Aquaculture 388391 (2013) 7688

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Aquaculture
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Ontogeny of the digestive system of meagre Argyrosomus regius reared in a mesocosm, and quantitative changes of lipids in the liver from hatching to juvenile
Ioannis E. Papadakis a, b,, Maroudio Kentouri b, Pascal Divanach a, Constantinos C. Mylonas a
a b

Institute of Marine Biology, Biotechnology and Aquaculture, Hellenic Center for Marine Research, P.O. Box 2214, Iraklion, Crete 71003, Greece Biology Department, University of Crete, P.O. Box 2208, Iraklion, Crete 70013, Greece

a r t i c l e

i n f o

a b s t r a c t
Histological development of the digestive system was studied in association with feeding preferences in meagre (Argyrosomus regius) from hatching to juvenile (44 days after hatching, dah), using a mesocosm system. In addition, the liver lipid content was evaluated using histological methods (area covered with lipid vacuoles, ACLV%). The ontogeny of the digestive system was completed by 19 dah (or 444 degree days) with the gastric glands appearing at 15 dah (361 degree days), the pyloric ceaca at 17 dah (404 degree days) and the Y-shaped stomach formed at 19 dah (444 degree days). The rearing period was characterized rst by relatively slow growth until tail exion (~15 dah) and fast growth thereafter (meanSD total length of 45.144.00 mm at 44 dah). When the feeding protocol included exclusively rotifers, mean ACLV remained low (2.390.34%) while feeding on Artemia nauplii and copepods increased liver ACLV to 47.186.56% at 20 dah. Changes in the feeding protocol were reected in feeding preferences (stomach content), and variations of liver lipid content and the occurrence of vacuoles in the intestine. During transition from live prey to articial feed (~28 dah), ACLV decreased signicantly indicating a malnutrition period concomitant with a delay in the acceptance of articial feed of 8 days. Thereafter, consumption of articial feed resulted in an increased ACLV to 56.37.6% at 36 dah. The results indicate that during early development meagre is a fast growing species, developing rapidly the structures and basic organs of the digestive system required to overcome successfully the critical stages of larval rearing. The study also shows that histological evaluation of liver lipid content using the ACLV may be a valuable tool in commercial aquaculture to improve larval rearing protocols, and production efciency. 2013 Elsevier B.V. All rights reserved.

Article history: Received 25 October 2012 Received in revised form 9 January 2013 Accepted 10 January 2013 Available online 23 January 2013 Keywords: Argyrosomus regius Meagre Digestive system Larvae Liver

1. Introduction Meagre (Argyrosomus regius) is a scienid species distributed in the Mediterranean Sea, Black Sea and the Atlantic coast of Europe, with maximum reported weight and total length of 103 kg and 182 cm, respectively (Chao, 1986; Quro and Vayne, 1987). This species inhabits coastal ecosystems near the continental shelf, and can be found in lagoons or river Deltas (Grifths and Heemstra, 1995) where it spawns in the spring. Flesh quality is considered exceptional and highly nutritious, and the species name regius (i.e., royal) was given because of its highly esteemed esh quality (Poli et al., 2003). Meagre has been considered a good species for the diversication of n-sh aquaculture in the Mediterranean cage-culture industry (Qumner, 2002). Growth rates of >1 kg year 1 have been reported (Monfort, 2010), which is many-times more than the growth of currently cultured species such as the gilthead seabream (Sparus aurata) and the European sea bass (Dicentrarchus labrax). In addition, meagre mature at a large size > 4 kg (Schuchardt et al., 2007), which is larger
Corresponding author. Tel.: +30 2810 337876; fax: +30 2810 337875. E-mail address: papad@hcmr.gr (I.E. Papadakis). 0044-8486/$ see front matter 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aquaculture.2013.01.012

than the harvest size, thus avoiding problems of reduced growth associated with maturation. Although meagre fail to undergo oocyte maturation spontaneously in captivity, recently developed hormonal induction methods have proven effective in controlling spawning (Duncan et al., 2012; Fernndez-Palacios et al., 2009a; Mylonas et al., 2011) and producing eggs of sufcient quality and quantity for commercial hatchery production. Larvae and juveniles have been reared with similar facilities and methodologies to other marine shes using rotifers, Artemia nauplii and inert feeds (Estvez et al., 2007; Fernndez-Palacios et al., 2007, 2009b; Hernndez -Cruz et al., 2007; Roo et al., 2007, 2009, 2010; Valls and Estvez, 2009). Therefore, the introduction of meagre in commercial aquaculture in the Mediterranean region may contribute positively to the further growth of the industry. Meagre was rst produced in 1997 in France, and once juveniles became available outside France, European production grew from 103 mtn in 2003 to 2377 mtn in 2010 (FAO, 2012). To optimize larval rearing technology, in order to achieve better production results (i.e., higher survival, less variable growth, reduced cannibalism, lower skeletal deformities, better welfare and reduced cost), it is essential to study the ontogeny of the digestive system in relation to the rearing method and employed feeding protocol. The

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most critical rearing period is during the early developmental stages, especially during the transition from endogenous to exogenous feeding (Watanabe and Kiron, 1994), and during weaning and the transition from live to articial feeds (Garcia-Ortega et al., 2003; Hamlin et al., 2000). A number of genetically programmed, species-specic morphological and functional changes are observed in the digestive system of sh at the early developmental stages until metamorphosis to juvenile (Zambonino-Infante et al., 2008). However, the ontogeny of the digestive system exhibits signicant plasticity, with temperature (Kamler, 2002) and larval rearing protocol (Papadakis et al., 2009) being the most inuential environmental factors affecting the appearance and development of the various structures of the digestive system. Histological examination of the timing of ontogenetic development of the digestive tract and accessory glands is considered the most accurate method for the evaluation of proper function and, thus, nutritional status of sh larvae (Hamlin et al., 2000). For example, hepatocyte morphology and vacuole formation in the cytoplasm are related to the nutritional status of the larvae (Chen et al., 2007; Gisbert et al., 2004a; O'Connell and Paloma, 1981). Understanding the changes of the digestive system occurring during ontogeny under specic rearing conditions is essential and may provide indicators of the nutritional status of the sh during early stages (Chen et al., 2007; Gisber et al., 2008; Trevio et al., 2011). These indicators, then may aid in the identication of periods of malnutrition of the reared organism and provide information for the optimization of commercial rearing technologies and feeding protocols (Zambonino-Infante et al., 2008). Although the ontogeny of the digestive system is basically similar among teleost shes, the existence of signicant species-specic differences related to the timing of appearance and complete functionality of the various structures or organs, makes it necessary to study each potential species in order to develop optimal rearing protocols (Conceio et al., 2007; Gisber et al., 2008; Zambonino-Infante et al., 2008). Such information is not yet available for the meagre. The aim of the present study was to describe the ontogeny of the digestive system in meagre from hatching to metamorphosis using the mesocosm rearing system, and relate changes occurring in lipid deposition in the liver with the feeding protocol during rearing of meagre using the mesocosm method. The use of the mesocosm is advantageous in n-sh larval rearing of species with little information of their nutritional requirements (Divanach and Kentouri, 2000; Koumoundouros et al., 2004) as this rearing system is more similar to the natural conditions. It is expected that the obtained information will aid in the improvement of other more intensive commercial larval rearing protocols for this species. 2. Materials and methods Larval rearing was performed at the facilities of the Institute of Marine Biology, Biotechnology and Aquaculture of the Hellenic Centre for Marine Research (HCMR), Iraklion, Crete, Greece. Rearing was based on the mesocosm technology (Divanach, 1985; Divanach and Kentouri, 2000; Kentouri, 1985) as described below. 2.1. Mesocosm larval rearing and sampling One hundred thousand meagre eggs were stocked in one 40-m3 tank. Unltered water was pumped initially directly from the sea (salinity of 38 psu) and then was renewed with well-water (salinity of 32 psu). Daily renewal of water increased gradually from 20% rst day after hatching (dah) to 400% at the end of the rearing period of 48 d. During daytime, the tank was exposed to ambient light and during the night articial light (600 lx) was used. Oxygen saturation ranged between 90% and 95% and temperature between 19 and 23 C from the start to the end of the rearing period. Surface skimmers were used between 4 and 15 dah to remove the surface oil and assist in swim bladder ination. From 2 to 27 dah, larvae were provided with daily additions of

microalgae Chlorella minutissima. Rotifers (Brachionus plicatilis), enriched previously (DHA Protein Selco, INVE S.A., Belgium) for 6 h in enrichment tanks (200250 ind ml 1) with aeration, were added daily from 3 to 8 dah at a concentration in the rearing tank of 23 ind ml 1. Enriched (A1 DHA Selco, INVE S.A., Belgium) A. nauplii (Artemia) also remained in an enrichment tank (280 nauplii ml 1) for 6 h with high aeration, and were then offered to the larvae from 7 to 30 dah at a starting concentration of 0.05 at 7 dah, increasing to 0.35 nauplii ml 1 at the beginning of the weaning stage. Articial feeds were added progressively from 15 to 44 dah. The feed size was adjusted gradually according to sh size (R1 100 and Proton 2/3 grain size 200300 m, NRD 3/5 grain size 300500 m, NRD 5/8 grain size 500800 m, INVE S.A., Belgium). Naturally produced copepods mainly of the families Harpacticoidae and Tisbidae appeared in the tank between 18 and 23 dah at water column concentrations ranging from 0.01 to 0.08 individuals ml 1. The overall copepod concentration may have been underestimated since the majority of them were attached to the tank walls. At 48 dah sh were counted and transferred to on-growing tanks. Random samples of larvae (n = 10) were taken at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 13, 14, 15, 17, 19, 20, 23, 26, 28, 30, 33, 36, 40 and 44 dah (day 0 was the day of hatching). Sampling was performed 2 h after feeding. Fish were measured in total length, examined with the aid of stereoscope for external morphology and were preserved for histology in buffered 4F:1G, containing 4% formaldehyde: 1% gluteraldehyde for at least 24 h (McDowell and Trump, 1976). 2.2. Histological analysis Before embedding in methacrylate resin (Technovit 7100, Heraeus Kulzer, Germany) larvae were dehydrated in gradually increased ethanol solutions (7096%). Serial sections of 3 m were obtained with a microtome (Reichert Jung, Biocut 2035, Germany). Sections were stained with Methylene Blue (Sigma, Germany)/Azure II (Sigma, Germany)/Basic Fuchsin (Polysciences, USA) according to (Bennett et al., 1976). In order to describe the ontogeny of the digestive system and accessory glands all the sections were examined using compound microscope. Stomach content was examined using microphotographs taken from histological sections from 4 larvae of each daily sampling, at 20 and 40 magnication. The ontogeny results were presented in relation to the age of the sh in dah as well as in temperature degree-days after hatching. The latter is estimated by the summation of the mean daily temperatures during rearing and allows for the inuence of temperature to be factored in the rates of growth and development processes in poikilothermic animals. 2.3. Area covered with lipids vacuoles in the liver (ACLV %) Four additional larvae were sampled at 6, 8, 11, 13, 15, 17, 20, 23, 26, 28, 30, 33, 36, 40 and 44 dah and processed histologically for the estimation of liver lipid content according to the methodology of Papadakis et al. (2009). Prior to 6 dah it was not possible to measure ACLV, due to the very small size of the liver. For each larva, 6 microphotographs were obtained at 100 magnication from sections obtained from different areas of the liver. A total number of 384 photographs were analyzed. Photographs were converted to gray scale in order to convert the area occupied with lipid vacuoles in white, and the total area covered with lipid vacuoles was calculated using an image analysis software (Image J, NIH, USA). Other tissues that could be confused by the software as lipid vacuoles (e.g., blood capillaries) were manually excluded from the analysis. Measurement of the lipid vacuole-covered area was performed automatically following manual delineation. The results are presented as the percentage of the total area of the hepatic tissue of the photograph (without other non-hepatic elements) covered with lipid vacuoles.

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2.4. Statistical analysis Statistical analyses of the ACLV (%) during the course of the larval rearing period were performed using two-way ANOVA (Sigma Stat statistical package) with ACLV and time as factors, followed by Duncan's New Multiple Range test, at P b 0.05. The ACLV values were transformed prior to analysis using the arcsine of the square root transformation, but for presentation the original data were used. Data are presented as meanSD. For the description of growth performance of total length as a function of time, linear regression was used. 3. Results Growth was best described by the equation TL=0.3711 dah+2.265 (R 2 = 0.9551) between 0 and 17 dah and by the equation TL = 1.2854 dah 10.047 (R2 = 0.9884) between 17 and 44 dah until the end of the experimental rearing (Fig. 1). 3.1. External morphology and development The pre-larval stage lasted until 3 dah (Fig. 2). Meagre larvae consumed most of their yolk reserves by 3 dah. During this period, the buccal cavity opened at the rostral area, corresponding to the mouth. This incidence concurred with the development of the lower jaw. Body transparency allowed identication of liver and swim bladder formation during this stage (later conrmed by histology). The eyes were pigmented between 2 and 3 dah and the ureter was visible posterior to the intestine. A primary marginal fold, which extended from the dorsal part of the head to the ventral limit of the stomach, was interrupted locally at the anus. The pectoral ns at this time were visible as a pair of strands. Between 5 and 6 dah the yolk sac was absorbed rapidly, followed by the lipid droplet (Fig. 2) at the same time that rotifers were offered to and consumed by the larvae. Thus, between 3 and 6 dah, both

endogenous and exogenous feeding occurred. Initially the notochord was straight (35 dah) and the head area was free from the yolk sac. At 5 dah the swim bladder was visible ventrally to the spinal cord, and anterior to the stomach and posterior to the acoustic vesicles. The swim bladder was formed at the pre-larval stage, however it was inated at 4 to 6 dah after mouth opening. The pre-exion larval stage lasted from 9 to 14 dah, and it was characterized by the complete absorption of the yolk sac and the lipid droplet (Fig. 2). Completion of tail exion the dorsal bending of the notochord tip was achieved at 1415 dah. During the larval post-exion stage, the caudal, dorsal and anal ns differentiated from the primary marginal folds between 14 and 19 dah. The pelvic ns appeared at 18 to 19 dah. The completion of the main external developmental stages occurred at 34 dah with the appearance of the scales and the nal pigmentation, at which time the sh looked similar to adults and were considered juveniles. 3.2. Digestive system ontogeny From hatching to 1 dah, the digestive tract appeared as a closed straight tube located dorsal to the yolk sac. The single-layer epithelium consisted of simple cuboidal and columnar cells. The rst differentiation event occurred with the opening of the mouth and the anus at 3 dah, which was followed by many important differentiations of the digestive tract (Fig. 3). The buccal cavity at 3 dah was covered by a single layer of epithelial cells. The taste buds were formed at 4 dah (Fig. 4A). At 5 dah, goblet cells rst appeared at the esophagus (Fig. 4B), at the same time that the various folds of the esophagus began to form (Fig. 4B), increasing in number and size over time. The rst pharyngeal teeth appeared at 6 dah (Fig. 4C). At 3 dah, intestinal segmentation was apparent since the intestinal sphincter that separates the middle and posterior intestine was

Prelarva
Flexion

Larva

Juvenile

50 45 40

Total length (mm)

35 30 25 20 15 10 5 0 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44

y = 0.3711x + 2.265 R2 = 0.9551

y = 1.2854x 10.047 R2 = 0.9884

Days after hatching

Phytoplankton Rotifers Artemia nauplii Artificial feeds Copepods
Fig. 1. Growth performance of meagre (Argyrosomus regius) larvae in total length (mean SD) during larval rearing, in relation to time (days after hatching). The type and relative amount of different feeding items offered during rearing are shown schematically below the graph. An increase or decrease in thickness of the bar representing the rotifers, Artemia nauplii and articial feeds, indicates higher or lower, respectively, amounts of feeding items offered. Copepods appeared in the rearing medium naturally. The linear regressions describe the best-t growth curve of meagre larvae separated in two periods.

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11 dah Eggs 1.14 0.01 mm Larva pre-flexion 5.83 0.70 mm

1dah Prelarva 2.95 0.03 mm

14 dah Larva flexion 7.38 0.48 mm

3dah Larva (open mouth) 3.01 0.03 mm

20 dah Larva post-flexion 16.00 1.82 mm

5 dah Larva 3.66 0.45 mm Juvenile 32.22 1.92 mm

34 dah

Fig. 2. Macrophotographs of meagre at representative developmental stages during larval rearing (days after hatching, dah). The mean (SD) total length for each sampling (n=10 randomly sampled individuals) is indicated below each photograph.

formed, and the different characteristic areas were all present, including the area where the stomach would be later formed (Fig. 5A). The pyloric sphincter appeared at 7 dah (Fig. 5B) and the gastric glands appeared at 15 dah (Fig. 5C). The rst intestinal lobe appeared at 3 dah (Fig. 5D). Intestinal villi appeared initially at 2 dah, but they were more obvious from 3 dah. At the same time, the rst vacuoles appeared at the posterior part of the intestine (Fig. 5D). The size of the villi increased progressively thereafter. The syncytium layer appeared to enclose the remaining of the yolk sac and the lipid droplet, the latter being the last to be absorbed, thus completing the endogenous feeding of the larva (Fig. 5D). The vacuoles observed at the foregut at 3 dah were no longer present at 5 dah, but reappeared at 11 dah when they increased progressively in number until 23 dah (Fig. 5E). From 23 dah onwards, their number was reduced until they disappeared completely by 33 dah. At 17 dah a small number of vacuoles were present in the middle part of the intestine, which disappeared at 20 dah. The rst goblet cells appeared at the mid gut at 11 dah together with the appearance of rodlet cells (Fig. 5F). During ontogeny, goblet cells were low in number compared to rodlet cells, the latter increasing over time (data not shown). Rodlet cells appeared in the posterior part of the intestine between 13 and 15 dah, at the boundaries of the intestinal sphincter (especially at the area that separates the anus and the middle part of the intestine). At 17 dah, the rst pyloric ceaca appeared (Fig. 5G) and by 26 dah already 5 pyloric ceaca had formed (Fig. 5G). The undifferentiated pancreas appeared initially at 3 dah (Fig. 5H) and was organized posterior to the liver, attached to the middle intestine and progressively increasing in size and surrounding the anterior intestine. Differentiation to endocrine and exocrine pancreas began at 4 dah (Fig. 5I). The liver

developed rapidly early hepatic cells appeared at 1 dah located initially behind the yolk sac under the anterior intestine and later surrounding the anterior part of the intestine. The rst lipid vacuoles in the liver appeared at 4 dah (not shown) and fat deposition in the liver increased with time (see below). 3.3. ACLV and stomach content The evolution of ACLV in the liver was characterized by four periods (Fig. 6A). In the rst period, the ACLV increased progressively from 6 to 20 dah (Fig. 6A), with the ACLV at 20 dah being signicantly higher than the previous days (P b 0.05). Until 11 dah, the ACLV was low and unchanged (Figs. 6A and 7.1A) compared to the following days. At this time the feeding protocol was based mainly on rotifers (Fig. 6B), and the larvae had only rotifers in their stomach (Fig. 7.1B and C). After 8 dah and until 13 dah, Artemia were offered in progressively larger numbers (Fig. 6B). During this period the ACLV increased (Fig. 7.2A) and based on stomach content analysis, the larvae consumed mostly Artemia (Fig. 7.2B and C). From 13 to 20 dah the ACLV increased further (Figs. 6.A and 7.3A), at the time that the feeding protocol included not only Artemia and articial feed, but also copepods that appeared in the mesocosm tank between 18 and 23 dah (Fig. 6B). Copepods were observed only in this specic period within the stomach content of the larvae together with Artemia (Fig. 7.3B and C). In the second period, between 20 and 28 dah, there was a depletion in ACLV (Figs. 6A and 7.4A) with the values at 26 dah already being signicantly lower than at 20 dah (P b 0.05). The concentration of Artemia in the protocol was stable at this time, while articial feed increased

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Days after hatching (dah)

0 2 4 6 8 10 12 14 16 18 20 22 24

Buccopharynx
Mouth opening Taste buds Pharyngeal teeth (91) (114) (160)

Esophagus
Esophagus folds Goblet cells (91) (137)

Stomach
Cardiac sphincter Gastric glands Y-Shape (114) (361) (444)

Intestine
Pyloric caeca . Rodlet cells MG Goblet cells MG Supranuclaer bodies in MG Rodlet cells FG Ileorectal valve Supranuclear bodies in FG. Anus opening (91) (91) (91) (91) (91) (137) (274) (722) (274) (274) (404) (361) (464) (404)

Liver Pancreas

Larva (3-9)

Pre-flexion (9-14)

Flexion (14-15)

Post-flexion (15-33)

Prelarva

Larva

Fig. 3. Schematic representation of the appearance (circles) of the main developmental structures examined in meagre larvae, as a function of days after hatching (dah, horizontal axis) and degree days (number in parentheses). Horizontal bars indicate the period that supranuclear bodies (vacuoles) were present in the anteriormedian intestine (mid gut, MG) and foregut (FG). The arrowhead indicates that the supranuclear bodies still remained in the FG at 33 dah or 722 degree days.

substantially (Fig. 6B). Stomach content analysis indicated that between 20 and 28 dah the articial feed consumed by the larvae increased (initially with very low rates) and at the same period the amount of Artemia decreased progressively and at 28 dah, the stomach appeared to be almost full of articial feed (Fig. 7.4B and C). In the third period, between 28 and 36 dah, there was an increase again in ACLV (Figs. 6A and 7.5A) with 36 dah being higher than 28 dah (P b 0.05). During this period, the feeding protocol included mostly articial feed, as Artemia was no longer offered after 31 dah (Fig. 6B), as could also be seen in the stomach content (Fig. 7.5B and C). During the last period, between 36 and 44 dah, there was no statistically signicant difference in ACLV (Figs. 6A and 7.6A), at a time that

the feeding protocol included only articial feed (Fig. 6B), which was found in abundance in the stomach (Fig. 7.6B and C). 4. Discussion The use of the mesocosm rearing system is advantageous in n-sh larval rearing, especially in species for which little is known of their nutritional requirements (Divanach and Kentouri, 2000; Koumoundouros et al., 2004). Better production results are achieved mainly due to the greater variety of prey items available, coming from the unltered sea water that is used to ll the tank initially (Papandroulakis et al., 2004). This variety of zooplankton that grows in a mesocosm in conjunction

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A
BC TB

GA

B
GC Kd Es F

C
AV

Br

BC PT Kd

L
Fig. 4. Microphotographs of histological sections of meagre larvae at 4 (A), 5 (B) and 6 (C) days after hatching, showing differentiation of the buccal cavity. AV=acoustic vesicles, GA=gill arch, BC=buccopharynx, Br=brain, GC=goblet cells, EsF=longitudinal fold at esophagus, Kd=kidney, L=liver, PT=pharyngeal teeth, and TB=taste buds. Bars represent 0.1 mm.

with external feeding makes this rearing system versatile and more similar to the natural conditions. To our knowledge, this is the rst study on the ontogeny of the digestive system in meagre under culture conditions. The growth data suggests that meagre can be considered a fast growing species at these early developmental stages, when compared to other species reared in comparable rearing systems and temperatures. For example, the shi drum (Umbrina cirrosa) also a member of the Sciaenidae family reached 322 mm in total length in 41 dah (Zaiss et al., 2006), while meagre under the same conditions reached 43.71.94 mm in the same period.

Other fast growing species cultured with the mesocosm technology in similar temperatures, such as the greater amberjack (Seriola dumerilii) reached 39.95.4 mm at 40 dah (Papandroulakis et al., 2005). Regarding the ontogeny of the digestive system, the present results indicate that the basic structures of the digestive system of meagre developed very rapidly (within 19 dah or 444 degree days), similar to the shi drum (U. cirrosa) that also developed at the same degree days (Zaiss et al., 2006). In comparison, the gilthead seabream which is widely cultured in the Mediterranean area completed its digestive system development in 6069 dah (Elbal et al., 2004) or between 1170 and 1345 degree days (our estimation from the results of Elbal et al., 2004). The appearance of the alimentary canal of meagre at hatching as a straight undifferentiated tube, located dorsally to the yolk sac without formed mouth or anus is consistent with the descriptions made in other teleostean shes, such as the spotted sand bass (Paralabrax maculatofasciatus) (Pea et al., 2003), the shi drum (Zaiss et al., 2006), the European sea bass (Beccaria et al., 1991), the common sole (Solea solea) (Boulhic and Gabaudan, 1992), the gilthead seabream (Sarasquete et al., 1995), the common dentex (Dendex dentex) (Santamara et al., 2004) and the red porgy (Pagrus pagrus) (Roo et al., 1999). During the endogenous feeding phase and prior to 3 dah, nutrient acquisition relied exclusively on the reserves of the yolk sac and the lipid droplet assimilated through the syncytium layer (Balon, 1986; Diaz et al., 2002; Heming and Buddington, 1988), which is formed during epiboly (Heming and Buddington, 1988). Yolk sac and lipid absorption is the result of the intracellular activity of this layer, whose function is assisted by the blood circulation that begins with the onset of cardiac function (Rnnestad et al., 1992). The opening of the buccal cavity and anus at 3 dah in meagre was one of the rst major developments of the digestive system, underscoring the rapid preparation for the transition of the larvae to the exogenous feeding phase. Similarly, the early appearance of pharyngeal teeth and the formation of longitudinal folds and appearance of goblet cells in the esophagus were also directly linked to the ability of meagre for early commencement of exogenous feeding (Abol-Muna et al., 2006). The presence of pharyngeal teeth early on and their rapid growth, suggests the ability of this species for mechanical processing of the ingested rotifers, which helps further the autolysis process (Luizi et al., 1999). The timing of the appearance of the pharyngeal teeth is species-specic, and in other species characterized also by rapid growth, such as the yellowtail kingsh (Seriola lalandi) (Chen et al., 2006) or common dentex (Santamara et al., 2004) pharyngeal teeth appeared at 8 and 7 dah, respectively. The sense of taste and the ability to test and select food was also acquired very early in meagre (4 dah). In comparison, in the bay snook (Petenia splendida) and marble goby (Oxyeleotris marmoratus), the taste buds appeared at 3 dah (Abol-Muna et al., 2006), in shi drum at 8 dah (Zaiss et al., 2006), in yellowtail kingsh at 8 dah (Chen et al., 2006), in common dentex at 14 dah (Santamara et al., 2004) and in spotted sand bass at 10 dah (Pea et al., 2003). At the esophagus goblet cells appeared as early as 5 dah together with mouth opening. The goblet cells produce a signicant mucous layer that prevents mechanical injuries and/or bacterial infections (Gisber et al., 1999; Humbert et al., 1984), facilitates food ingestion (Abol-Muna et al., 2006; Kapoor et al., 1975) and protects the intestinal mucosa from proteolytic degradation by neutralizing stomach acidity (Scocco et al., 1996; Smith, 1989). The appearance of the longitudinal folds on the esophagus at the same period relates to its ability for enlargement that can aid in the transition of food particles to the subsequent sections of the digestive system (Diaz et al., 2008). The appearance of the longitudinal folds in haddock (Melanogrammus aeglenus) has been associated also with the initiation of the exotrophic feeding phase (Hamlin et al., 2000). The rapid segmentation of the intestine of meagre occurred during the overlap of the autotrophic and exotrophic feeding phase (from 3 dah) and correlated with mouth opening, as described in other shes (Elbal et al., 2004). The ileorectal valve that separates the middle from the

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Kd Br Es

MG

B
L FG

AV BC PT Es L PS MG P Kd

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GA

EsF GA

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C
BC EsF GA L St MG GG Art

D
Rot P MG P L MG SV L Y

Syn

FG P FG

OD Syn

E
SV

F
RC

FG

GC

FG

G
BC CS L H P

SB

H
MG Art St P

P ex P end

PS PC MG
Fig. 5. Microphotographs of histological sections of meagre larvae at different developmental stages. Sections (sagittal) are oriented with the anterior side on the left. (A) At 3 days after hatching (dah) showing the different characteristic areas of the digestive system. (B) Larvae at 7 dah when the pyloric sphincter appeared. (C) At 15 dah when the gastric glands rst appeared (insert). (D) At 3 dah when the syncytium was visible surrounding the yolk and oil droplet (broken line). The insert comes from another section, but from the same larva and shows the syncytium layer around the oil droplet. (E) At 24 dah when numerous vacuoles were visible in the foregut. (F) At 11 dah when the rodlet and goblet cells were visible. (G) At 26 dah when 5 pyloric caeca were developed. (H) At 3 dah showing the formation of the pancreas. (I) At 4 dah when the pancreas differentiated to endocrine and exocrine parts. AV=acoustic vesicles, Art=Artemia nauplii, BC=buccopharynx, Br=brain, CS=cardiac sphincter, Es=esophagus, Es F=longitudinal fold at esophagus, FG=fore gut, GA=gill arch, =heart, IL=ileo-rectal valve, GG=gastric glands, GC=goblet cells, Kd=kidney, L=liver, G=anteriormedian intestine, OD=oil droblet, PT=pharyngeal teeth, PS=pyloric sphincter, P=pancreas, P end=endocrine pancreas, P ex=exocrine pancreas, RC=rodlet cells, Rot=Rotifers, St=stomach, Syn=syncytium, SB=swim bladder, SV= supranuclear vacuoles, Y=yolk. The black bars represent 0.1 mm, the broken black bar in G represents 1 mm and the white bars in D and F represent 0.05 mm.

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83

100

Area covered with lipids vacoules %

90 80 70 60 50 40 30 20 10 0

i h, i g, h f, g c, d c a a, b b
11 13 15 17 20 23 26 28 30 33 36 40 44 2300

d, e

e, f f, g f, g

6 8

Number of Rotifer - Artemia (millions)

200 180 160 140 120 100 80 60 40 20 0 0 4 8 12 16 20 24 28 32 36 40 44

B
1800

1300

800

300

-200

Days after hatching

Fig. 6. (A) Mean (SD) area covered by lipid vacuoles (ACLV, %) in the liver of meagre larvae at different times during larval rearing. Statistically signicant differences between means are indicated with different letters above the means (ANOVA, Duncan's New Multiple Range test, P b 0.05). Four periods with changes in ACLV were identied (1 to 4, above graph). (B) Presentation of the dietary protocol and correlation with the time of appearance of each type of food item in the stomach contents of the larvae. The circles and triangles indicate the number (individuals) of rotifers and Artemia nauplii offered, respectively, while the squares indicate the amount of articial feed (g) offered during the rearing. Open symbols of any food item indicate times that this item was not identied in the stomach content (though it was offered), whereas a solid symbol indicates times that the item was present in the stomach contents of the examined larvae. The horizontal bar represents the period during which copepods appeared in the rearing tank and the solid section of the bar represents the period during which copepods were identied also in the stomach contents.

posterior part of the intestine appeared at the end of the autotrophic phase in other species, such as the gilthead seabream (Calzada et al., 1998; Elbal et al., 2004), green sturgeon (Acipenser medirostris) (Gisbert and Doroshov, 2003), turbot (Scophthalmus maximus) (Segner et al., 1994) and European sea bass (Garca Hernndez et al., 2001). The rapid growth of the intestine in length, but also the appearance of the rst lobe concomitantly with the intestinal valve, increase the residence time of food in the intestine and restrict enzymes in separate sections (Kowalska et al., 2006; Pedersen and Hjelmeland, 1988). Thus their activity is more effective in the digestive system that just begins to develop and mature progressively. The concurrent development of the intestinal villi, especially at the posterior segment, from 2 to 3 dah, indicates a further increase in the absorptive capacity of the intestine and suggests that the posterior part is more active than the other segments in terms of absorption during the developmental stages before the formation of the stomach (Ostaszewska, 2005). The presence of vacuoles at the posterior part of the intestine at 2 and 3 dah, immediately after the start of exogenous feeding was an indication of pinocytic assimilation of proteins (Iwai, 1968), which are later digested intracellularly. The reappearance of the vacuoles at 11 dah at the posterior part of the intestine, which was 45 days before the appearance of the gastric glands, correlates with pinocytic activity of the intestinal cells, where big molecules and proteins are assimilated (Deplano et al., 1991; Kurokawa et al., 1996; Watanabe, 1984b).

This pinocytic action compensates for the typically low proteolytic activity in the digestive tract during these early developmental stages (Rojas-Garca and Rnnestad, 2003). The presence of vacuoles at the posterior part of the intestine in meagre, even after the appearance of the gastric glands at 15 dah is not unusual (Watanabe, 1984a). The decrease in number and size of these vacuoles from 23 to 30 dah is a result of the start of extracellular protein digestion linked to the development of the gastric glands (Chen et al., 2006; Gisbert et al., 2004b) and the digestive enzymes they secrete (Gordon and Hecht, 2002). The gastric glands are not considered functional immediately upon their appearance in the digestive tract, and there is a delay in their maturation (Zaiss et al., 2006). Between 23 and 30 dah, there was a period of underfeeding, identied by examination of stomach contents, which presumably contributed to the reduction and nally disappearance of the intestinal vacuoles, as reported elsewhere (Chen et al., 2007). The vacuoles (lipids) observed at 17 dah in the mid-gut are connected to the hydrolysis of lipids in fatty acids and monoglycerides, which later are assimilated and stored in the intestinal cells (Ostaszewska, 2005). Fish larvae are capable of digesting and assimilating lipids from the start of the exogenous feeding phase (Morais et al., 2007). The rapid growth of the intestinal cells during larval development is associated with an increased synthesis of lipoproteins, which is accompanied with a reduction of the number of big lipid vacuoles in the intestine (Sarasquete et al., 1995).

Artificial feed (g)

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1. (8 dah) FG MG

Rot

MG

2. (14 dah)

St

Art

Art Cop 3. (20 dah) St Cop

4. (28 dah) St AF

5. (36 dah)

AF St

6. (44 dah) MG FG Liver St

AF

Stomach contend

Fig. 7. Microphotographs of histological sections of meagre at different times (days after hatching, dah) during rearing, showing sections from the liver (A), and the stomach (mid and fore gut at 8 dah) at low (B) and high magnication (C). Art= Artemia nauplii, AF=articial feed, Cop=copepod, FG=fore gut, MG=anteriormedian intestine, Rot=rotifer, St=stomach. In column A the bars represent 0.05 mm, while in column B and C the bars represent 0.2 mm.

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The gastric glands appeared at 15 dah, heralding the preparation for extracellular digestion of consumed food, through the actions of the secreted HCl and other enzymes, such as pepsin (Zambonino-Infante et al., 2008). With the appearance of the gastric glands begins the development of a functional stomach (Stroband and Kroon, 1981), and it is considered as a dening moment for the physiology of nutrition of the larvae leading to the transition from larval to juvenile function of the digestive system (Kolkovski, 2001; Sarasquete et al., 1995; Tanaka, 1971). The change from intracellular to extracellular intestinal digestion of food allows the digestion of complex proteins into free amino acids, mainly under the action of pepsin in an acidic environment (Segner et al., 1994). In this way, the range of foods that can be utilized by the larva at this stage is wider (Segner et al., 1994) and provide the organism with more nutrients to meet its energy requirements of growth and development (Zambonino-Infante et al., 2008). Also development and differentiation of the stomach with the appearance of the gastric glands can be used as an indicator for the ability of the larva to move from live preys to dry food (Segner et al., 1993; Verreth et al., 1992). The appearance time of the gastric glands is species-specic (Chen et al., 2006) and in slow-growing species the gastric glands appear rst as late as 36 dah in yellowtail ounder (Pleuronectes ferruginea) (Baglole et al., 1997) and 60 dah in gilthead seabream (Elbal et al., 2004). In white seabream (Diplodus sargus) the gastric glands appeared at 20 dah (Cara et al., 2003) and the stomach was fully functional at 30 dah (Ortiz-Delgado et al., 2003). In fast-growing species the gastric glands appear earlier; for example at 16 dah in spotted sand bass (Pea et al., 2003), in Pacic bluen tuna (Thunnus orientalis) at 11 dah (Kaji et al., 1996), in yellowtail kingsh at 15 dah (Chen et al., 2006) and in shi drum at 14 dah (Zaiss et al., 2006). The appearance of pyloric ceaca at 17 dah in meagre is associated with an increase in the absorptive area of the intestine (Buddington and Diamond, 1989; Hossain and Dutta, 1998; Zaiss et al., 2006), but also with the development of a basic area for the production of enzymes (Kim et al., 2001) for the enhancement of food assimilation. Together with the gastric glands, the appearance of the pyloric ceaca is probably, at least partly, related to the faster growth rate that was observed in meagre after 17 dah. Goblet cells that appeared at 11 dah at the mid-gut of meagre have a protective role for the digestive tract (Kowalska et al., 2006) by producing mucus. A small number of rodlet cells appeared at 11 dah at the mid-gut and at 13 and 15 dah at the foregut. These cells are considered enigmatic, since their origin and function has yet to be revealed (Kramer and Potter, 2003; Manera and Dezfuli, 2004). Rodlet cells in the intestinal epithelium have been described in other species, such as trout (Oncorhynchus mykiss), freshwater eel (Anquilla anquilla), chub (Leuciscus cephalus) and angelsh (Pterophyllum scalare scalare) (Bielek, 2002; Dezfuli et al., 1998, 2003; Smith et al., 1995a,b). Some authors believe that these are host cells that occur normally (Dezfuli et al., 1998, 2000; Leino, 1996). The liver and pancreas develop rst from cells of mesodermic origin (Ostaszewska, 2005) and appeared 1 dah in meagre. The differentiation of exocrine and endocrine pancreas began as early as 3 dah, pointing to the capacity of the larva for enzymatic function. The exocrine pancreas produces and secretes digestive enzymes in the intestine (Ostaszewska, 2005; Zambonino Infante and Cahu, 2001), with the quantity of enzymes increasing with the size of the larva (Zambonino Infante and Cahu, 2001). Similarly the function of the liver started from 3 dah in meagre, as reported in other species such as the common sole (Boulhic and Gabaudan, 1992) and summer ounder (Paralichthys dentatus) (Bisbal and Bengston, 1995). The presence at this early stage of glycogen in the liver simultaneously with the differentiation of the hepatic cells (Diaz and Connes, 1991) has been connected with maternal matter in mammals and with yolk reserves in sh and birds (Diaz and Connes, 1991). Signicant changes in lipid content in the liver of meagre (as estimated by ACLV) were observed as the larvae advanced through development. Initially, when consuming low energy rotifers, liver lipid content was low, increasing gradually, particularly when consumption

of Artemia increased, as described also for pike-perch (Sander lucioperca) (Ostaszewska et al., 2005). The increased mobility of Artemia together with some chemical attractants (mainly free amino acids) that they release (Cahu and Infante, 2001; Hart and Purser, 1996; Kolkovski, 2001; Kolkovski et al., 1997b, 2004), probably explains the high preference of meagre for Artemia, as observed from the stomach content analysis. From 13 dah, meagre larvae consumed large quantities of enriched Artemia of high energy content, a fact that was reected in the increase in liver lipid content. The maximum liver lipid content was observed some days later when the larvae consumed copepods that began to develop in the tank. Similarly, in a histological analysis of Atlantic halibut (Hippoglossus hippoglossus) liver, lipid deposition was greater when larvae were fed with copepods than enriched Artemia (Shields et al., 1999). A signicant reduction in liver lipid content was observed around 28 dah during the transition of meagre larvae from live prey to dry feeds. This dietary transition is the most critical phase in early developmental stages in sh larvae (Garcia-Ortega et al., 2003; Hamlin et al., 2000), and high mortalities are observed due to a decreased feed intake and low assimilation of the dry feeds (Watanabe and Kiron, 1994). Although the total quantity of food ingested was not altered during this phase, since together with the decrease of Artemia there was an increase of dry feed provided to the larvae, the observed reduction of liver lipid content was presumably because larvae rejected dry feed initially. As a result, a period of malnourishment was observed, as concluded by the reduced liver lipid content. A similar period of reduced liver lipid content was observed at the transition from live prey to dry feed when shi drum larvae were reared using a feeding protocol of only rotifers as live prey (Papadakis et al., 2009). One of the many impacts of malnourishment in juvenile and adult sh is the reduction of lipid content of hepatic cells (Power et al., 2000), which has been observed to occur also during early stages in other sh (Papadakis et al., 2009). The vacuoles of the hepatocytes store lipid and glycogen that are mobilized rapidly when needed, and eventually are depleted during problematic feeding periods (Ehrlich et al., 1976; Margulies, 1993; O'Connell, 1976; Theilacker, 1978). The reason for the existence of this malnourishment period observed around 28 dah in meagre, was presumably related to an adjustment period required for the transition from live prey to dry feeds. Contrary to live prey, articial feeds do not move in different directions in the water column, therefore they do not stimulate feeding activity by sh larvae (Hart and Purser, 1996; Kolkovski, 2001; Kolkovski et al., 1997a,b, 2004). Therefore, sh do not feed as much during early weaning. Furthermore, the signicantly lower water content of articial feeds (710%) compared to live prey (95%) (Buddington et al., 1997) may cause additional difculties in digestion (e.g., hydration of digesta), passage time through the gut, nutrient release and digestibility (Papadakis et al., 2008), as sh larvae encounter this type of feed item for the rst time. In conclusion, the present study on the ontogeny of the digestive system of meagre indicates that during early development meagre is a fast growing species, developing rapidly the structures and basic organs of the digestive system required to overcome successfully the critical stages of larval rearing. This characteristic is a major advantage for a reared sh, and makes meagre a very promising species for commercial aquaculture. Also, the present study conrms earlier works pointing to changes in the liver lipid content as precise and rapid indicators for periods of malnutrition in sh larvae. Evaluation of liver lipid content in commercial aquaculture may be a valuable tool for the improvement of larval rearing protocols and improvement of production efciency. Further studies focusing on the physiology of the digestion of this species with the study of digestive enzymes, but also other processes that are connected to the feeding acquisition such as vision, smell and taste will complete the necessary information required for the optimization of the production results.

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I.E. Papadakis et al. / Aquaculture 388391 (2013) 7688 mormyrus, Puntazzo puntazzo (Poissons Teleosteens). Thse de Doctorat s Science, Universite des Sciences et Techniques de Languedoc, Montpellier, pp. 479. Divanach, P., Kentouri, M., 2000. Hatchery techniques for specic diversication in Mediterranean nsh larviculture. Recent Advances in Mediterranean Aquaculture Finsh Species Diversication. Cahiers Options Mditerranennes, 47. CIHEAM, Instituto Agronomico de Zaragoza, Zaragoza Spain, pp. 7587. Duncan, N., Estvez, A., Porta, J., Carazo, I., Norambuena, F., Aguilera, C., Gairin, I., Bucci, F., Valles, R., Mylonas, C., 2012. Reproductive development, GnRHa-induced spawning and egg quality of wild meagre (Argyrosomus regius) acclimatised to captivity. Fish Physiology and Biochemistry 114. Ehrlich, K.F., Blaxter, J.H.S., Pemberton, R., 1976. Morphological and histological changes during the growth and starvation of herring and plaice larvae. Marine Biology 35, 105118. 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Acknowledgments Financial support for this study has been provided by the Ministry of Education, Lifelong Learning and Religious Affairs, General Secretariat for Research and Technology, Greece under the Call Cooperation of the National Strategic Reference Framework 2007 2013 (SYN09-24-424) to CCM, titled Development of methods for reproduction and rearing of meagre (Argyrosomus regius) as a means for the enhancement of the competitiveness of aquaculture, with the introduction of new species. References
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