Histological development of the digestive system of Mayan cichlid Cichlasomaurophthalmus (Gunther 1862)

By C. A. Cuenca-Soria1,2, C. A. �Alvarez-Gonz�alez3, J. L. Ortiz-Galindo1, D. Tovar-Ram�ırez4,R. Guerrero-Z�arate3, S. Aguilar-Hern�andez2, M. A. Perera-Garc�ıa2, R. Hern�andez-G�omez2 and E. Gisbert5

1Centro Interdisciplinario de Ciencias Marinas, La Paz, M�exico; 2Divisi�on Acad�emica Multidisciplinaria de los R�ıos,Universidad Ju�arez Aut�onoma de Tabasco, Tabasco, Tenosique, M�exico; 3Laboratorio de Acuicultura TropicalUJAT-DACBIOL, Tabasco, M�exico; 4Centro de Investigaciones Biol�ogicas del Noroeste, La Paz, M�exico; 5Institut de Recercai Tecnologia Agroaliment�aries (IRTA), Sant Carles de la R�apita, Spain

Summary

The ontogeny of the digestive tract in Cichlasoma urophthal-mus was studied by means of optical microscopy from hatch-

ing to 30 days post-hatching (dph; 855 degree days, dd). Thedevelopment of the digestive system in this precocial specieswas a very intense and asynchronous process, which pro-

ceeded from both distal ends interiorly. At hatching, thedigestive tract consisted of a straight tube with a smoothlumen dorsally attached to the yolk-sac. The digestive acces-

sory glands were already differentiated and eosinophiliczymogen granules were visible in the exocrine pancreas. Atthe onset of exogenous feeding between 5 and 6 dph

(142.5–171.0 cumulative thermal units, CTU), the buccophar-ynx, oesophagus, intestine, liver and pancreas were almostcompletely differentiated, with the exception of the gastricstomach that completed its differentiation between 11 and

14 dph (313.5–399.0 CTU). The development of gastricglands at 14 dph and the differentiation of the stomach in thefundic, cardiac and pyloric regions at 19 dph (541.5 CTU)

were the last major events in digestive tract development anddesignated the onset of the juvenile period. Remnants of yolkwere still detected until 16 dph (456.0 CTU), indicating a

long period of mixed nutrition that lasted between 10 and11 days (285.0–313.5 CTU). The results of the organogenesisof larvae complement previous data on the functionality ofthe digestive system and represent a useful tool for establish-

ing the functional systemic capabilities and physiologicalrequirements of larvae to ensure optimal welfare and growthunder aquaculture conditions, which might be useful for

improving current larval rearing practices for this cichlidspecies.

Introduction

Nowadays, the rearing of fish larvae is perhaps one of the

most critical processes in aquaculture due to high larval mor-tality, frequently associated with nutritional deficiencies and/or deficient feeding protocols, among other factors. Growthand survival of larval fish depend primarily on the feeding

success and an effective digestion and absorption of nutrients(Tanaka et al., 1995). In this context, one of the aspects toconsider when designing feeding protocols based on live prey

and/or inert diets for fish larvae is the morphology and phys-iology of their digestive system. The larval fish may be mor-phologically capable of capturing food items at first feeding,

but the digestive system needs a series of developmental

changes before being fully functional (Govoni et al., 1986;Canino and Bailey, 1995). In this sense, several studies have

highlighted the importance of conducting species-specificstudies on larval fish organogenesis of the digestive tract inorder to better understand its functionality and nutritional

physiology (see review in Lazo et al., 2011). Thus, knowledgeregarding the morphological and functional changes duringearly ontogeny in fishes is of capital importance to adapt rear-

ing technology according to digestive ability of larvae fish.Large numbers of published studies describe the morphologi-cal and histological changes of the digestive system duringearly ontogeny in fishes (see reviews in Rønnestad and

Morais, 2008; Zambonino-Infante et al., 2008). Although fishas a group show a remarkable diversity of structure and func-tion in their nutritional physiology, the basic mechanisms of

organ and system development are similar in all teleosts, eventhough there are considerable interspecific differences in therelative timing of their differentiation, development, and func-

tionality during early ontogeny (Lazo et al., 2011). However,there are considerable differences regarding the timing ofmorphological and cytological changes during early ontogeny

in fishes depending on several factors, with temperature, foodavailability and composition as well as water quality beingsome of the most important (Zambonino-Infante and Cahu,2007). The timing of development of organ and physiological

function is affected by the general life history and reproduc-tive strategy of each species (Lazo et al., 2011). In this sense,there are species-specific differences in the timing of differenti-

ation of organs within cichlid species related to their repro-ductive guild, whereby Trevi~no et al. (2011) recommendedthat the reproductive guild (Balon, 1975) of the fish must be

considered when designing intensive larval rearing and feed-ing protocols for different species of cichlids or for other fishspecies with similar characteristics.The Mayan cichlid Cichlasoma urophthalmus (G€unther,

1862), or ‘mojarra castarrica’ in the native Mexican lan-guage, is one of the native Neotropical species that hasreceived more attention within recent years due to its poten-

tial for aquaculture diversification in Central America. Thiscichlid species is distributed along the Atlantic slope of tropi-cal Central America, ranging from Mexico southward to

Nicaragua (Espinosa-P�erez et al., 1993). This species has anexcellent flavour and is of high demand in local markets,additional to its high adaptability in captivity and high fertil-

ity, growth and survival rates, making this species a promis-ing candidate for intensive freshwater aquaculture that

U.S. Copyright Clearance Centre Code Statement: 0175-8659/2013/2906–1304$15.00/0

J. Appl. Ichthyol. 29 (2013), 1304–1312© 2013 Blackwell Verlag GmbHISSN 0175–8659

Received: March 29, 2013Accepted: June 6, 2013doi: 10.1111/jai.12307

Applied IchthyologyJournal of

otherwise is currently mostly based on the culture of tilapinespecies (P�erez-S�anchez and P�aramo-Delgadillo, 2008;Trevi~no et al., 2011). However, the culture of C. urophthal-mus is still limited by the fragmentary knowledge of its early

ontogeny and the processes affecting it. In this sense,although there is some information regarding the functional-ity of the digestive system of C. urophthalmus during its lar-

val development (L�opez-Ram�ırez et al., 2011), little is knownabout histomorphological changes occurring in the digestivesystem of C. urophthalmus larvae. Such information would

help improve actual feeding practices, allowing the synchro-nization of the rearing procedures with the development ofthe larvae, as well as providing support for designing artifi-

cial larvae feeds for this cichlid species. The aim of this studywas to describe the histological changes and the organogene-sis during early ontogeny, from hatching to the early juvenilestages of C. urophthalmus.

Materials and methods

Larvae of C. urophthalmus were collected from a singlespontaneous spawning from a broodstock composed of 3females and 1 male kept in captivity in the facilities of the

Laboratorio de Acuicultura Tropical of the Divsi�onAcad�emica de Ciencias Biol�ogicas (DACBIOL), UniversidadJu�arez Aut�onoma de Tabasco (UJAT), Mexico (17°59′22.96″N, 92°58′33.98″W). Broodstock was maintained in an

open system of 9 9 2000-L circular plastic tanks with aratio of two females per male (250 and 300 g, respectively).Adult fish were maintained in natural photoperiod (12:12

light: darkness), with daily 200% water exchange and fedcontinuously with frozen tilapia for 30 days until the repro-ductive behaviour began. Thirty days after recording the

first spawning, one spontaneous spawning was obtainedfrom one of the tanks. Egg incubation lasted for 36 h.Hatchlings (ca. 2500) were collected by siphoning and

placed in a new 1.3 m3 open-flow circular tank (1.5 m Ø)(daily 200% water exchange). Eggs were incubated and thelarvae reared in the environmental conditions: 28.5 � 1.1°C,5.2 � 0.95 mg L�1 of dissolved oxygen and pH 7.5 � 0.2

(n = 32). Water parameters were measured daily with anoxymeter (YSI 55; CA) and a pH-meter (Denver InstrumentUB-10, Denver, CO). Photoperiod was 12:12 (light:darkness)

during larval rearing. Larval development was scaled to age(days post hatch, dph) and thermal units (cumulativedegree-days post hatch, CTU). Cichlasoma urophthalmus lar-

vae were fed ad libitum with non-enriched Artemia nauplii(INVE Aquaculture Nutrition, Belgium) three times per dayfrom 6 days post hatch (dph) until 15 dph. Afterwards to30 dph larvae were fed with Silver Cup trout compound

feed (45% protein and 16% lipids; Nelson and Sons, Inc.)as previously described (L�opez-Ram�ırez et al., 2011). Feed-ing ration was 10% of body weight, and particle size was

adjusted during the growing period (125–250 lm from 15until 25 dph, and 250–500 lm from 25 until 30 dph). On adaily regular basis (from 1 to 30 dph), two sets of six larvae

were sampled, sacrificed with an overdose of anaesthetic(tricaine methanesulphonate, MS-222; Argent Laboratories,Redmont, WA) and fixed in either formalin or Bouin fixa-

tives for 24 and 4 h, respectively. Larvae were then washedin fresh water (four times), and dehydrated in a graded ser-ies of ethanol (30, 40 and 50%) to eliminate any remainderof fixing liquids and finally, preserved in ethanol 70% until

their use for histological studies.

Prior to their fixation, larvae were photographed under abinocular microscope equipped with a magnifying glassNikon SM800 (Yokohama, Japan) and a digital cameraCCTV WV-CP240 (Netherlands); the images (600 dpi) were

used to measure larval standard length and maximum andminimum yolk-sac lengths. Measurements of larvae (n = 15per sampling date) were taken with an image analysis soft-

ware package (ANALYSISTM Soft Imaging Systems GmbH,M€unster, Germany). Yolk-sac volume was calculatedaccording to the formula of Heming and Buddington

(1988): YSV = (1/6) LH2, where YSV is the volume of anellipsoid (yolk sac), and L and H are the maximum andminimum yolk sac lengths, respectively. For histological

purposes, five larvae per sampling date were dehydratedwith graded series of ethanol and embedded in paraffin withan automatic tissue processor Histolab ZX-60 Myr (Espe-cialidades M�edicas MYR SL, Tarragona, Spain). Paraffin

blocks were then prepared in AP280-2 Myr station and cutinto serial sagittal sections (3 lm thick) with an automaticmicrotome Microm HM (Leica Microsystems Nussloch

GmbH, Germany). Paraffin larvae cuts were kept at 40°C,overnight. Samples were then deparafined with graded seriesof xylene and stained by means of Hematoxylin & Eosin

(H & E) for general histomorphological observations. Peri-odic acid-Schiff (PAS) and Alcian Blue (AB) at pH 2.5, 1.0and 0.5 were used to detect neutral and carboxyl-rich andsulphated glycoconjugates in mucous cells (Pearse, 1985).

Histological preparations were observed in a microscopyLeica DMLB equipped with a digital camera OlympusDP70 (Leica Microsystems Nussloch GmbH). Measurements

on histological slides were performed with an image analysissoftware package (ANALYSISTM) on five fish and dataexpressed as mean � SE.

Results

Figure 1 shows the growth in SL of C. urophthalmus fromhatching to the juvenile stage. Larval growth (dph or CTU)under present rearing conditions followed a potential curverepresented by the regression equation: SL (mm) = 3.179 *Ln (Age in dph or CTU) + 0.342 (R2 = 0.99, n = 110;P < 0.05).

Histological development of the digestive system

At hatching, the digestive system of C. urophthalmus con-

sisted of a straight tube (rudimentary intestine) lying dorsallyto the yolk sac. At this stage, the mouth and anal pore werestill closed and did not open to the exterior until age 2 dph(57.0 dd), when larvae measured 2.6 � 0.2 mm SL

(mean � SE; n = 15). Histological observations revealed thatlarvae started feeding on live prey between 5 and 6 dph(142.5 and 171.0 dd) when they measured 4.0 � 0.1 mm SL

(n = 30). During the endogenous feeding period, the diges-tive system of C. urophthalmus experienced a dramatic trans-formation with the almost complete development and

differentiation of all digestive organs, with the exception ofthe gastric stomach that completed its differentiationbetween 11 and 14 dph (313.5 and 399.0 CTU, respectively),

when fish size was between 5.7 and 6.3 mm SL. Thereafterand until the end of the study at 30 dph (855.0 CTU), mostchanges in the digestive system were associated with changesin the tissue complexity and organ size due to the increase in

larvae size.

Digestive system of Mayan cichlid 1305

Yolk-sac

At hatching, the yolk-sac occupied most of the abdominalcavity with an average volume of 1.08 � 0.05 mm3 (n = 15),whereas the volume was reduced by 59.3% (0.44 �0.07 mm3) after 3 days (85.5 CTU; 3.1 � 0.01 mm SL,n = 15) and 81.2% at 5 dph (142.5 CTU; 3.9 � 0.1 mm SL,n = 15). Yolk-sac consumption was linearly correlated with

larval age and described by the equation: YSV = �0.18 (Agein dph or CTU) + 1.08 (R2 = 0.98, P < 0.01; n = 100). Mac-roscopically, the yolk-sac was no longer visible after 6 dph(171.0 CTU), whereas remnants of yolk platelets were micro-

scopically visible next to the hepatic tissue and surroundedby it until 16 dph (456.0 CTU), when larvae measured6.6 � 0.4 mm SL (n = 15). Microscopically, the yolk-sac was

surrounded by a syncytial epithelium and consisted of a largeaccumulation of ovoid eosinophlic and PAS-positive yolkplatelets with scattered spherical non-stained vacuoles scat-

tered throughout the yolk-sac matrix (Fig. 2a,b). These vacu-oles corresponded to lipids that were washed out during theparaffin embedding process of the samples. Yolk platelets

were also slightly stained in light blue with AB (pH 0.5, 1and 2.5), which indicated that they also contained acidic(carboxylated and sulphated) glycoproteins (Fig. 3f). At14 dph (399.0 CTU; 6.2 � 0.3 mm SL, n = 15), remnants of

yolk, mainly lipid droplets and very few small platelets, wereobserved surrounded by hepatic tissue and in contact withthe venous circulation (Fig. 2e,f).

Swim bladder

The primordial swim bladder was differentiated from thedorsal wall of the digestive tract at the time of mouth open-ing at 1 dph (28.5 CTU; 2.1 � 0.1 mm SL, n = 15), appear-

ing as a compressed ovoid structure formed by two layers

of cubical cells with a very narrow lumen between them;however, neither a pneumatic duct nor rete mirabile wasdistinguishable at this age. Between 2 and 3 dph(57.0–85.5 CTU), the swim bladder inflated in all the exam-

ined specimens, and the epithelium lining is flattened. At thisage, the pneumatic duct with a narrow lumen connected theswim bladder with the posterior oesophagus, and a mass of

undifferentiated cubical cells on the posterior area of thechamber formed the rete mirabile. No remarkable differenceswere observed in this organ thereafter with the exception of

its increase in size.

Buccopharynx

At hatching, the mouth was closed and did not open until2 dph (57.0 CTU) when larvae measured 2.6 � 0.2 mm SL(n = 15). The buccopharyngeal cavity was short and lined

by a simple and flat epithelium with scattered round gobletcells. These secretory cells stained blue and purple with PASand AB pH 2.5, 1.0 and 0.5, indicating the presence of a

combination of neutral and acidic (carboxylated and sulph-ated) mucins. Scattered round-shape ionocytes cells wereobserved in the anterior part of the oral cavity, as well as

in the simple and flat epithelium that covered the branchialcavity, although they were no longer visible after 6 dph(171.0 CTU) when larvae were 4.0 � 0.1 mm in SL(n = 15).

At 3 dph (85.5 CTU; 3.1 � 0.1 mm SL, n = 15), the oralcavity grew in length and the first taste buds appeared alongthe mid and posterior regions of the buccopharynx, con-

comitant with a prominent proliferation of goblet cellsalong the entire buccopharyngeal epithelium. Canine-liketeeth were visible in the connective tissue underlying the

pharyngeal submucosa close to the oesophageal opening,but they did not protrude into the pharyngeal lumenuntil 6 dph (171.0 CTU). At 7 dph (199.5 CTU; 4.5 �0.2 mm SL, n = 15), the first pharyngeal papillae wereobserved as a wide folding of the pharyngeal mucosa anteri-orly to the pharyngeal teeth and the oesophageal opening.The number of oral and pharyngeal canine-like teeth, goblet

cells and taste buds tended to increase as fish larvae grewuntil the end of the study, although the most prominentincreases in the number of the above-mentioned structures

were observed between 14 dph (399.0 CTU; 6.7 � 0.5mm SL, n = 15) and 24 dph (684.0 CTU; 7.2 � 0.4 mm SL,n = 15).

Oesophagus

Between 1 and 2 dph (28.5–57.0 CTU), the oesophagus

started to differentiate as a short duct lined by a simple cubi-cal epithelium with scattered goblet cells (18 � 4 cells in100 lm of epithelium) connecting the pharyngeal cavity with

the anterior intestine (Fig. 2a). Histochemically, most of thegoblet cells were stained in magenta (PAS-positive), whereasa few stained light blue (AB pH 0.5, 1.0, 2.5 positive) indi-

cating their content of neutral and acidic (carboxylated andsulphated) glycoproteins, respectively. Between 3 and 4 dph(85.5–114.0 CTU), the oesophagus grew in length and two

layers of circular and longitudinal muscle fibres were clearlydistinguishable forming part of the oesophageal mucosa, aswell as a thin layer of connective tissue surrounding them.At this age, most of the oesophageal goblet cells were stained

purple, indicating that they produced a mixture of neutral

Fig. 1. Standard length growth of Cichlasoma urophthalmus rearedfrom hatching to 30 days post hatch (rearing temp. 28.5 � 1.1°C).Inner image shows macroscopic changes in yolk-sac volume (mm3).At each sampling point, data represent mean value and respectiveSE calculated from 15 specimens. Scale bar below fishimages = 500 µm

1306 C. A. Cuenca-Soria et al.

(PAS-positive) and acidic (AB pH 0.5, 1.0 and 2.5 positive)glycoproteins (Fig. 3f).As the oesophagus grew in length, the oesophageal mucosa

also grew in depth, resulting in an increase in the height ofepithelial and goblet cells lining the oesophagus, and a thick-ening of the circular and longitudinal layers composing themucosa. In larvae measuring 7.2 � 0.4 mm SL and aged

24 dph (684.0 CTU), the entire oesophageal epithelium wascovered by goblet cells (21.3 � 2.5 cells in 100 lm of epithe-lium) secreting a mixture of neutral and acidic (carboxylated

and sulphated) mucins (Fig. 4e). At latter stages of develop-ment, there were no further important histological changesin the oesophagus.

Stomach

Between 3 and 4 dph (85.5–114.0 CTU), the cardiac stomachstarted to form as a dilatation between the oesophagus andanterior intestine. This area was lined by a pseudostratified

columnar epithelium deprived of goblet cells and an incipientmucosal fold separating the future stomach from the intes-tine was already distinguishable (Fig. 3f).

At 9 dph (256.5 CTU; 4.9 � 0.1 mm SL, n = 15), the firstclusters of cubic cells forming the gastric glands were observedin the mid-posterior region of the developing stomach. At11 dph (313.5 CTU; 5.7 � 0.4 mm SL, n = 15), the first

mucous cells secreting neutral glycoconjugates (PAS-positive)were visible in the gastric mucosa (Fig. 4e), whereas at 14 dph

(a) (b)

(c)

(e) (f)

(d)

Fig. 2. Longitudinal section, Cichlasoma urophthalmus, at different stages of development. (a) General view, 4-day-old post hatch (dph)larvae body mid-part showing a large yolk-sac, a well differentiated intestine and an oesophagus with goblet cells in differentiation (stainingtechnique: Periodic acid-Schiff). (b) Yolk sac detail of 3 dph larvae; note decreasing size of yolk platelets toward periphery of the yolk sac asthey are in contact with the surrounding syncithium. Exocrine and endocrine parts of the pancreas are well developed, as seen by large isletof Langerhans size and presence of eosinophilic zymogen granules in pancreocytes (staining technique: haematoxylin-eosin, H & E). (c) Gen-eral view of 5 dph larvae at onset of exogenous feeding. Note large size of the yolk and large number of goblet cells in the oesophagus andinflated swim bladder (staining technique: Periodic acid-Schiff). (d) General view of 8 dph larvae; note reduction of the yolk (mainly com-posed of large lipid vacuoles) and oesophagus epithelium completely covered by goblet cells (staining technique: H & E). (e) General view ofmid- and posterior regions of abdominal cavity. Note important reduction of yolk remnants, large development of the liver occupying one-third of the abdominal cavity, and intermediate intestine devoid of mucosal folds (staining technique: H & E). (f) Yolk residue surrounded byhepatic tissue in larva aged 16 dph; note yolk connection with the venous circulation and accumulation of pigment granules (hydrogen perox-ide bleached; Gisbert and Sarasquete, 2000) around the yolk remnants of (staining technique: H & E). AI, anterior intestine; BC, branchialcavity; E, eye; EP, exocrine pancreas; GC, goblet cell; H, heart; HS, hepatic sinusoid; IL, islet of Langerhans (endocrine pancreas); L, liver;M, trunk musculature; MD, melanin deposit; MN, metanephros (posterior kidney); OE, oesophagus; P, pharynx; PI, posterior intestine;PN, pronephros (anterior kidney); R, rectum; SB, swim bladder; YP, yolk platelet; YS, yolk sac; ZG, zymogen granule

Digestive system of Mayan cichlid 1307

(399.0 CTU; 6.3 � 0.2 mm SL, n = 15), their number andPAS-positive reactivity had increased remarkably with regards

to younger ages (Fig. 4f). At this age, some moderate foldingof the gastric mucosa was observed in the cardiac (anterior)stomach, whereas no remarkable changes were observed until

19 dph (541.5 CTU; 7.0 � 0.2 mm SL, n = 15) when threedifferent regions were clearly distinguishable in the stomach:the cardias, fundus and pylorus. The cardiac region of the

stomach consisted of a wide cavity lined by a simple ciliatedcolumnar epithelium with basal nuclei and a thin foldedmucosa with a layer of smooth circular musculature. The fun-dic region occupied most of the stomach and was lined with a

simple tall ciliated columnar epithelium with scattered PAS-positive mucous cells and a large number of tubular gastricglands. The pyloric region of the stomach was short and lined

by a columnar epithelium devoid of gastric glands. The stom-ach was separated from the intestine by the pyloric sphincter

that was distinguishable as folding of the gastric mucosa sepa-rating both parts of the digestive tract. The histological orga-nization of the stomach did not experience any remarkable

change until the end of the study, with the exception of itsgrowth in size and the increase in the number of gastricglands.

Intestine

At hatching, the intestine was a straight closed tube lined by

a simple columnar epithelium with eosinophilic microvilliand lacking goblet cells. At 2 dph (57.0 CTU), the intestinalmucosa started to form incipient folds and the first goblet

(a) (b)

(c)

(e) (f)

(d)

Fig. 3. Longitudinal paraffin sections of buccopharynx and oesophagus, Cichlasoma urophthalmus, at different stages of development. (a) Gen-eral view of buccopharynx in 2 dph larva covered by a flat epithelium with very low number of goblet cells and devoid of papillae stainingtechnique: haematoxylin-eosin, H & E). (b) Detail of buccopharynx in 7 dph larva; note large number of goblet cells lining the epitheliumcovering the buccopharyngeal lumen (staining technique: Alcian blue ph 2.5 + periodic acid-Schiff, AB pH 2.5 + PAS). Inner image showsepithelium detail containing two goblet cells containing PAS-positive mucins and a taste bud (staining technique: PAS). (c) Detail of pharynxand anterior oesophagus in 12 dph larva having a large number of goblet cells containing a mixture of neutral and acidic glycoproteins, asshown by their purple colour (staining technique: AB pH 2.5 + PAS). (d) Detail of buccopharyngeal mucosa and submucosa; note PAS-posi-tive staining of the dentine in developing teeth, as well as the neutral glycoprotein content (PAS-positive) in goblet cells in the buccopharyn-geal epithelium (staining technique: PAS). (e) Detail of posterior part of buccopharynx (pharynx) with round-flattened papillae in the upperpharyngeal mucosa and pointed teeth protruding into the pharyngeal lumen. Teeth shape is typical of carnivorous species with a broad baseand a pointed apex (staining technique: H & E). (f) Detail of connection between oesophagus and stomach in development of 5 dph larva.Note strong intensity of the microvilli (PAS-positive) covering the stomach epithelium (staining technique: AB pH 2.5 + PAS). BC, bucco-pharynx; BCv, branchial cavity; BP, buccal papilla; E, eye; En, enamel; Dn, dentine; GC, goblet cells; IP, immature tooth; L, liver; LV, lipidvacuole; MC, Meckel’s cartilage; O, operculum; OE, oesophagus; OM, oesophageal musculature; OV, oral valve; P, pharynx; Pc, pulp core;S, stomach; SM, sumucosa; T, canine-like tooth; TB, taste bud; TF, thyroideal follicle; YS, yolk sac

1308 C. A. Cuenca-Soria et al.

cells appeared in the intestinal epithelium among enterocytes.Most parts of intestinal goblet cells stained magenta

(PAS-positive), whereas some others were light blue (AB pH0.5, 1.0, 2.5 positive), indicating a different content of neutraland acidic (carboxylated and sulphated) glycoproteins,

respectively. At this age, the posterior region of the intestinestarted to differentiate into the rectum, as the absence offolding of the mucosa and flattening of enterocytes indicated.At 4 dph (114.0 CTU; 3.9 � 0.03 mm SL, n = 15), the

intestine bent and the intestinal valve separating the anteriorand mid intestinal regions from the posterior one was clearlyvisible. At this age, no relevant histomorphological differ-

ences were obtained between the two intestinal regions sepa-rated by the intestinal valve; both regions were lined by asimple columnar epithelium with basal nuclei, slightly baso-

philic cytoplasm, and prominent eosinophilic microvilli.Coinciding with the onset of exogenous feeding between5 and 6 dph (142.5–171.0 CTU), the first lipid vacuoles were

observed within the enterocytes in both anterior and

posterior regions of the intestine. Goblet cells mainly stainedpurple, indicating that their content was a mixture of neutral

(PAS-positive) and acidic (AB pH 0.5, 1.0 and 2.5 positive)glycoproteins, while a few of them only contained neutral oracidic glycoproteins. Eosinophilic supranuclear inclusion

bodies in enterocytes from the anterior intestine weredetected between 5 dph (142.5 CTU) and 10 dph (285.0CTU). Between 6 and 8 dph (171.0–228.0 CTU; 4.5 �0.1 mm SL, n = 30), several histomorphological differences

were distinguishable between the anterior and posterior intes-tinal segments. In particular, lipid vacuoles were moreabundant in the anterior than in the posterior intestine, indi-

cating that this region was the primary site of lipids absorp-tion in the intestine. In addition, mucosal folds were largerand more numerous in the posterior than in tge anterior

intestinal regions and goblet cells were three times moreabundant in the posterior than in the anterior intestine(12.3 � 1.9 vs 2.7 � 0.4 cells in 100 lm of epithelium).

Folding of the intestinal mucosa increased markably between

(a) (b)

(c)

(e) (f)

(d)

Fig. 4. Longitudinal paraffin sections of intestine and stomach, Cichlasoma urophthalmus, at different stages of development. (a) Detail ofpyloric sphincter in differentiation that separates the future gastric region from anterior intestine (staining technique: haematoxylin-eosin, H& E). (b) Detail of spiral valve separating intermediate from posterior intestine in 4 dph larva (staining technique: H & E). (c) Detail of pos-terior intestine in 12 dph larva containing almost-intact decapsulated Artemia cysts (staining technique: periodic acid-Schiff counterstainedwith methylene blue, PAS-MB). (d) Detail of anterior intestine mucosa showing moderate level of lipid inclusions in enterocytes and PAS-positive microvilli (staining technique: PAS-MB). (e) General view of anterior region of digestive tract in 9 dph larva showing connection ofthe oesophagus covered by abundant goblet cells with the stomach in differentiation. Note presence of first gastric glands in anterior regionsof the stomach (staining technique: H & E). Inner image: detail of oesophageal goblet cells containing neutral glycoproteins stained pink(staining technique: PAS-MB). (f) Detail of stomach in 10 dph larva showing presence of mucin-producing cells (arrow) that contain neutralmucins (PAS-positive) and the microvilli of epithelial cells lining the gastric mucosa containing neutral mucins (asterisk) for protective pur-poses (staining technique: AB pH 2.5 + PAS). AC, Artemia cyst; AI, anterior intestine; EP, exocrine pancreas; GG, gastric gland; IL, islet ofLangerhans (endocrine pancreas); IV, intestinal valve; L, liver; M, trunk musculature; OE, oesophagus; PI, posterior intestine; PS, pyloricsphincter; S, stomach; SB, swim bladder; SLV, supranuclear lipid vacuole; YS, yolk sac

Digestive system of Mayan cichlid 1309

11 and 14 dph (313.5 and 399.0 CTU, respectively), coincid-ing with the full development of gastric glands in the stom-ach.No significant morphoanatomical changes were observed

in the intestinal mucosa after the age of 11 dph (313.5 CTU)until the end of the study, with the exception of the progres-sive increase in length of the intestine and thickening of the

mucosa and increase in length and size of the intestinal foldsas larvae grew in size.

Accessory digestive glands

At hatching, the liver was already developed in C. urophthal-

mus larvae and appeared as a lobular mass running along theentire abdominal cavity almost to the anal pore. Hepatic tissuewas arranged along sinusoids and consisted of polyhedralhepatocytes with centrally located nuclei, reduced eosinophilic

cytoplasm and a few and small lipid inclusions (10.7 � 1.5 lmin diameter, n = 20). Biliary ducts were already visible between2 and 3 dph (57.0–85.5 CTU) and were lined by short and cili-

ated columnar epithelium with basal nuclei occupying most ofthe cytoplasm. At 4 dph (114.0 CTU), hepatocytes started toaccumulate a larger quantity of lipids, as indicated by the

increase in diameter of fat vacuoles (16.8 � 2.2 lm, n = 20).Coinciding with the onset of exogenous feeding, lipid and glyco-gen (PAS-positive) deposits increased progressively withinhepatocytes, while after 16 dph (456.0 CTU; 6.6� 0.4 mm SL,

n = 10) lipid vacuoles occupied most of the cytoplasm (71.1 �5.3 lm, n = 30), displacing the nucleus to the periphery of thehepatocyte, which resulted in a decrease in the glycogen (PAS-

positive) storage in the liver.Similar to the liver, the exocrine pancreas was already dif-

ferentiated in newly hatched larvae. The pancreas was orga-

nized in polyhedral basophilic cells arranged in acini groupedin rosette patterns, containing round-shaped eosinophilic andPAS-positive eosinophilic zymogen granules. Between 2 and

4 dph (57.0 and 114.0 CTU, respectively), the PAS-positivestaining intensity of zymogen granules contained in acinarcells increased, denoting an increase in the synthesis of theprecursors of digestive pancreatic enzymes. At 3 dph

(85.5 CTU), the endocrine pancreas was visible and endo-crine cells were arranged around many capillaries forming asingle islet of Langerhans. The quantitative growth after dif-

ferentiation of the endocrine and exocrine pancreas includedan increase in tissue size, as well as an increase in the contentof zymogen granules, while no new structural elements devel-

oped at latter stages.

Discussion

The developmental timetable of organ appearance and matu-ration depends on the phylogenetic status and reproductive/life history strategy of each fish species. Precocial species

allocate a large amount of resources to the production of asmall number of eggs, and the young hatch at an advancedstage of development. In contrast, altricial species allocate

far fewer resources, releasing a large number of small eggsthat hatch at a less developed stage (Balon, 1986). Fisheswith precocial development are typically larger, as well as

further developed at hatching than are fishes with altricialdevelopment (Govoni, 2004). Regardless of the strategy, fishlarvae need to develop their feeding and digestive sys-tems rapidly, as once the endogenous reserves contained in

their yolk sac are depleted, they have to begin successfully

capturing, ingesting and digesting food in order to providefuel for the intense metabolic and growth processes takingplace during early ontogeny. In the present study, the devel-opment of the digestive system was a very intense process.

At the onset of exogenous feeding (142.5–171.0 CTU;3.8–4.1 mm SL), the buccopharynx, oesophagus, intestineand accessory digestive glands (liver and pancreas) were

almost completely differentiated, whereas the gastric stomachcompleted its differentiation later on (313.5–399.0 CTU;5.8–6.4 mm in SL). Similar results have been reported in

other cichlid species (Morrison et al., 2001; Trevi~no et al.,2011), and other groups of freshwater species such as salmo-nids (Rust, 2002), acipenserids (Gisbert et al., 1998; Gisbert

and Doroshov, 2003; Wegner et al., 2009) and siluriformes(Kozaric et al., 2008; Yang et al., 2010; Saelee et al., 2011;Pradhan et al., 2012).At hatching, the most prelevant feature of the histological

organization of the digestive system in C. urophthalmus wasthe presence of eosinophilic zymogen granules in the exocrinepancreas. Zymogen granules in newly hatched larvae may

correspond to chymotrypsinogen, as the assessment of theontogenic changes in the activity of digestive enzymes in thisspecies conducted by L�opez-Ram�ırez et al. (2011) revealed

that alkaline proteases, especially chymotrypsin, were alreadydetected in newly hatched larvae, whereas trypsin activitywas mainly detected after the onset of exogenous feeding at228.0 CTU. The early presence of zymogen granules in this

species is similar to other cichlid species such as Oreochromisniloticus (Morrison et al., 2001) and Petenia splendida(Trevi~no et al., 2011), and other freshwater species Danio

rerio (Kamaci et al., 2010) and Ompok bimaculatus (Pradhanet al., 2012). In contrast, zymogen granules were not detecteduntil the transition to exogenous feeding in Clarias gariepinus

(Verreth et al., 1992), Aspius aspius (Ostaszewska andWezgiel, 2002), Sander lucioperca (Ostaszewska, 2005), Chond-rostoma nasus (Sysa et al., 2006), Silurus glanis (Kozaric

et al., 2008), and Pelteobagrus fulvidraco (Yang et al., 2010).The opening of the mouth and the macroscopical depletion

of yolk-sac are morphoanatomical events generally consid-ered as markers for the beginning of feeding in fish larvae

(Gisbert and Williot, 2002). Under present rearing condi-tions, mouth opening in C. urophthalmus was detected at57.0 CTU (2.4–2.8 mm in SL), although the onset of exoge-

nous feeding was not observed until 171.0 CTU (3.8–4.0 mmin SL). Remnants of yolk were still detected microscopicallyuntil 456.0 CTU (6.2–7.0 mm in SL), indicating a period of

mixed nutrition that lasted between 285.0 and 313.5 CTU.Similar results have been reported in P. splendida thatshowed an even longer period of mixed nutrition(504–540 CTU) (Trevi~no et al., 2011). This long period of

mixed nutrition indicated that these species might be not vul-nerable to starvation at the onset of exogenous feeding;whereas in aquaculture practices, the presence of yolk

reserves for such a long period might be an advantage for asuccessful transition to exogenous feeding or delaying timeof first feeding without affecting larval growth performance

(Gisbert and Williot, 1997). These results contrast to mostother freshwater species where this period is either non-exis-tent or lasts just a few days (Kamler, 1992; Ostaszewska,

2005; Sysa et al., 2006; Kozaric et al., 2008, among others).Cichlasom urophthalmus is considered to be a generalist

predator, consuming small fishes, macroinvertebrates andsome plant materials that are ingested incidentally with

the consumption of a primary prey item such as benthonic

1310 C. A. Cuenca-Soria et al.

invertebrates (Mart�ınez-Palacios and Ross, 1988; Porter-Whitaker et al., 2012). Thus, the increase in the density ofoesophageal goblet cells observed between 6.2 and7.6 mm SL (399.0–684.0 CTU) might be related to an onto-

genetic change in fish feeding habits, shifting from a dietbased on small zooplankton preys to a juvenile-type dietbased on larger prey such as other fishes and benthic inverte-

brates (Mart�ınez-Palacios and Ross, 1988). Thus, the above-mentioned changes might be age-dependent and dietaryinduced, since mucins produced by goblet cells may serve as

lubricants for protecting the oesophageal mucosa from physicaland chemical damage resulting from the ingestion of prey, or asa protection against bacterial infections (Sarasquete et al.,

2001).Although the stomach anlagen in C. urophthalmus appeared

as a dilatation between the oesophagus and anterior intestinebefore the onset of exogenous feeding, the complete morpho-

anatomical differentiation of this organ was not achieved until541.5 CTU (6.8–7.3 mm in SL). These results are differentfrom those reported in P. splendida where stomach differentia-

tion coincided with the transition to exogenous feeding(Trevi~no et al., 2011). The assessment of different digestiveenzyme activities conducted in C. urophthalmus (L�opez-Ram�ırez et al., 2011) revealed that acid proteases were detectedin this species after the onset of exogenous feeding, coincidingwith the appearance of the first clusters of gastric glands (pres-ent results), whereas their activity progressively increased until

age 551–609 CTU, when they became maximal coinciding withthe complete morphoanatomical differentiation of the stomach(present study). The early functional development of the stom-

ach coupled with the differentiation of the pancreas (produc-tion of zymogen granules and pancreatic enzymes) and theliver (bile salts production) indicated that C. urophthalmus

might be capable of satisfactorily digesting different types offeeds soon after the transition to exogenous feeding.In this study, lipid vacuoles were observed in the liver and

intestine of C. urophthalmus larvae after the onset of exoge-nous feeding. In this context, the accumulation of lipids inthe intestinal mucosa is considered as an indicator of luminalabsorption and temporal storage of lipids (Gisbert et al.,

2008), reflecting the functional development of the intestine(L�opez-Ram�ırez et al., 2011). In addition, accumulation oflipids in the intestine was positively correlated with changes

in the degree of lipid deposits in the liver of C. urophthalmusat first feeding. At this early development stage (142.5–228.0 CTU; 3.8–4.6 mm in SL), the existence of acidophilic

supranuclear bodies in the postvalvular intestine of C. uroph-thalmus larvae indicated the presence of pinocytotic absorp-tion and intracellular protein digestion during thedevelopmental period, characterized by the absence of a

functional stomach (Govoni et al., 1986). Thus, during thisperiod, the anterior intestine has been described as the mainsite of the larval digestive tract for extracellular proteolytic

digestion due to its alkaline pH and the presence of trypsinsecreted from exocrine pancreas (Zambonino-Infante andCahu, 2001). It is generally accepted that a progressive

reduction in the presence of intestinal supranuclear vacuolesin the larval intestine after the acquisition of the gastricfunction would result from a change in the protein digestion

mechanisms, as the secretion of hydrochloric acid andenzymes produced in gastric glands might reduce pinocytoticactivity and intracellular digestion by cytosolic enzymes(Cahu and Zambonino-Infante, 2001). In this study, the

reduction in number and size of intestinal supranuclear

vacuoles was consistent with the maturation of enterocytesand the acquisition of an adult mode of digestion observedbetween 377 and 493 CTU (L�opez-Ram�ırez et al., 2011),although their presence after the complete development of

the digestive system between 598.5 and 684 CTU remainsunclear. Histological observations showed that Artemia-fedlarvae had almost intact nauplii in the postvalvular intestinal

lumen and rectum. Although the presence of intact food inthe intestine could be attributed to a low level of functional-ity of the digestive system, histological results (present study)

coupled with enzymatic data (L�opez-Ram�ırez et al., 2011)indicated that fish had a completely differentiated and func-tional digestive system. Thus, the presence of intact prey

inside the gut of fish may be attributed to voracious appetitesand high feeding rates that result in a rapid digestive transit(food items may be expelled before being completelydigested), which may result in a limited availability of nutri-

ents from this type of food item. Thus, it is recommended toadjust live prey distribution throughout the day in order toreduce prey density in larval rearing tanks and force larvae

to continuously forage during daylight hours but at a lowerintensity. This would result in longer digestion times andpotentially higher growth performances, as larvae would

make more efficient use of dietary nutrients (Rønnestad andMorais, 2008). In C. urophthalmus larvae, fat accumulationin the anterior and posterior intestine and liver remained lowduring the Artemia feeding period, which suggested a correct

balance of dietary fat, larval nutritional requirements andtheir digestive capacities (L�opez-Ram�ırez et al., 2011;Boglino et al., 2012). However, the level of hepatic fat depos-

its increased dramatically after larval weaning (456 CTU;6.2–7.0 mm in SL), although intestinal fat deposits did notvary. These observations revealed that the level of lipid

absorption by enterocytes did not exceed their rate oflipoprotein synthesis and lipid transport to the liver (Olsenet al., 1999), which resulted in a fatty liver (Gisbert et al.,

2008). These results are similar to those already reported inP. splendida (Trevi~no et al., 2011), as both cichlid species aregenerally fed with diets formulated for rainbow trout. Thesediets might cover the basic nutritional requirements of the

species, but they are not especially designed to matchthe species-specific nutritional needs of these two cichlidspecies.

In conclusion, the developmental pattern of pancreatic,gastric and intestinal enzymes described by L�opez-Ram�ırezet al. (2011) was closely related to the histological develop-

ment of the digestive system reported in this study. Findingson the development of the digestive system in C. urophthal-mus coupled with those on its functionality could lead to abetter understanding of the digestive physiology of this spe-

cies. These results on the organogenesis of larvae are a usefultool for establishing the functional systemic capabilities andphysiological requirements of larvae to ensure optimal wel-

fare and growth under aquaculture conditions, which mightbe useful for improving current larval rearing practices forthis species.

Acknowledgements

This study was funded by the project “Identificaci�on de ingre-dientes en alimentos balanceados y su digestibilidad para elcultivo de peces en Tabasco” of the Government of the Stateof Tabasco and CONACYT (Mexico). The research stage of

C. A. Cuenca-Soria at IRTA was funded by CONACYT.

Digestive system of Mayan cichlid 1311

Authors thanks O. Bellot (IRTA) for her excellent technicalassistance in preparing histological slides.

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Author’s address: Enric Gisbert, Institut de Recerca i TecnologiaAgroaliment�aries (IRTA), Centre de Sant Carlesde la R�apita, E-43540 Sant Carles de la R�apita,Spain.E-mail: enric.gisbert@irta.cat

1312 C. A. Cuenca-Soria et al.