrane(Fig.7).Similar structures weredescribed byNoaillac-Depeyre andGas H.W.J. Stroband and F.M.H. Debets 184 anus «^ .è #•' ... «, . i • * j> -I *r- <. > $ , i551t .. •v>; f .•• i ... V _ _ À ' l :'• ;4A>.*V •t v >- •'t1-."'*•• « HA» • >/ yt. t -* %i;'~ ' \t,-' • - . 'i * % % . ' • - • • 2b 2a ,* ? " .' iV- *.t '% ** ••'•.••• . ** ^ t, • •ƒ•• *•<;'?-w t' % <• . -'.#. ' • ' ^ >. * * < •/,;- ' , . « - . ». - • v. j, ••••* %'.. . _ • - #* **.. ..', $': >••" • . -..''* 1? '• \' A-I *- m A l ; t •• X. ! Grasscarp Intestinal Epithelium 185 im 10096 "T"l à 3h rb > < 24h < 5d < lOd > < I5d Fig.4.Areasofthemucosal foldscontaininglabelledcells.Eachvaluerepresentsthemean of3animals; 5-8 folds have been measured per fish. Ordinate: % of fold height. Abscissa: time after injection (1973b)incellsofthe"rectum"ofthecarp,anditwassuggestedthattheymayplay a role in ion transport. Between the microvilli, pinocytotic vesicles may be present (Fig.9), which do not contain fat droplets, most membranes of which have a coated appearance. A terminal webispresent below the microvillous border (Fig.5). In this area cytoplasmicorganellesarescarce,andribosomesandsomevesiclesmaybepresent also. Mitochondria, located in the basal parts of the cells(Fig.13),are generally closely connected with the lamellar structures. Thenucleusoftheabsorptivecellisovalandlocatedinthebasalpartofthecell at about one third of the height.A few ribosomes may be attached to the outer membrane of the nuclear envelope, which isgenerally smooth (Fig. 10). Rough-(RER) and smooth-(SER) endoplasmic reticulum are overall present, except in the terminal web area. In the upper part of the cells, the SER is predominant. Many small fat particles, approximately 700 in diameter, are present in the SER, RER, Golgi apparatus (which is located in the perinuclear cytoplasm) and intercellular spaces (Figs.9,10,11,12). Large droplets of fat are present too(Fig.10),especially in thecytoplasm above thenucleus,and theseare surrounded by a membrane. b) InFastedFish.Themoststrikingchangeintheabsorptivecellsafter fastingisthe absenceoffat particlesand fat dropletsinthecellsand intheintercellular spaces. Fig.1. Schemeoftheposition ofthegrasscarpintestine.Theoesophagus(O)opensdirectlyinto the gut. In the wide first limb, or intestinal bulb (IB), the ductus choledochus (DC) opens a few mm from the oesophagus (O). From theend of the first limb theintestine proper (IP) narrows and leads to the anus. Thearrowsindicatethelocationsofthetissuesusedinthisstudy.A andZJrepresenttheboundariesofthe middle segment. GB gallbladder Fig.2aandb. Radioautograph of the middle segment of the intestine 3h after injection with 3 Hthymidine.TheDNA precursor ispresentinnucleiincellsofthebasalpart ofthemucosalfolds, x 125.b As Fig.2a. x250 Fig.3. Radioautograph of the middle segment 10days after injection. Labelled cells have reached the apex of the mucosal folds, x 125 Figs.5-8.Absorptive cellsin the proximal segment Grasscarp Intestinal Epithelium Mitochondriawerenotonlyfoundinlargenumbersinthebasalpartofthecells, asin fedfish,but they areconcentrated in theupper third of theabsorptivecells also.Thissuggeststhat severalofthemitochondria havemigrated tomoreapical parts of the cellsduring the fasting period. This type of apical concentration of mitochondria isnot noticed three hours after feeding, following a long period of fasting. The matrix of mitochondria of fasted specimens (Fig.5) contains dense granules, which disappear after feeding. c) CellsatDifferentLocationsoftheMucosalFolds.Allcellorganellesappeartobe welldeveloped,eveninthemostbasalpartofthefolds.Adistinctindicationofcells at thebaseofthefoldsbeinglessdifferentiated istheirnarrow shapecompared to theshapeinotherareasofthefold(theaveragenumberofmicrovillipercellis15in sectionsatthebaseofthefoldsand 30insectionsatthetopofthemucosalfoldsin theproximal segment) and thegreat number offree polyribosomes(Fig.15).One typeofcolumnar cellisrestricted to thebaseofthefolds. Thiscellcontainsmany large mitochondria with electron dense granules intermingled with ER and free ribosomes (Fig.17). This type may be found in groups of two or three cells. Lymphocytes arepresentin thebasal part ofthemucosal folds, generally inlarge numbers, most of which are located near the basement membrane. After feeding, thecellsinthebasalpartofthefoldsaresimilartocellsatother locations. The ER (Fig.16)and Golgi vesicles are filled with lipid particles, and mostmitochondria arelocatedinthebasalpart ofthecells,butthisislessdistinct than at other locations of the fold. After fasting neither lipid particles nor lipid droplets have been observed. After feeding thecellsat the topofthefolds aredifferent from thoseat other locations. Next to structures mentioned above, several large lipid droplets(up to 4u) are present, most of them in the supranuclear cytoplasm (Fig.14). The extracellular spaces are very large in this area and the different location of the mitochondria in fed and fasted fish ismost conspicuous in theseparts. Fig.5. Theapicalpartofsomecellsinthemiddleareaofamucosalfoldinafastedfish.Manyvesiclesof SERwithoutlipidparticlesbelowtheterminalweb(TfV). Notetheabundantpresenceofmitochondria (M)in thispart of thecells,containing densegranules x16,600 Fig.6.Thelateralplasmamembranes(LM)ofadjacent cellsinthenuclear(NU)area.Themembranes form infoldings(I)containinganelectrondensematerial.Themembranesareasymmetricand thicker than the lateral plasma membrane, x81,500 Fig.7.Basalportionofacellwithinfoldingsofthebasalplasmamembrane(arrows),closelyconnected withmitochondria(M).Betweenthebasalcellmembraneandthebasementmembrane(BM)anarrow spacecan be noticed, x41,000 Fig.8.Transverse section through microvilli. Many fibrils form the core of the microvilli. The unit membrane isasymmetric. Themicrovilli are covered with a thin fuzzy coating, x104,000 1 187 « •«•»w n om. .£vi^y >A ^«*<fe^W^iASBrî 200 H.W.J. Stroband and F.M.H. Debets Noaillac-Depeyre, J., Gas, N.: Absorption of protein macromolecules by the enterocytes of the carp (Cyprinus carpio L.). Z. Zellforsch. Mikrosk. Anat. 146, 525-541 (1973a) Noaillac-Depeyre,J.,Gas,N.:Miseenévidenced'une zoneadaptéeautransport desionsdans l'intestine decarpcommune(Cyprinus carpioL.).C.R.Hebd. Séance.Acad. Sei.(Paris), 276, 773-776(1973b) Noaillac-Depeyre, J., Gas, N.: Fat absorption by the enterocytes of the carp(Cyprinus carpioL.). Cell Tiss. Res. 155, 353-365 (1974) Noaillac-Depeyre,J., Gas,N.: Electronmicroscopicstudyongutepithelium ofthetench(TineatineaL.) with respect to its absorptive functions. Tissue & Cell 8, 511-530 (1976) Palay, S.L., Karlin, L.J.: An electron microscopic study of the intestinal villus II. The pathway of fat absorption. J. biophys. biochem. Cytol. 5, 373-383 (1959) Reynolds, E.S.: The use of lead at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208-212 (1963) Rombout, J.H.W.M.: Enteroendocrine cells in the digestive tract of Barbus conchonius (Teleostei, Cyprinidae). Cell. Tiss. Res. 185, 435^150 (1977) Sarbahi,D.S.:Studiesofthedigestivetracts and thedigestiveenzymesof thegoldfish, Carassiusauratus (L) and the largemouth black bass, Micropterus salmoides (Lacépède) Biol. Bull. mar. biol. lab. Woods Hole, 100, 244-257 (1951) Strauss, E.W.: Electron microscopic study of intestinal fat absorption in vitro from mixed micelles containing linolic acid, monolein and bile salt. J. Lipid Res. 7, 307-323 (1966) Stroband, H.W.J.: Growth and diet dependant structural adaptations of the digestive tract in juvenile grasscarp (Ctenopharyngodon idella, Val.). J. Fish. Biol. 11, 167-174 (1977) Toner, P.G.: Cytology of intestinal epithelial cells. Int. Rev. Cytol. 24, 233-243 (1968) Trier,J.S.,Rubin, C E .: Electronmicroscopy ofthesmallintestine.Areview.Gastroenterology 49,574603 (1965) Vickers, T.:A study of the intestinal epithelium of the goldfish Carassiusauratus: its normal structure, thedynamics ofcellreplacement, and thechanges induced by saltsofcobalt and manganese. Quart. J. Micr. Sei. 103,93-110(1962) Yamamoto, T.:An electron microscope study of the columnar epithelial cell in the intestine of fresh water teleosts:goldfish (Carassius auratus)and rainbow trout(Salmo irideus).Z. Zellforsch. 72,6687 (1966) Accepted October 28, 1977 "»••' r'-fi-WJfm-'1,'-' "^f>F"ISJJ**" ï ' • ï -< , »? ' IA- * \ > «.« »'S *.~,i i.*li4f 3 t *• • ( .;>* ±LL Hl stoche ml str y 64, 235-249 (1979) Histochemistry © by Springer-Verlag 1979 Regional Functional Differentiation in the Gut of the Grasscarp, Ctenopharyngodonidella (Val.) H.W.J. Stroband, H. v.d. Meer, and Lucy P.M. Timmermans Agricultural University, Department Experimental Animal Morphology and Cell Biology, Zodiac, Marijkeweg 40, Wageningen, The Netherlands Summary. A regional differentiation - reflecting structural differences - of the intestine of larval andjuvenile grasscarps can be illustrated by studying the activity of alkaline phosphatase and the uptake of orally administered horseradish peroxidase. Pinocytosis of peroxidase takes place in a welldefined area of about 23% of the length of the gut (segment II). Neither the rostral ±68% (segment I)nor the caudal ±9% (segment III) shows absorption of the enzyme. Alkaline phosphatase activity, mainly localized at the microvilli of the enterocytes is high in the first segment of the gut and low in the second segment. In larvae, the activity decreases sharply at the transition from segment I to segment II. The activity is weak or absent in the caudal third segment.Quantitativehistochemical data areconfirmed bybiochemical analyses. Alkaline phosphatase activity is found all over the mucosal folds of the first segment, with relatively weak activity at the base and at the tip of the folds. This may be related to a renewal of the epithelium. Our results suggest that active absorption of digested food takes place mainly in the rostral first segment, while the uptake of macromolecules by pinocytosis is a function of the second segment. Comparison of the results with information available in literature leads to a rejection of the hypothesis that the uptake of protein macromolecules in Cyprinids is to be attributed to the absence of a stomach and therefore to an inefficient digestion of proteins. Introduction An understanding ofabsorption processes and related morphological and physiologicalcharacteristics in larval,juvenile and adult stomachless teleosts isvaluable for fish culture. This is especially true because most teleosts pass through a larval period (Balon, 1975)in which exogenous food is used when a stomach 0301-5564/79/0064/0235/$03.00 LOCALIZATION OF PROTEIN ABSORPTION DURING TRANSPORT OF FOOD IN THE INTESTINE OF THE GRASSCARP, CTENOPHARYNGODON IDELLA (VAL.) H.W.J.StrobandandF.H.vanderVeen, AgriculturalUniversity,DepartmentofExperimentalAnimalMorphologyand CellBiology,Zodiac,Marijkeweg 40,Wageningen,The Netherlands SUMMARY Theuptake of proteinand aminoacids in the intestine of young (18months old) and adult grasscarp has been studied with the inertmarker (Cr„0~)method. Theabsorption ofprotein takesplace in theanterior 40-50% of thegut. The second intestinal segment (+25%of gut length)has the ability to absorbmacromolecules ofprotein.Ferritin (MW300.000)was found inpinocytotic vesicles inenterocytes of this second segment.On thebasis ofmorphological and histochemical evidence,ithas previously been suggested that the pinocytotic uptake ofwholeproteinmoleculesmight be related to the lack of pepticdigestion instomachless fishes.Amajor quantitative function inprotein absorption however cannotbe attributed to the second gut segment,and proteins appear tobe digested adequately in these stomachless animals.The function of this partof thegut and therole of the stomach inprotein digestion inother vertebrates remain tobe explained. The absorption of individual aminoacids in the intestine of the grasscarp suggeststhatessential aminoacids arepreferentially absorbed. The rapid disappearance of lysine and arginine from the chyme points toa tryptic breakdown of proteins. I REFERENCES Austreng,E.(1978).Digestibilitydeterminationinfishusingchromicoxide markingandanalysisofcontentsfromdifferentsegmentsofthegastro intestinaltract.AquacultureJ3^265-272. Barrington,E.J.W. (1957).Thealimentarycanalanddigestion.In:"ThePhysiologyofFishes"(M.E.Brown,ed.),VolI109-161.AcademicPress,NewYork, Bell,G.H.,Davidson,J.N.&Emslie4smith,D. (1972)."Textbookofphysiology andbiochemistry"ChurchillLivingstone,Edinburgh,London. Ben-Ghedalia,D.,Tagari,H.,Bondi,H.&Tadmor,A. (1974).Proteindigestion intheintestineofsheep.Br.J.Nutr.3J_>125-142. Bergot,P. (1976).Demonstrationwithrutheniumredofdeepinvaginationsinthe apicalplasmamembraneofenterocytesinrainbowtroutintestine. Ann.Biol.Anim.Biochem.Biophys.16,37-43. Cowey,C.B.&Sargent,J.R. (1979).Nutrition.In:"FishPhysiology",HoarW.S., Randall,D.J.&Brett,J.R. (ed|s).Vol.VIII,1-69.Acad.Press,NewYork, London. Crampton,R.F. (1972).Nutritionalandmetabolicaspectsofpeptidetransport. In:"Peptidetransportinbacteriaandmammaliangut",CibaFoundation symposium 11nov.1971.Ass.Sei.publ.Amsterdam. Creach,P.V. (1963).Lesenzymesprotéolytiquesdespoissons.Ann.Nutr.Alim. J2.375-471. Davenport,H.W. (1977)."Physiologyofthedigestivetract".Yearbookmedical publishers,Inc.,Chicago. Dijke,J.M.van&Sutton,D.L. (1977).Digestionofduckweed (Lemmaspp)bythe grasscarp (Ctenopharyngodon idella) J.Fish.Biol.JJ_,273-278. Farmanfarmaian,A.,Ross,A.&Mazal,D. (1972).Invivointestinalabsorption ofsugarinthetoadfish (Marineteleost, Opsanus tau). Biol.bull.142, 427-455. Furakawa,A.&Tsukahara,H.(1966).Ontheaciddigestionmethodforthedeterminationofchromicoxideasanindexsubstanceinthestudyofdigestibilityoffishfeed.Bull.Jap.Soc.Sei.fish.32,502-506. Gauthier,G.F.&Landis,S.C. (1972).Therelationshipofultrastructuraland cytochemicalfeaturestoabsorptiveactivityinthegoldfishintestine. Anat.Rec.172,675-701. Hickling,C F . (1966).OnthefeedingprocessinthewhiteAmur, Ctenopharyngodon idella. J.Zool.140,408-419. Hsu,Y.L.&Wu,J.L. (1979).Therelationshipbetweenfeedinghabitsanddigestiveproteasesofsomefreshwaterfishes.Bull.Inst.Zool.Acad.Sin.18, 45-53. Iwai,T.(1968).Thecomparativestudyofthedigestivetractofteleostlarvae Vfatabsorptioninthegutepitheliumofgoldfishlarvae.Bull.Jap.Soc. Sei.Fish34,973-978. Iwai,T.(1969).Finestructureofgutepithelialcellsoflarvalandjuvenile carpduringabsorptionoffatandprotein.Arch,histol.jap.30_,183-189. Jacobshagen,E.(1937).DarmsystemIVMittelundEnddarm.Rumpfdarm.In: "HandbuchderVergleichendenAnatomiederWirbeltiere"(L.Bolk,E.Göppert E.KalliusandW.Lubosch,eds).Vol.Illpp.563-724. UrbanandSchwärzenberg,BerlinundWien. Jany,K.D.(1976).Studiesonthedigestiveenzymesofthestomachlessbonefish Carassius auratus Gibelio (Bloch):Endoptidases.Comp.Biochem.Physiol. 53B,31-38. Kawai,S.&Ikeda,S.(1973).Studiesondigestiveenzymesoffishes-IVdevelopmentofthedigestiveenzymesofcarpandblackseabreamafterhatching. Bull.Jap.Soc.Sei.Fish.39,877-881. 14 Krause,W.J.,Cutts,J.H.&Leeson,C R . (1977).Thepostnataldevelopmentof thealimentarycanalintheopossum:IIIsmallintestineandcolon. J.Anat.123,21-46. Krementz,A.B.&Chapman,G.B. (1974).Ultrastructureoftheposteriorhalfof theintestineofthechannalcatfish, Iotalurus punotatus. J.Morph._U5,441-482. tfepham,T.B.&Smith,M.W. (1966).Aminoacid transportinthegoldfishintestine. J.Physiol.184,673-684. Noaillac-Depeyre,J.&Gas,N.(1973)• Absorptionofproteinmacromoleculesby theenterocytesofthecarp {Cyprinus aarpio L.).Z.Zellforsch.146, 525-541. Noaillac-Depeyre,J.&Gas,N.(1974).Fatabsorptionbytheenterocytesofthe carp {Cyprinus aarpio L.)CellTiss.Res.155,353-365. foaillac-Depeyre,J.&Gas,N. (1976).Electronmicroscopicstudyongutepitheliumofthetench {Tinaa tinea L)withrespecttoitsabsorptivefunctions. Tissue&Cell8,511-530. Joaillac-Depeyre,J.&Gas,N. (1979).Structureandfunctionoftheintestinal epithelialcellsintheperch {Peraa fluviatilis L).Anat.Rec.195, 621-640. )nishi,T.,Murayama,S.&Takeuchi,M. (1976).Changesindigestiveenzyme levelsincarpafterfeeding:IIIresponseofproteaseandamylasetotwice adayfeeding.Bull.Jap.Soc.Sei.Fish42,921-929. )rten,A.U.(1963).Intestinalphaseofaminoacidnutrition.Fed.Proc.22, 1103-1109. ihcherbina,M.A. (1973).Astudyofdigestiveprocessesinthecarp {Cyprinus aarpio) CommunicationIabsorptionofcrudefatintheintestinefrom syntheticdiets.Vopr.Ikhtiol.J_3,119-127. ihcherbina,M.&Sorvatchev,K. (1969).Afewdataconcerningtheabsorptionof aminoacidsinthedigestivetractoftwoyearsoldcommoncarp. Vopr.Prud.Rubov. \6, 315-322. Ihcherbina,M.A.etal. (1976).Trofimova,L.N.&Kazlauskene,O.P.Theactivity ofproteaseandtheresorptionintensityofproteinwith theintroduction ofdifferentquantitiesoffatintothefoodofthecarp Cyprinus aarpio J.Ichthyol.J6^632-636. ihcherbina,M.A.,Shcherbina,T.V.&Kazlauskene,O.P. (1977).Amylaseactivity andrateofcarbohydrateresorptionwiththeintroductionofvariousamounts offatintothedietofthecarp, Cyprinus aarpio. J.Ichthyol.17, 327-331. mith,M.W. (1966).Sodium-glucoseinteractioninthegoldfishintestine J.Physiol.182,559-573. taley,T.E.,Corley,L.D.,Bush,L.J.&Jones,E.W. (1972).Theultrastructure ofneonatalcalfintestineandabsorptionofheterologousprotein. Anat.Rec.172,559-579. troband,H.W.J. (1977).Growthanddietdependantstructuraladaptationsof thedigestivetractinjuvenilegrasscarp {Ctenopharyngodon idella Val.) J.Fish.Biol. H.»167-174. troband,H.W.J.&Debets,F.M.H. (1978).Theultrastructureandrenewalofthe intestinalepitheliumofthejuvenilegrasscarp Ctenopharyngodon idella (Val.).Cell.tiss.Res.187,181-200. troband,H.W.J.,Meer,H.v.d.&Timmermans,L.P.M.(1979).Regionalfunction differentationinthegutofthegrasscarp Ctenopharyngodon idella (Val.). Histochemistry64,235-249. troband,H.W.J.&Dabrowski,K.R.Morphologicalandphysiologicalaspectsofthe digestivesystemandfeedinginfreshwaterfishlarvae.In:"LaNutrition desPoissons"CNERNA(Paris),inpress. 15 Stroband,H.W.J.&AnnetG.Kroon.Thedevelopmentofthestomachin Clarias lazera andtheintestinalabsorptionofproteinmacromolecules. SubmittedtoCell.tiss.Res.June1980. Tanaka,M. (1969).Studiesonthestructureandfunctionofthedigestivesyste inteleostlarvaeII.Characteristicsofthedigestivesysteminlarvaeat thestageoffirstfeeding.Jpn.J.Ichthyol.J_6,141-149. Tanaka,M. (1970).Studiesonthestructureandfunctionofthedigestivesyste inteleostlarvaeIII.Developmentofthedigestivesystemduringpostlarv stage.Jap.J.ofIchthyologyJ_8,164-174. Walter,W.A.&Isselbacker,K.J. (1974).Uptakeandtransportofmacromolecules bytheintestine.Possibleroleinclinicaldisorders.GastroEnterol.67, 531-550. Yamamoto,T. (1966).Anelectronmicroscopestudyofthecolumnarepithelialce intheintestineoffreshwaterteleosts:goldfish (Cavassius auratus) and rainbowtrout (Salmo irideus). Z.Zeilforsch.72,66-78. 16 THE DEVELOPMENT OF THE STOMACH IN CLARIAS LAZERA AND THE INTESTINAL ABSORPTION OF PROTEIN MACROMOLECULES H.W.J.Stroband andAnnetG. Kroon Agriculture University, Department of Experimental Animal Morphology and Cell Biology,Wageningen, The Netherlands. SUMMARY The development of the stomach of the teleost Clarias lazera is described for theearly posthatching period.A comparison ismade to the stomach of adult Clarices. The stomach develops in two distinct parts:the corpus,which is theearliestdifferentiated part,and the pylorus.The corpus contains amucous surface epithelium, arranged infolds,and atubular gland system containing only one type of gland cell, towhich thesecretion of pepsinogen and HCl is attributed. Thepyloric region does not contain tubular glands. From theultrastructure of thegland cells,the H thymidine labeling index, and theonset of acid production (asdetermined with pH indicators) it isconcluded that afunctional stomach ispresent in juvenileswith astandard length of +_ 11mm. (approximately 12days after fertilization at 23-24C). Inaddition to the stomach, theultrastructure of the intestinal epithelium hasbeen studied. The gut shows three segments,similar asdescribed for stomachless teleosts and anumber of fish larvae. In larvae aswell as injuveniles,the enterocytes of the second segment showpinocytosis ofhorse radish peroxidase, although in thejuveniles thestomachhas already developed. This second segment has the same relative length inall studied larvae and juveniles and isalso present inadult Clarias• Therefore itmust be concluded that the ability of absorbing proteinmacromolecules isnot definitely related to the absence of a functional stomach in this teleost species. Key words: Stomach - Intestine -Epithelium -Teleost -larvae. INTRODUCTION Instomachless teleosts the intestine shows aregional differentiation. The anterior segment has themorphological characteristics of lipid absorption, while the shorter second segment has the ability toabsorb proteinmacromoleculesby pinocytosis (Yamamoto,1966;Iwai,1969;Gauthier&Landis,1972;Noaillac-Depeyre&Gas,1973a,1976;Stroband,1977;Stroband&Debets,1978;Strobandet al.,1979).Thethirdsegmentisprobablyinvolvedinionandwatertransport (Noaillac-Depeyre&Gas,1973b;Stroband&Debets,1978). Itwaspostulatedbytheabovementionedauthorsthattheabsorptionof macromoleculesmightberelatedtothelackofastomachandtoaninadequate proteindigestion.Thisisalsoknowninsucklingmammals,inwhichpinocytosis ofproteinmacromoleculestakesplaceinthesmallintestinebeforethestomach isfullyfunctional(Cornell&Padykula,1965;Kraehenbühl&Campiche,1969; Krauseetal.,1977).Inourviewtherearetwowaysfortestingthementioned hypothesis.Ifnoappreciablequantitiesofproteinareabsorbedbythesecond gutsegmentinstomachlessfish,thehypothesiswouldbewithoutanyfoundation. Experimentsareinprogressandthefirstresultswillbepublishedelsewhere (Stroband&VanderVeen,inpreparation).Thesecondwayistostudythepost hatchingdevelopmentofthedigestivetractofateleostwithastomach.Usuall) teleostlarvaelackafunctionalstomachuntilseveralweeksafterthestartof exogenousfeeding (Tanaka,1972).Duringthisperiodtheintestineshowssimilai morphologicalcharacteristicsasfoundinthestomachlessfishes.Consequently, thesecondsegmentoftheintestineofteleostlarvaeislikelytoabsorbprote: macromoleculesalso. Theprincipalobjectiveofthepresentstudyistoascertainwhetherchange occurintheformandfunctionoftheintestine,especiallyinthesecondgut segment,whenthestomachbecomesfunctional.Therefore,thedevelopmentofthe digestivetracthasbeenstudiedwithlight-andelectronmicroscopicalmethods. 3 Theperiodofdevelopmentofthestomachwasalsodeterminedwith Hthymidineasamarkerofcellproliferation.Moreoveritwasexpectedthatthelabelingindexdecreaseswhencellsbecomefunctionallyactive. pHIndicatorshavebeenusedtofindoutwhenacidityreachespH5-4,as cathepsinaswellaspepsinhasanoptimumpHbelow5.Theuptakeofhorseradi: peroxidaseservedasamarkerformacromoleculeabsorption. MATERIALANDMETHODS Twobatchesofnew-hatched Clarias lazera (Cuv.andVal.)larvaeand6adu: specimenwerekindlyprovidedbytheDepartmentofFishCultureandInland FisheriesoftheAgriculturalUniversity.Althoughthelarvaewererearedin exactlythesameway,thetwobatchesshowedsomewhatdifferentgrowthrates (fig. 1). g.1.Growthratesofthetwobatchesof Clavias larvae,(a)and(b). E z 10. Thelarvaewererearedat23-24C.At5,6,8,9,12and15daysafterfer.lizationanumberoffishesofbath (a)wereinjectedwith+30-40nl.ofa Ithymidinesolution (1Ci/L,spec.act.24Ci/mmol,RadiochemicalCentre, ïersham,England).After3-4hincubationsomespecimenswerefixedinBouin's jcativeandsubsequentlyembeddedinparaffin.4pSectionswerestainedwith emalumandeosinorwithAlcianBlueandSchiffsreagens (Rcmeis,1968). dioautographsweremadewithKodakNTB-2liquidemulsion(Eastman-Kodak, ehester,NewYork),diluted1 : 1 . Theslideswereexposedfortwoweeks.For eexperimentsdescribedbelow,thesecondbatch (b)ofClariaslarvaewasused, ecimensof4,7,9,10,11,12and15daysoldwerepreparedforelectron croscopy:theywerefixedinamixturecontaining 2%osmiumtetroxide, 2%gluraldehydeand M potassiumbichromate,bufferedwith0,05MNa-cacodylate H7.2)at0C.1 \iSectionswerepreparedforradioautography.UltrathinseconsweremadeonaReichertUltratomeIVmicrotome,contrastedwithuranyl etateandleadcitrateaccordingtoReynolds (1963),andphotographedwitha ilips300electronmicroscope. Furthermore,specimensof4to21dayswerefedwith Artemia satina ina horseradishperoxidasesolution(Sigma,St.Louis;typeII,mol.Wt.+ .000).Theyswallowedtheenzymetogetherwiththefood.After2hthefishes refixedfor2hin3%glutaraldehydebufferedwith0,05MNa-cacodylate(pH 2),rinsedwithwaterfor3h,frozeninliquidnitrogenandfreezedriedfor h.Afterembeddinginparaffin,5ysectionswerepreparedandincubatedat Dmtemperaturefor10-30minutesinamediumaftervanDuyn (Pearse,1972). 1 2 3 4 5 Fig. 2.a. schematic drawings of thedigestive tract of Clarias (batch a)at t stages ofdevelopment.Dorsalview: o= oesophagus; s= stomach; i= intestin« a= anus.A and Brepresent theboundaries of the second gut segment =wall of thecorpus of the stomach. = wall of thepyloric part of the stomach. b. ordinate:Mean percentages of H thymidine labeled epithelial cells three fishes at the same 6stages of development as fig.2a. abscissa: 1,2, 4 and 5represent the caudal oesophagus,themost rostral,mid and caudal (pyloric)part of the stomach,and the intestine respectively. For thestomacl 2 slideshavebeen counted per location ineach fish.In the intestine thepel centage of labelled cellswas counted at 7locations over theentire length. Duringtheexperiments,everydaysomespecimensofbath (b)wereanaestheLzedwith0,1 /ooMS222immediatelyafterfeeding.Asmalldoseofmethyl-red rcongo-redwasinjectedintothestomachthroughaglasscapillary. Partsofthedigestivetractsofadult Clarias lazera werefixedandpre- iredforlightandelectronmicroscopyasdescribedabove. 5SULTS , The morphology and regional differentation of the digestive tract. Fig.2ashowsthemorphologyofthedigestivetractasreconstructedfrom Lghtmicroscopicpreparations.Beforeday5,an"Anlage"ofthefuturestomach )uldnotbedetected.Theintestineshowedthethreesegmentsasdescribedfor :omachlessfish.Theesophagusisdirectlyfollowedbythefirstsegment,and iebileductentersthisfirstsegmentveryclosetotheesophagus.Asfromday ,themostcaudalpartofthe"esophagus"graduallydevelopsintothestomach, liehiseasilyrecognizedhistologicallybytheglandulartissueinthethickened .11(figs.3,4,5).Betweenday9andday15thepyloricpartofthestomachis veloped. Afterthisstagenoimportantchangesseemtotakeplaceinthemorphology thedigestivetract,apartfromaproportionalgrowthofthevariousparts, erelativelengthofthegut(ascomparedtobodylength)andtherelative ngthsoftheintestinalsegmentsremainthesame(table 1). Inadultsthethird gmentisrelativelyshort,butthesecondsegmenthasthesamelengthasin rvae. Table1.Ratioofthelengthoftheintestinetothestandardbodylength,andthe relativelengthofthethreegutsegmentsasdetermined for3specimensofeach age(batcha). age standardlength (days) (mm) lengthintestine bodylength relativelengthofgutsegments (%) I II III 5 8,0* 0,36 68,3+1,6 22,0+1,6 6 8,4 + 0,5 0,44 73,0+4,0 16,7+_3,8 10,3+1,5 9,8+2,4 8 10,4* 0,34 67,7+3,5 20,0+2,6 12,3+1,5 9 11,9 + 0,5 0,29 69,7+2,1 22,0+1,0 12 14,3 + 0,6 0,35 64,3+5,5 20,7+4,0 12,0+1,0 8,3+1,5 15 15,2 + 0,8 0,37 66,0+1,0 23,7+0,6 10,1+1,5 adult 257,8 + 15,7 0,45 70,8+3,1 23,9+2,4 5,2+0,5 * :extrapolationsfromfig.1. 2. The differentiation of the digestive 3 H-thymidine labeling index. tract epithelium as determined by its 3 Thepercentageof H-thymidine labeledcellsinseveralpartsofthedigestivetractandatdifferentstagesofdevelopmentarepresentedinfig.2b.In larvaeof5days,21to231-oftheepithelialcellswerelabeledovertheentire lengthoftheintestine,butlargevariationswerenoticedineachspecimen. Labeledcellswerefoundallovertheepithelium,butmostarepresentinthe lowerpartsofthemucosalfolds (fig.6).Duringdevelopmentthepercentageof labeledcellsinthegutgraduallydimishedto+ 31onday15,whenlabeledcells 3 Fig.3-6:Incorporationof Hthymidineinthegutof Clarias lazera. Fig.3.Larva,4daysold.Lightmicroscopicradioautographofa1yeponsection stainedwithtoluidineblue.Notethehighlabelingindexofthetissues. S=surfaceepithelium;Gl=undifferentiatedglandularcells,(x500) Fig.4.1ueponsectionthroughthecorpus (C)pylorus (P)androstralintestine (Int)ofa7daysold Clarias. Notetherelativelyhighlabelingindexinthe pyloricareaandtheverylowpercentagesoflabeledcellsinthegut (x250) Fig.5.Developingstomachofa12daysoldlarva.Afewlabeledcellsinthe anteriorcorpusofthestomach (C)andahighlabelingindexintheposterior pyloricarea(P). x250 Fig.6.Mucosalfoldofthesecondintestinalsegmentofa4daysoldlarva. Manysupranuclearvacuolesintheenterocytesandmostlabeledcellsatthebase ofthefolds,x500 irerestrictedtothebasalpartofthemucosal folds.Intheareaofthedevelo)ingstomach,thelabeling indexdiminishes later,firstintheanteriorpartand atermorecaudally (figs.2-5).Asfromday9thepyloricareacanberecognized. tslabelingindexof+30°sdecreasesbetweenday12andday15tosimilarvalues .sfoundinotherareasofthedigestive tract.Asintheintestinal epithelium, iroliferativesurfacemucus cellsincorpusandpylorusaremerelyfoundinthe asalpartofthefoldsafterthedropofthelabeling index.Glandularcells emain labeledatrandominallstages. Fortheproliferationoftheepitheliumofthesuccessivepartsofthediestive tract,thefollowingisofinterest.Acomparisonofradioautographsof psectionswithultrathin sectionsdidnotshowanydistinctdifferencesbetween 3 hecytologyof H-thymidinelabeledcellsandothercells,neitherinthesurface pithelium (fig.21)andglandularepitheliumofthestomachnorintheepithelium ftheintestineofthestudied fishes (upto10days old). FunctionalcellsappaentlyhavethefacilitytosynthesizeDNA;consequently theyareabletodivide. . Ultrastructure and regional differentiation of the intestine. Inlarvaeof4daystheesophagus,mainly linedwithmucous cells,enters irectlyintothefirstsegment.Thisfirstsegmentisinvolvedinlipidabsorpion.Absorptive enterocytes,recognizedbytheapicalborderofmicrovilli,conainchylomicrons intheirendoplasmaticreticulum (ER)andGolgi apparatus, irgelipiddropletsarealsopresent (fig.7).Chylomicronsarealsofoundin ieintercellular spacesbetweentheenterocytes.Pinocytotic vesiclesarescarce rabsent.MitochondriaandstrandsofERarepresent throughoutthecytoplasm Lgs. 7-9: Electronmicrographs of gut absorptive cells in Clarias lazera. Lg.7.Apical part of an absorptive cell in the caudal part of the first itestinal segment of a4days old larva.MV =microvilli;TW= terminalweb; L=mitochondria; L = lipid droplet;Ch = chylomicrons;G =Golgi apparatus Lthchylomicrons,x 17.000 Lg. 8. Some absorptive cells in the second intestinal segment.Many pinocytotic :sicles (arrows)are present beneath themicrovillous border (MV),Supranuclear icuoles (SV),probably originating from fusing pinocytotic vesicles and lyso>mes.4Days old larva,x 26.000 .g.9.Enterocytes in the third segment of thegut of a7days old larva.Many imellar infoldings of theplasmamembrane in stacks (arrows)and afew small .crovilli (MV).x 13.000 10 exceptinthatoftheterminalweb.Lamellarinfoldingsofthebasalandlateral plasmamembranesoccur,justasinenterocytesofotherteleosts. Absorptiveenterocytesofthesecondsegmentshowheavypinocytosisbeneath theirmicrovillousborder (fig.8).Moreovermanypinocytoticvesiclesandone01 morelargesupranuclearvacuolesmaybepresent (figs.6, 8).Chylomicronshave notbeenfoundintheER,intheGolgiapparatus,andintheintercellularspaces butsomefatdropletsmaybepresentinthecytoplasm. Themostcaudalsegmentdoesnothavethecharacteristicsofthefirstand secondsegments;itlackslipiddroplets,chylomicrons,pinocytoticvesicles,and supranuclearvacuolesintheenterocytes.Themicrovilliarerelativelyshort andfewinnumber.Asinotherteleosts,foodabsorptiondoesnotseemtobean importantfunctionofthesecells.Lamellarinfoldings,however,arefoundin largenumbers,generallyconcentratedinstacks (fig. 9). Duringdevelopmentnoimportantchangescanbenoticedinthemorphologyof theabsorptiveenterocytes.Itisnoteworthythatthesecondsegmentremainsof thesamelengthinallstages.Inadult Clarias thissegmenthasthesamecharacteristicsasinlarvae (table 1). 4. Ultrastrueture of the developing stomach. Thefirst"Anlage"ofthestomachcanberecognizedonday4withtheelectronmicroscopebythepresenceofafewlobulesoffutureglandularcells,forme byinvaginationsofthesurfaceepitheliumofwhatseemstobethemostcaudal partoftheesophagus. Epithelialcells (fig.10)areelongate,andthenucleus (withlargenucleolus)issituatedinthebasalhalfofthecells.Neighbouringcellshavecomplex interdigitationsofthelateralcellmembranes.AdistinctGolgiapparatusis presentandmanystrandsofRERarefoundthroughoutthecells.Mucoidgranules maybefoundinthecytoplasm,butareaccumulatedintheapexofthecell.The roundtoovoidgranulesaremembranebound (fig.10).TheyprovedtobeP.A.S. positiveafterAlcian-blue-P.A.S.reactiononparaffinmaterial;thereforethese granulesprobablycontainneutralmucopolysaccharides.Thecytologyoftheepitheliumliningofthelumendoesnotchangemuchinlaterstagesofdevelopment,but Fig. 10.Epithelial cells from thestomach of an 11days old larva. NU =nucleus;Int.= complex interdigitations of the lateral plasmamembranes; G =Golgi apparatus;M =Mucoid granules;Mi =mitochondria; RER= rough endoplasmatic reticulum,x 28.000 11 moregranulesandstrandsofendoplasmicreticulumandfewerfreeribosomesare foundinolderspecimens.Afewveryshortmicrovillimaybepresentonthesurfaceoftheepitheliumlining.Inadultspecimensthesecretiongranulesare flatterandmorenumerous (fig.19);theyaremoreelectrondensethaninlarvae. Thetransitionfromsurfaceepitheliallayertotubularglandepitheliumis veryabrupt,withoutatransitionor"neck"zone. Theglandularcellscanbeeasilydistinguishedfromtheepitheliumlining cellsbythelackofmucousgranulesandthepresenceofanumberofsmooth vesiclesintheapicalarea(figs.11-16).Thecellsarepyramidalandshowcomplexinterdigitationsofthelateralcellmembranes.Thenucleicontainprominent nucleoli.Manymitochondriaarepresentinthecytoplasm.AwelldevelopedGolgi apparatusisfoundnearthenucleus.At4daysonlyafewstrandsofRERhave beennoticed.Atthatagethecytoplasmisfilledwithfreeribosomes (fig. 11). Apartfromtheliningepitheliumonlyonetypeofstomachglandularcell wasfoundinthevariousstages.Theapicaltubularsystem,inearlystagesconsistingofanumberofvesicles,developsrapidly (fig.13-14)andfillsupthe greaterpartofthecellsinspecimensof11days.Sincethemembranesofthe vesiclesstronglyresembletheapicalplasmamembrane (fig.14),thesemembranes mayhavethesameorigin.Importantchangeshavebeennoticedinthemorphology ofthetubularsystemduringlaterontogeny.Inlarvaefrom11daysoldthe vesiclesweremoreelongated,possiblybecauseofmutualfusion.Inadultspecimensthemoreelongatetubulesaremerelyextendinginlongitudinaldirection (fig. 16). Asfromtheageof7dayssmallsecretiongranulesappearinthecytoplasm oftheglandularcells (fig.12).Inthefollowingdayslargezymogengranules areformed (figs.4,13)andonday11thefirstindicationsofexocytosiscan beobserved (fig. 15). InadultspecimenstheRERfillsupthebasalpartoftheglandularcells (fig.17).Adifferenceinthemorphologyofglandularcellsofyounganimals andadultsisthepresenceofmanymicrofilamentsinadults,runningfromthe junctionalcomplexintothecytoplasm (fig.18).Glandularcellsareabsent beneaththeepitheliumlininginthepyloricareaofthestomach,whichisalso Fig. 11. Corpus gland cells of a4days old larva.T=apical tubular system; Nu =nucleus;Mi =mitochondria; G =Golgi apparatus x 22.000 Fig. 12.Corpus gland cells ina 7days old larva. Some small secretion granules (arrows).Legends as fig. 11.x 22.000 13 14 'ecognizedbyarelativelywelldevelopedmuscularwall.Thecellsoftheepihelial liningaresimilartothose inthecorpusregion. . pHindicator test. ThepHindicatormethyl-red (colourchange fromyellowtoredbetweenpH nd4,4)onlyshowsaredcolourwhen injected intothestomachof13daysan< lderlarvae.However,afterinjectingalargedoseofindicator this colour hangewasnotnoticedat13days.Inlarvaeof14daysandolderthecolour hangewasdistinctlyvisible,evenwith largerquantitiesofindicator.Conge ed (pH5,2to3,3)changestoblueinlarvaeof15days.Duringthenextfew aysthereactionwasevenmoredistinct.This shows thattheacidityoftheÏ achcontents afterfeedingishigherthanpH5inyoung larvaeofupto11,5 tandardlength.DuringthenextfewdaysthepHdropstovaluesof5,2-3,3. Anumberofolder larvae survivedthetreatmentwith indicator.Theday£ heexperimentthedyeswere foundinthegutlumen.Theircolourswereyellov ndred(methyl-red,resp.Congo-red),which indicates thatthepHinthegut jmenishigherthan5. . Uptake of horse radish peroxidase by enterocytes. Aftertheingestionofhorseradishperoxidase,enzymeactivityisnotice ienterocytesofthesecondsegmentonly (fig.20).Apositive reactionwas i ialllarvaeandjuvenileswestudied (4to15days).Thelocalizationofthe îroxidaseactivitysuggests thattheenzymeispresentinthesupranuclearpa Etheabsorptive cells,evidentlywithinthepinocytoticvesiclesandthelar ipranuclearvacuoles.Thelengthofthesegmentwithperoxidaseabsorptionan s locationcorrespondtothesecondsegmentasdetermined histologically. g. 13.Corpus glandular cells ina12daysoldlarva.Many large zymogengra îles(Zy)especially inthebasalpartofthecells.Theapical partisfille thtubules.Legendsasfig.11.x 15.000 g. 14.Theapical portionofacorpus gland cellofa12daysoldlarva.The ructureoftheapicalplasmamembraneissimilar tothatofthemembranesof emany apical tubules,sincebothareasymmetrical,x52.000 g. 15.Apical partofacorpus gland cellofa12daysoldlarva,showing ocytosisofasecretion granule,x26.000 15 16 DISCUSSION 1. Regional differentiation of the Intheintestineoflarval Clarias intestine. lazera aregionaldifferentiationin threesegmentscanbemade,similartotheoneinstomachlessfish.In Clarias larvaetheenterocytesinthedifferentsegmentshavethesamehistological characteristicsas instomachlessfish(Yamamoto,1966;Iwai,1969;Gauthier& Landis,1972;NoaillacDepeyre&Gas,1974,1976;Stroband&Debets,1978). Similarfeatures,includingthepresenceof asecondsegmentwithpinocytosisof macromolecules,havebeendescribedfortheintestineofotherinitiallystomachlesslarvaeoffishspecieswhichpossessastomachinthejuvenileandadult stages(Iwai&Tanaka,1968;Tanaka,1972). 2. Generalmorphology of the epitheliwn of the stomach. Forthemorphologyofthestomachepithelium,theresultsaresimilar to thoseobtainedbyothersinultrastructuralstudies (Ling&Tan,197SinChelmon rostratus; Huebner&Chee,1978in Hoplosternum sp.;jäs&Noaillac-Depeyre,1978 in Ameiurus sp.andNoaillac-Depeyre&Gas,1978,inperch).Alsoinmanylightmicroscopicalstudiesonfishes,onlyonetypeofglandularcellwasfoundinthe corpus,apartfromthesurfacemucouscells (AlHussaini,1946;Weinreb&Bilstad, 1955;Verma&Tyagi,1974;Moitra&Ray,1977;Reifel&Travill,1978;Weisel, 1973).Thedifferentiationoftheglandularcellsintotwotypes,oneofwhich producespepsinogenandtheotherhydrochloricacid,isonlyknownforthemammalianstomach (Smit,1968).Specialneckcells,asfoundin Mulloides sp.(AlHussaini,1946),stickleback (Hale,1965)andperch (Gas&Noaillac-Depeyre,1978), havenotbeenfoundinClarias lazera, justas inthesearobin(Blake,1936)in Colisa sp.(Moitra&Ray,1977),andin anumberofotherteleostspecies(Reifel &Travill,1978). Figs. 16-18.Stomachglandcellsofadult Clarias lazera. Fig.16.Apicaltubularsystem.Mi =mitochondrium;Zy=zymogengranule,x23.000 Fig. 17.RERinthebasalpartofacell,x40.000 Fig.18.Desmosomesandmanymicrofibrillsintheapicalpartofaglandular cell,x40.000 Fig.19.Adult Cl. lazera',secretion granuleslocatedintheapexofasurface mucouscell,x18.000 17 Fig. 20.Lightmicrograph ofpart of the gut after horse radish peroxidase ingestion.Black deposits of reaction product indicate the presence of enzyme activity. 1= segment 1;3= segment 3;A = anus. Fig. 21.Functional cell of the epithelial lining of the stomach,x 10.000 The inset shows a lightmicroscopical radioautogram. The arrowpoints to the H-thymidine labeled nucleus of the cell in the electronmicrograph,x 400 18 Thecellsoftheepitheliallininghavethesameappearanceinallstudied shspecies.UsuallythesecellsshowPASpositivityintheirapicesafter cianBlue-PASstaining,andthissuggeststhattheycontainneutralmucopolyccharides.Inpike,however,Reifel&Travill (1978)foundacidcarbohydrates thesecells. Development of stomach functions. Itisdifficulttodeterminewhenthestomachbecomesactually"functional", evolumeincreasesrapidlyasfromday8,andoneoftheimportantstomach nctions,thestorageoffoodmaybeginasofthatday.Thedigestivefunction thestomachclearlydevelopslater,betweentheformationofsecretiongranules thecorpusglandcellsandtheproductionofasmuchhydrochloricacidasrearedforreachinglowpHvaluesinthestomachlumen. Fromthemorphologicalevidenceitcanbeconcludedthatin Clarias lazera ecorpusofthestomachisformedbetweenday4whenthefirstglandularcells efound,andday12whenexocytosisofsecretiongranulesisfirstobserved. Anacidenvironmentinthestomachasfromday13allowscathepticorpeptic gestion.Fromday12theapicalvesicularsystemshowsatendencytodevelop bules,whicharedistinctinadults.Thesevesiclesandtubulespossessmemaneswithacoatingattheinnerside.Thiscoating,alsopresentonthememanesofthemicrovilli,consistofglycoproteinsin Ameiurus nebulosus (Gas& aillac-Depeyre,1978)andin Perca fluviatilis (Noaillac-Depeyre&Gas,1978). similartubulo-vesicularsystemisalsopresentinstomachglandcellsof liernon-mammalianvertebratesandapparentlyplaysacertainroleinhydrochloc-acidproduction (Sedar,1961).Inmammalsthevolumeofthetubulo-vesicular stem,asonlyfoundinparietalcells,isininverseratiotothevolumeof 3socalledintracellularcanaliculi,whicharemaximallydevelopedwhenmuch irochloricacidisproduced (Padykula,1977).Intracellularcanaliculi,however, reneitherbeenfoundin Clarias i Chelmon rostratus lazera, norin A. nebulosus, P. fluviatilis, (Ling&Tan,1975). Afterday13theacidityincreases.Thepyloricsphincterispresentas 3mday12.Theseresultsareingeneralaccordancewiththeradioautographic suits;thelabelingindexdecreasedsharplyinthestomachcorpusglandsbetween f9andday12andinthepyloricareabetweenday12and15.Differentiation theepitheliummusttakeplacefirstintheintestine,thenintherostral rtofthestomach,andfinallyinthepylorus. 19 Grizzle&Curd (1978)foundalarvalperiodofapproximately35daysin logperch (Percina eaprodes), rearedat22C.Thefirstgastricglandswerefoi onday27,whereasthepyloricsphincterdevelopedsomedayslater.Thisiso: interest,asTanaka(1971)concludedforalargenumberofteleostspeciesth; gastricglandsdevelopafterabout3/4ofthetotallarvalstage.Thestomach developsveryearlyin Clarias lazera andthelarvalstageisevidentlyvery shortwhenthefisharerearedat23-24C.Thelatterisbeginningwhen,at+ 30hafterfertilization,exogenousfeedingstarts.Thecompletionofthestoi isusuallyconsideredtocoincidewiththeendofthelarvalstage (Tanaka,1! 4. Physiological relevance of the second gut segment. Themorphologyoftheintestinedoesnotchangeafterthetimewhenthe: machbecomesfunctional.Asthesecondsegmentwiththefacilityforabsorbin; proteinmacromoleculesremainstobepresentbeyondthisperiod,thisfacilit; cannotberelatedtothelackofastomachin Clarias lazera. Thisisunderlii bythefactthatthelengthofthesegmentremainsthesameandalsobythep senceofpinocytoticenterocytesintheposteriorintestineofadult Clarias. lastargumentisinaccordingwiththeresultsofKrementz&Chapman (1974), Bergot (1976)andNoaillac-Depeyre&Gas(1979),whofoundpinocytosisinthe posteriorpartofthegutofadultcatfish {Ictalurus punctatus), in15cmla trout,andinadultperch,respectively.However,supranuclearvacuoleshave: beenfoundinadult Ictalurus . Thismightbeattributedtothelongtimeinte betweenfeedingandfixingofthetissues;starvationcausesthedisappearanc supranuclearvacuolesbutnotofpinocytosis,aswasfoundin Coregonus larva (Stroband&Dabrowski,inpress).Intheadultperchthesecondsegmentseeme< relativelyshort(+111ofgutlength,but_+20%in Clarias lazera larvae,ju nilesandadults).Tanaka(1972)studied15fishspecies.In5oftheseheno cedatthetransitionalstagefromlarvatojuvenile,whenthegastricglands becomefunctional,thegradualdisappearanceofacidophylicgranules (i.e.su nuclearvacuoles)intheposteriorintestine.Thiswasnotobservedintheot' 10species.Inhisexperimentssomefixationswerepossiblycarriedoutlong afterfeeding,andultrastructuralstudiesmightshowpinocytosisintheseca segmentofthegut. Thefacilityofabsorbingmacromoleculesisprobablyageneralfeatureo theteleosteanintestine,butitmightbemorecommoninlarvaeandstomachle speciesthaninspecimenswithafunctionalstomach.Thephysiologicalroleo 20 "secondsegment"infishlarvaemightbedifferentfromthatinadults.In irvaethefoodreachesthecaudalintestineinaveryshorttime,andabsorption :macromoleculesmightbeofquantitativeimportance (Iwai,1968).Inadultspe.mens,includingstomachlessfish,manyhoursgobybeforethefoodreachesthe »steriorgut,anddigestionofproteinthenmaybeinanadvancedstage.That (sorptionofundigestedfoodinthesecondgutsegmentplaysaroleinfishlarieisalsoindicatedbytheabsenceofsupranuclearvacuolesinstarved Coregonus irvae(O.C.).TheobservationofIwai(1969)thatafterfeedingthenumberof 'sosomesincarplarvaeincreasestogetherwiththesupranuclearvacuolespoints iintracellulardigestionofabsorbedmaterial. Inadults,eveninstomachlessfish,absorptionofproteintakesplacemain'intheanteriorpartoftheintestine (Shcherbina&Sorvatchev,1969;Stroband jVanderVeen,inpress;studyingcarpandgrasscarprespectively).Thereforeit emslikelythatadultstomachlessfishareabletodigestproteinsefficiently thoughapepsin-hydrochloricacidsystemisabsent (Creach,1963;Jany,1976). fferenceshavenotyetbeenfoundinthepartoffoodproteinabsorbedbyfishes thorwithoutastomach.Thequestioniswhyotherspeciesdevelopedastomach dapepsin-HClsystem.Otherstomachfunctions,e.g.storageoffood,partioningoffooditems,ordefenceagainstmicroorganismsmayhavetobeconsidered thisrespect. Renewal of the epithelium. 3 Therandomdistributionof H-thymidine labeledcellsinthedigestivetract itheliumintheearlystagesofdevelopmentisinaccordancewiththeresults Romboutetal.(inpress)obtainedin Barbus aonohonius larvae,inwhichthe Delingindexsharplydeclinedbeforethefirstexogenousfeeding.In Clarias zera thedecreasewasnoticedtwodayslater.Inbothspecies,thelocationof jeledcellsduringthisdecreasechangestowardsthebasalpartsoftheinteslalmucosalfolds.Thepresenceofthymidinelabelinnucleioffunctional Lis(glandularandsurfacestomachcellsaswellasabsorptivecellsin xrias lazera larvae)wasobservedalsobyRomboutetal.andbyStroband& jets(1978)intheintestinalabsorptiveenterocytesof Barbus larvaeand senilegrasscarp,respectively.Theabilitytoproliferatewasalsofoundin ictionalsmall-intestinalcellsin Xenopus laevislarvaebyMarshall&Dixon )78). 21 Itisinteresting thatnotwithstanding thedecreasing labeling index,wh< thetissueshavedifferentiated andbecome functionally active,onlyfunction; proliferating cellsappeartobepresent.This incontrast tothemammaliansi intestine,whereonlyundifferentiated cryptcellsare abletoproliferate (Lipkip,1973).Theresults indicatethat fishenterocytes andstomach cells loosetheirproliferative activityinarelatively latestageofdifferentiat: TheauthorswishtothankProf.Dr.LucyP.M. Timmermans andProf.Dr.J.W.M.0: fortheirconstructive comments,MissJ.J. Thiele fortechnical assistance in electronmicroscopy,andMessrs.W.ValenenH.G.Eleri (T.F.D.L.)forprepar: the illustrations.Special thanksaredueDr.L.Boomgaartfor correcting linguisticerrors. 22 REFERENCES Al-Hussaini,A.H.The anatomy andhistology of the alimentary tract of the bottomfeeder Mulloides auriflamma (Forsk.). J.Morph. 78.» 121-153 (1946). Bergot,P. Demonstrationwith ruthenium red of deep invaginations in the apical plasmamembrane of enterocytes in rainbow trout intestine. Ann. Biol.Anim. Biochem. Biophys.J_6>37-43 (1976). Blake,I.H.Studies on the comparative histology of thedigestive tube of certain teleost fishes.J.Morph. 60,77-102 (1936). Creach,P.V. Les enzymes protéolytiques des poissons.Ann.Nutr.Alim. 17, 375-471 (1963). Cornell,R. &Padykula,H.A. A cytochemical study of intestinal absorption in thepostnatal Rat.Anat.Rec. L5_i> 139 (1965). Gas,N. &Noaillac-Depeyre,J. 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'erma,S.R.&Tyagi,M.P.Acomparativehistologicalstudyofthestomachofa fewteleostfishes.Morph.Jahrb.J_20,244-253 (1974). (einreb,E.L.&Bilstad,N.M.Histologyofthedigestivetractandadjacent structuresoftherainbowtrout, Salmo gairdneri irideus. Copeia3^ 194-204 (1955). 'eisel,G.F.Anatomyandhistologyofthedigestivesystemofthepaddlefish (Polyodon spathula). J.Morph.140,243-256 (1973). amamoto,T.Anelectronmicroscopestudyofthecolumnarepithelialcellinthe intestineoffreshwaterteleosts:Goldfish (Carassius auratus) andrainbow trout {Salmo irideus). Z.Zeilforsch.72,66-87 (1966). 25 CURRICULUM VITAE Het levenslichtwerd doormijaanschouwd in Zeistop 31 december 1946. Hoewel naar ikmeen nooit doormijn ouders opgejaagd,voelde ik,als ieder kind,al snel dat ik geborenwas ineen samenlevingwaarin "het presteren" vangrotebetekeniswordt geacht.Grootwas dan ook mijn frustratie toenik naeenvliegende start (ikwerd pas eind februari 1947verwacht)al op de openbare kleuterschool "Griffensteijn" in Zeist mijn eerste doubleren te verwerken kreeg.Achteraf heeft 3 jaar fröbelen stellig inpositieve zin aan mijnvormingbijdragen. Later kon ik aan demaatschappelijke verwachtingen voldoen doorhet slagenvoorhet toelatingsexamen,na daarop uitstekend voorbereid te zijn op dejongensschool derEvangelische Broedergemeente. Hetvoorbereidend wetenschappelijk onderwijswerd aangevangen in 1959 aanhet IeChristelijk Lyceum te Zeist.Eenpoging tothetvolgen vanhet Gymnasiumwas totmislukken gedoemd,maar in 1965werd het einddiploma H.B.S.-B verworven. Na eenkorte periode vanarbeidbijPhilips telecommunicatie industrie teHilversum volgdeeenoproep voormilitaire dienst. Dezewerd vervuld van januari 1966 tot juli 1967,grotendeels bij een infanterie bataljon teErmelo. Hoewel strepenvoormijnietwarenweggelegd,behaalde ikhet diploma "militaire lichamelijke vaardigheid" in 1967,ietswaarop ikvanzelfsprekend nóg trotsben. In september 1967ben ik,naenig "voorwerk" in diensttijd (aulapockets e.d.) gestart met de studieBiologie aan de Rijksuniversiteit te Utrecht.Hetkandidaatsexamen werd, totniet alleen mijn genoegen,behaald in mei 1970en indecember 1972studeerde ik "met lof"afmet als hoofdvak Vergelijkende Endocrinologie (prof.v.Oordt)en alsbijvakken Plantenfysiologie (Prof.v. Die) enDidaktiek der Biologie (Drs. Saaltink). Vanaf januari 1973ben ikwerkzaambij de afdeling Dierkunde,later vakgroep Experimentele Diermorfologie en Celbiologie aan de Landbouwhogeschool teWageningen. Naast vele andere aktiviteiten zoals onderwijs aanpre-en postkandidatenvanverschillende studierichtingen enhetbehalen vanhet diploma "C-deskundige"aanhet ITAL teWageningen in 1978,bleef er tijd beschikbaar voorhet doenvanhet onderzoek waarvan deresultaten indit proefschrift zijn neergelegd.