Lipidic and proteic absorption in digestive tract of tropical fat snook (Centropomus parallelus, POEY 1860)

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Lipidic and proteic absorption in digestive tract of tropical fat snook(Centropomus parallelus, POEY 1860)

João Carlos Shimada Borges a,c,⁎, Leandro Nogueira Pressinotti c,Vicente Gomes b, José Roberto Machado Cunha da Silva c

a Faculdade de Medicina Veterinária – Faculdades Metropolitanas Unidas – FMU, Brazilb Departamento de Oceanografia Biológica – Instituto Oceanográfico – Universidade de São Paulo – USP, Brazilc Departamento de Biologia Celular e do Desenvolvimento – Instituto de Ciências Biomédicas – Universidade de São Paulo – USP, Brazil

a b s t r a c ta r t i c l e i n f o

Article history:Received 20 August 2009Received in revised form 12 February 2010Accepted 15 February 2010

Keywords:Centropomus parallelusFerritinPerl's methodSudan black staining

The tropical fat snook Centropomus parallelus is a species of recognized ecological importance and with a higheconomical potential for fisheries and aquaculture. The investigations of digestive tube morphology inassociation with their feeding abilities are fundamental to improve techniques for aquaculture feedingprocedures. Sudan black staining and Perl's method were used to evaluate their absorption capacity of fatand protein respectively. The Sudan black stain was performed 12 h after the ingestion of lipids. The lipidsare intensely absorbed in the ceca epithelium and less intensely in the intestine and rectum. The Perl'smethod was performed 12 h after the ingestion of ferritin. The proteins are absorbed only in the rectum. Thisis the first description of fat and protein absorption ability by the digestive tube of fat snook. These dataenhance the possibility of the addition of macromolecules in rations that can show a diversity ofphysiological effects. The histological implications of each segment of the digestive tube in association withfish biology are further discussed.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Fat snooks are coastal marine fishes that reproduce in estuarine andfreshwater systems. They can remain the entire life cycle at the lowerends of hydrographical watersheds or they may alternate betweenmarine and estuarine environments, depending on their reproductiveconditions (Gilmore et al., 1983; Alvarez-Lajonchere et al., 1982).Twelve species were identified at the Pacific and Atlantic coasts of theAmerican continent (Rivas, 1986), four of which are registered on theBrazilian coast. They are Centropomus undecimalis, Centropomusparallelus, Centropomus ensiferus and Centropomus pectinatus, withpredominance of C. parallelus at the southeastern region. Due to theirhigh commercial value individuals of this species are being intenselycaptured, diminishing their natural stocks (Aliaume et al., 2000).

The literature has described the feeding habits of fat snooksclassifying them as carnivores (Rojas, 1975). Studies of stomachcontents helped to identify that they feed mainly on fish, with nospecies selection, but also including crustaceans, molluscs, fish eggsand insects. Nevertheless, there are no data on the morphologicaldescription of the Centropomidae digestive tract.

In carnivorous fishes, the intestine is usually fairly short in relationto the body length. At the initial portion, there are numerous pyloricceca, isolated or in groups, opening at the duodenum. The limitbetween the posterior end of the intestine and the rectum is ofdifficult anatomical identification. Histologically, there are only threeportionswithwell defined and constantmicroscopical characteristics:the proximal segment, 60–75% of the total length, where the cells thatabsorb fat are conspicuous (Hernandez-Blazquez et al., 1989); themedium segment (20–25%), responsible for the absorption of proteicmacromolecules (Gauthier and Landis, 1972; Noaillac-Depeyre andGas, 1973; Stroband and Kroon, 1981; Iida and Yamamoto, 1985;Rombout et al., 1985; Nachi et al., 1998); and the distal segment, withcells containing ultrastructures for the transport of ions, water andelectrolyte absorption (Noaillac-Depeyre and Gas, 1976, 1979;Stroband et al., 1979).

The fat absorption in temperate and tropical teleosts takes place atthe cephalic regions of the intestine as in Perca fluviatilis (Noaillac-Depeyre and Gas, 1979), Ameiurus nebulosus (Noaillac-Depeyre andGas, 1983), Prochilodus scrofa (Nachi et al., 1998) and Nototheniacoriiceps (Hernandez-Blazquez et al., 2006). This region is known asproximal segment following histophysiological criteria.

Demonstrationof intact protein absorptionoccurs at theproximal andother segments as demonstrated by the presence of peroxidase in thecytoplasm of the enterocytes (Gauthier and Landis, 1972; Noaillac-Depeyre andGas, 1973; 1976, 1979), by the presence of ferritin (Strobandand Kroon, 1981; Iida and Yamamoto, 1985; Rombout et al., 1985;

Journal of Experimental Marine Biology and Ecology 386 (2010) 39–44

⁎ Corresponding author. Faculdades Metropolitanas Unidas – FMU, Av. Santo Amaro,1239, CEP: 04505-002 – São Paulo, SP – Brazil. Tel.: +55 11 3091 7223; fax: +55 113091 7402.

E-mail address: [email protected] (J.C.S. Borges).

0022-0981/$ – see front matter © 2010 Elsevier B.V. All rights reserved.doi:10.1016/j.jembe.2010.02.013

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Georgopoulou et al., 1985; Georgopoulou and Vernier, 1986; Nachi et al.,1998)andbacteria (Vibrioanguillarum) (Davinaet al., 1982;RomboutandVan Den Berg, 1989). The ultrastructure of different proteic markers arefound at the fish absorptive epithelium from first feeding at larval stagethrough the adult, in species with or without stomachs, herbivores,omnivores and carnivores (Sire and Vernier, 1992).

The ultrastructure of the enterocytes of the different intestinalsegments is variable, the majority have a tubuline–vesicular system fortransport and numerous lysosomes, of different sizes, at the supranuclear region (Noaillac-Depeyre and Gas, 1973, 1976, 1979; Ezeasorand Stroke, 1981; Stroband and VanDer Veen, 1981; Nachi et al., 1998).Large digestive vesicles are present in the enterocytes' cytoplasm ofsome species (Georgopoulou et al., 1985; Rombout et al., 1985;Rombout and Van Den Berg, 1989).

The present work aims to contribute to the understanding ofC. parallelus nutrition and to give support to future aquaculturepractices, presenting data on the lipidic and proteic macromolecularabsorption by the digestive tract of these fish.

2. Material and methods

Twenty juveniles of C. parallelus (weight 115.0±22.0 g, standardlength 25.0±5.0 cm), Centropomidae, were collected in the CananeiaEstuary, on the Southern Coast of São Paulo State, Brazil (25º 05′S–47°55′W). The fish were captured using a beach seine (4 m wide, 1.5 mhigh, mesh size of 0.5 mm) specially developed for this purpose. Theanimals were immediately placed in plastic boxes with aerated waterand transported to the laboratory, where they were acclimated in200 L tanks with artificial aeration for 30 days at salinity 35‰. Thetanks were monitored daily for fish feeding, were fed daily with acommercial trout ration ad libitum for 15 days and jejune for 15 daysbefore the experimentation, the wastewater was removed by waterreplacement. All the water used, both saltwater and freshwater werefrom natural sources and were filtered at 1 µm prior to its preparationas experimental water.

A total of 10 animals were used to demonstrate protein absorption.For that, 12 h before being sacrificed with medulla section afteranesthesia (immersion in ethyl benzoate at 50 ppm according to Silvaet al., 2005), each individual was inoculated orally with 1.0 mL ofhorse spleen ferritin (85 mg/mL, Sigma®) solution diluted 50% insterilized sea water filtered in 0.22 µm pore filter. To demonstratelipid absorption 10 fish were inoculated orally with 1.0 mL of corn oil,12 h before being sacrificed. As control to protein and lipid absorptiontwo fish without feeding or any inoculation were used.

Tissues were dissected before fixation so that they would notsurpass 5 mm in diameter for light microscopy and 3 mm for electrontransmission microscopy (Silva et al., 2005).

To evaluate protein absorption, samples were taken fromesophagus, stomach, caecum, anterior and posterior intestine, andrectum, 12 h after ferritin inoculation. Samples were fixed inMcDowell solution (4% paraformaldehyde in 1% glutaraldehyde inphosphate buffer solution 0.1 M pH 7.2) (McDowell and Trump, 1976)for 48 h. Histological preparations were stained by the Perl's method(Hernandez-Blazquez and Silva, 1998), in which the potassiumferrocyanide stains molecules containing iron ions, revealing ferritinabsorption. Safranine O was used for background staining.

To observe lipid absorption the same segments were sampled 12 hafter the inoculation of corn oil. To avoid lipid losses during thehistological procedures samples were fixed in Baker calcium formalde-hyde solution for 48 h (Hernandez-Blazquez et al., 1989) and alcoholicsolutions were never used to historesin embedding. Histologicalsections were stained in Sudan Black (Hernandez-Blazquez et al.,1989), which gives black color to the lipid containing regions.

The material was embedded in historesin in both groups (GlicolMetacrilate) (Junqueira, 1995), sections of 1 to 3 µm were cut in a

microtome (American Optical®), and slides were observed and photo-graphed under a Zeiss axiomat microscope.

Samples taken from each experimental group were processed forelectron transmissionmicroscopy. Tissues were fixed at 4 °C with 2.5%glutaraldehyde in phosphate buffer (0.1 M), pH 7.2, postfixed in 1.0%OsO4 (Hayat, 1981) and embedded in Spurr resin (Sigma). Ultra thinsections of 70 nm were gathered onto copper grids and stained with2% uranyl acetate in distilled water for 1 h, then washed in distilledwater and stained again in 0.5% lead citrate in distilled water.Ultrastructure was examined in a Jeol 100 CX-II electron microscope.

3. Results

Intestine represents nearly 70% of the total length of the digestivetube of C. parallelus. This species exhibits a voluminous stomach thatseems capable to accommodate a large quantity of food. At the cranialportion of the intestine, 4 to 6 digitiform appendages, denominatedpyloric caeca, are present, with about 0.3±0.1 cm width and 0.8±0.2 cm length. Intestine is coiled in the abdominal cavity formingthree segments. The first segment, connected with the caeca, wasdenominated as anterior intestine; the second segment, at the sacralportion, as posterior intestine. The final portion of the last segmentends in a sphincter, delimitating a short third segment denominatedrectum (Fig. 1).

The three different layers that compose the digestive tube wall ofC. parallelus are in conformity with those reported by Burnstock(1982). The mucosa has a conspicous muscularis mucosa at thepyloric ceca; the submucosa is thick at the rectum; the muscular layeris well defined all along the digestive tube, with a circular internal anda longitudinal external portions; a serosa layer lining the tube isidentifiable with a simple squamous epithelium sustained by a looseconnective tissue.

Twelve hours after corn oil was ingested, fat was already present inthe apical and basal cytoplasm of the enterocytes of all intestinalsegments and pyloric caeca. The lipids appear as little sudanophilicdroplets with 0.5–1 µm of diameter. The fat inclusions are seen fromthe subapical zone to the supranuclear zone (Fig. 2). Caecal villositieswere homogeneously and intensely distributed in all enterocytes 12hafter the ingestion of lipids (Fig. 3).

The lipids are absorbed by the enterocytes in the form of lipiddroplets. The ultra structural morphology of droplets is heteroge-neous and it may be round,multilobulated or segmented. The externalmembrane of the mitochondria is often seen in close association withthe droplets. In the subterminal zone the droplets are more frequentand smaller (Fig. 4). The size, distribution and form of the fat dropletsare very heterogeneous in fish that were sacrificed 12h after fatingestion.

Twelve hours after ingestion of ferritin, Perl's stain showed up asvery small quantity of bluish cytoplasmic granules in the supranuclear

Fig. 1. Photography of digestive tract of the Centropomus parallelus a — oesophagusb— stomach, c— caecum, d—proximal intestine, e—distal intestine, f— rectum, scale bar=4 cm.

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half of the apical enterocytes of the rectal segment, indicating thepresence of ferritin. Although all intesinal segments were examinedby both light and electron microscopy, ferritin was observed only inthe mucosa epithelium of the rectum. The bluish granules werepresent in any of the segments of the digestive tube in the controlgroups (Fig. 5).

Ultrastructurally the enterocytes exhibit microvillus. The surfacemembrane between themicrovilli formsdeep invaginations that detachthemselves from the surface, forming electron-dense vesicles. Thecytoplasmic region, where these huge vesicles appear, coincides withthe ferritin zone absorption, observed using light microscopy (Fig. 5).The electron density of cytoplasmic vesicles also increases from theapical to basal regions of the cells (Fig. 6). The fusion between vesicles offerritin with a granular matrix of different electron densities was alsoobserved. At higher magnification the vesicular matrix shows smallgranules of a uniform size with an electron-dense core and a light halo,identified as ferritin particles (Fig. 7). The localization of these electron-dense granules is coincident with the ferritin-positive cytoplasmic zoneobserved under light microscopy.

4. Discussion

The anatomy of the digestive system of C. parallelus is relativelysimple when compared to herbivorous fishes that ingest substancesthat are difficult to digest. These latter have a great number of

intestinal loops to increase the absorptive area (Hernandez-Blazquez,et al., 2006). It seems that fat snooks are adapted to a diet rich innutrients, as they have a voluminous stomach, short intestine and bigpyloric ceca in relation to the intestine. Their size is typical ofcarnivorous fishes (Hernandez-Blazquez et al., 1989) and fits to the fatsnook feeding habits. The size of the stomach can be usually relatedwith the intervals between meals and the size of the food particlesingested. Fishes that consume large prey at large intervals have biggerstomachs and those that feed on small particles, but constantly, have,in general, smaller or no stomachs, as the common carp (Noaillac-Depeyre and Gas, 1973).

Our data of three different histological layers agrees with literature.Burnstock (1982) reported that the histological structure of thedigestive tube of teleosts is organized in three layers. The inner one isa mucosa that contains an epithelium supported by a lamina propriathat, in turn, is over a compact layer of dense connective tissue. Thislayermay also be constituted by amuscularis mucosa and a submucosa.Further one can find the muscular layer composed of circular andlongitudinal fibers and, finally, a serosa layer lining the tube.

The lipid or osmiophilic droplets observed in the electron micro-graphies are composed of triglycerides as reported by other authors forother species of fishes using OTO method (Bergot and Vodovar, 1967;Bergot and Flechon, 1970; Noaillac-Depeyre and Gas, 1973). In the

Fig. 3. Cecal villosities of Centropomus parallelus 12 h after the ingestion of lipids. Theblack droplets are lipids. Staining: Sudan Black and hematoxylin. Scale bar=50 µm.

Fig. 4. Ultra structure of Centropomus parallelus enterocyte of the cecal villosities, 12 hafter the ingestion of lipids. The clear grey droplets are lipids, some of them fusing.Mitochondrias can be characterized near the dorplets. Scale bar=2 µm.

Fig. 5. Photomicrography of the villosities of the recto of Centropomus parallelus, 12 hafter the ingestion of ferritin. The apical region of the prismatic epithelium are stainedin dark in color due to the presence of ferritin vesicles. Staining: Perl"s and Safranine O.Scale bar=50 µm.

Fig. 2. Photomicrography of the proximal intestinal epithelium of Centropomus parallelus,12 h after the ingestion of lipids. The black droplets are lipids. Staining: Sudan Black andhematoxylin. Scale bar=20 µm.

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present work, the diet was exclusively lipidic, and the lipid dropletswere easily identified, only by their morphological aspect, due to somedegree osmiophily and the absence of a membrane. Some authors,however, described the presence of a trilaminar membrane lining thedroplets (Iwai, 1969; Stroband and Debets, 1979; Bauermeister et al.,1979).

In relation to majority of studied teleosts, a different trait of the lipidabsorption of C. parallelus is the extensiveness of the process over thelength of the intestine. Fat droplets are found only at the first segments ofthe intestine of many freshwater species, as Carassius auratus (Gauthierand Landis, 1972), P. fluviatilis (Noaillac-Depeyre andGas, 1979), Ameirus

nebulosus (Noaillac-Depeyre and Gas, 1983), Prochilodus scrofa (Nachiet al., 1998) and Pimelodus maculatus (Hernandez-Blazquez et al., 1989)andalsoofmarine species, suchasAnarhichas lupus (HellbergandBjerkås,2005). The accumulation of lipid in the enterocytes has been related astemporary storage, which may result from an inability to mobilize thelipid (Baskerville-Bridges and Kling, 2000) and for that reason not all thesegments are histologically observed within lipidic droplets. The lipidicdroplets are considered as a temporarymode of energy storage caused bya surplus income of fatty acid that overcomes the capacity of lipoproteinliberation or production (Sire and Vernier, 1992). Lipid absorption inC. parallelus occurs at every portion of the intestine. This feature is similarto that of the antarctic fish Notothenia coriiceps (=N. neglecta)(Hernandez-Blazquez et al., 2006). These authors ascribed the extensivelipid absorption to an adaptive cause, as fat may play a central role in theenergetic and respiratory metabolism of antarctic fish, entailing anintense oxidation of fatty acids (Crockett and Sidell, 1990) and retentionof oxygen by intracellular lipid droplets that are used for energy storage(Londraville and Sidell, 1995). The possible explanation for thesesimilarities between C parallelus and Notothenia neglecta could be relatedto the energetic metabolism. Fat snook is a euryhaline species that canexploremarine and freshwater environments, with high osmoregulatorycosts (Imsland et al., 2003). The necessary energy for osmotic regulationcould be supplied by lipid metabolism. A similar trait for lipid absorptionis reported for Salmo salar (Krogdahl et al., 1999), including the extensiveabsorptive area with higher intensity at the ceca. Like fat snook, theAtlantic salmon is also a species that can cope with hypo andhyperosmotic waters. Rocha et al. (2005) studied the energy budgets ofjuveniles of C. parallelus exposed to different salinities (5, 20 and 30). Theauthors concluded from O:N (oxygen:nitrogen ratio) that at highersalinities the energetic requirements were mainly supplied by lipidicoxidation. These data are in accordance with our findings, where intenselipid absorption occurred along the whole intestine and caecum inspecimens acclimated to salinity 35.

The incorporation of macromolecules by the intestine of otherfishes is a quantitatively significant absorptive phenomenon, due toits intensity and the extension where this proccess occurs. In specieswithout a stomach and in fish larvae, the absorption of macromole-cules takes place over 25% of the intestinal length, generally at themedium or at the distal segments (Noaillac-Depeyre and Gas, 1973,1983; Stroband et al., 1979; Stroband and Van Der Veen, 1981).Transepithelial transport is also observed in such species, as inP. fluviatilis (Noaillac-Depeyre and Gas, 1979), Clarias lazera (Strobandand Kroon, 1981) and Prochilodus scrofa (Nachi et al., 1998). Mai et al.(2005) observed in their study the presence of acidophilic supra-nuclear vacuoles in the post valvular intestine soon after the onset ofexogenous feeding that indicated the presence of pinocytic absorptionand intracellular protein digestion, proposed as the main mechanismfor protein absorption in larvae during the absence of a functionalstomach. In the present study the ferritin absorption was indicated bythe Perl's stain only in the rectum during adult life, confirming thatproteins are absolved only in the rectum of C. paralellus. The exclusiveabsorption in the region of the rectum coincided with the processdescribed for the antarctic fish, N. neglecta (=N.coriiceps), bothcarnivorous fishes. The authors suggested this reabsorption reincor-porate the antifreeze glycoproteins secreted in the digestive lumen toavoid freezing of the faeces under polar temperatures (Hernandez-Blazquez and Silva, 1998). We suggest that in fat snook, a tropical fish,the rectum absorption of macromolecules meaning may be explaineddue to the large enzymatic production of fat snook, and posteriorabsorption, since several works reported that the ratio of totalproteinase: total amylase activity in omnivorous and herbivorous fishwas lower than in carnivorous fish (M.Y. Tsuzuki et al., 2007). ThePyloric ceca, that can retain a large volume of material, are found inboth species, too. Enzymatic hydrolysis takes place at these ceca,similar to that of the intestine lumen, with the presence of trypsins,chymotrypsin A and B, and elastases (Sire and Vernier, 1992).

Fig. 6. Electronmicrography of the enterocytes of the recto of Centropomus parallelus.Some of the vesicles, ferritin appears as a central dark spot iron surrounded by a clearcircle region, that is a proteic covering. This can be better observed in the clear apicalvesicles. Scale bar=1 µm.

Fig. 7. Electronmicrography of the enterocytes of the Centropomus parallelus recto, 12hafter the ingestion of ferritin. The apical region of the enterocytes is filled with electron-dense vesicles, filled with ferritin. Scale bar=5 µm.

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Digestive proteolytic enzymes of antarctic fishes also demonstratedan elevated activity (Rehbein et al., 1986; Genicot et al., 1988). Indetritivorous and herbivorous fishes such as P. scrofa (Nachi et al.,1998) that depends on symbiotic microorganisms, the absorption ofmacromolecules is more intense as a compensatory mechanism forthe low proteolytic activity of the enzymes. Studies on nutrientabsorption in teleost fishes that lack a stomach suggest that theposterior rectum and medium portions of the intestine incorporatemacromolecules as a consequence of inadequate digestion of proteins(Gauthier and Landis, 1972; Noaillac-Depeyre and Gas, 1973, 1976;Stroband and Van Der Veen, 1981). Equivalent regions of the intestinethat can absorb macromolecules are also found in some teleosts thathave a stomach (Noaillac-Depeyre and Gas, 1979, 1983; Stroband andKroon, 1981).

The ultrastructural aspects of the enterocytes' positive region to theintracytoplasmic ferritin are typical of a cell with a high absorptivecapacity, indicating protein (macromolecules) absorption. This fact isnoticeable by the presence of long microvilli and by the activeendocytosis at the basal portion of these structures. Ferritin captureseems to be a non-selective procedure in C. parallelus, as it is found inclumps or scattered in the vesicles where it is accumulated until fusionin bigger vesicles. These results are comparable to those reported byRombout et al. (1985) and Nachi et al. (1998) who observed the non-selective absorption of ferritin by the intestinal epithelium of theherbivorous Cyprinus carpio and detritivorous Prochilodus scrofa of lowproteolytic enzymatic production. These data support the hypothesis ofthe reabsorption of the proteolytic enzymes by carnivorous fishes.

This work reveals, for the first time, the absorption of lipids and ofproteic macromolecules in C. parallelus, demonstrating that thisspecies has an excellent mechanism for lipid absorption and indicatesabsorption of macromolecules in the rectum. These data arefundamental to give support to future researches to understand thevariations of the absorptive processes of fat snook, thus enhancing thepossibility of the addition of macromolecules, in rations, that canshow a diversity of physiological effects.

Acknowledgments

The authors express their thanks to the Faculdades MetropolitanasUnidas — FMU for the financial support, to Emilia Ribeiro forhistological techniques; Edson Rocha de Oliveira for TEM techniquesand Yara Shimada Brotto for the English revision.[SS]

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