Ontogeny of the digestive tract in mud loach Misgurnus anguillicaudatus larvae

doi:10.1111/are.12574 Ontogeny of the digestive tract in mud loach Misgurnus anguillicaudatus larvae Jianye Zhang 1, Ruibin Yang 1,2, Xuefen Yang 1, Qixue Fan 1, Kaijian Wei 1,2 & Weimin Wang 1,2 1 Key Lab of Freshwater Animal Breeding Certificated by Ministry of Agriculture, College of Fisheries, Huazhong Agricultural University, Wuhan, China 2 Freshwater Aquaculture Collaborative innovation Center of Hubei Province, Wuhan, China Correspondence: R Yang, Key Lab of Freshwater Animal Breeding Certificated by Ministry of Agriculture, College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China. E-mail: rbyang@mail.hzau.edu.cn Abstract This study on histological and mucous histochemistry characteristics of the digestive system of loach (Misgurnus anguillicaudatus) was carried out from hatching (0 day after hatching, DAH) until 45 DAH. The peculiar development of both digestion processes and air-breathing functions of the intestine was revealed. At 3 DAH, both the mouth and anus opened along with the first feed. At 4 DAH, lipid vacuoles appeared in the anterior part of the intestine and at 5 DAH the acidophilic supranuclear vacuoles appeared in the posterior part of the intestine. Mucous cells occurred in the buccopharynx and oesophagus after mouth opening and grew both in number and size as larvae grew. At 15 DAH, blood capillaries were found in the posterior part of the intestine. At 20 DAH, as a valve appeared in the intestine, the whole intestine could be divided into anterior, mid and posterior parts. With large numbers of blood capillaries and a very thin wall, a gas blood barrier formed in the posterior intestine, which indicated that the dual roles of intestine were formed. These results suggested that the air-breathing function of the digestive tract formed from 15 to 20 DAH, which is a critical period for loach larvae. Keywords: Misgurnus anguillicaudatus, ontogeny, larvae, digestive tract, histology, mucous cells Introduction The mud loach (Misgurnus anguillicaudatus) is a small freshwater teleost belonging to the family Cobitididae of the order Cypriniformes. It is an important commercial species widely distributed in China, Japan, South Korea and Southeast Asia (McMahon & Burggren 1987; Park & Kim 2001). The mud loach is in great demand in Asia due to its high nutritional and medicinal value (Zhang, Jiang, Zhang, Pan & Zhang 2010). With the rapid growth in its demand and the shortage of its wild resource, artificial culture of this species has also gained popularity during recent years (Hu, Chu, Wang, Zhou, Chen, Liu, Li & Lian 2012; Zhu & Zhao 2014). Techniques for its large-scale artificial propagation has been developed (Wang, Hu, Wang & Cao 2009; Hu et al. 2012; Zhu & Zhao 2014), and widely applied in China. However, mass mortality in larvae rearing is a major bottleneck in seed production. The cause of high mortality remains unclear. This situation has greatly restricted the industrial development of loach culture. The loach is an interesting intestinal air-breathing fish that makes use of its hindgut or intestine as an accessory air-breathing organ (McMahon & Burggren 1987). In addition to a digestive function, the intestine of loach also affords to share a respiratory function (Moitra, Singh & Munshi 1989). Many studies were carried out to focus mainly on its larva culture (Wang et al. 2009), feeding habits and rhythm (Wang, Hu, Wang, Cao, Yang, Lu & Yao 2008), embryonic and larval development (Zheng, Ding, Liu & Yang 1992) and histology of the digestive tract in adult loach (Park & Kim 2001). However, the ontogeny of the digestive system of loach larvae and the development of intestinal dual functions have never been reported. Knowledge regarding the morphological and functional changes during early ontogeny in 1180 2014 The Authors. Aquaculture Research Published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Ontogeny of digestive tract in mud loach larvae J Zhang et al. fish is of great importance in order to adapt rearing technology according to the digestive ability of fish larvae (Pradhan, Jena, Mitra, Sood & Gisbert 2012; Cuenca-Soria, Alvarez-Gonzalez, Ortiz-Galindo, Tovar-Ramirez, Guerrero-Zarate, Aguilar-Hernandez, Perera-Garcia, Hernandez-Gomez & Gisbert 2013). The objective of this study was to understand the ontogeny of the digestive system of loach from hatching to 45 days after hatching (DAH), aiming to discover not only the sequence of the structural and functional development of the digestive organ but also the development of the intestine as an air-breathing organ. Materials and methods Larvae used in the present study were obtained from routine spawning and rearing procedures in the laboratory of the Fisheries College, Huangzhong Agriculture University, Wuhan, China (30 28 0 44 N, 114 21 0 49 E). Eggs were incubated in early summer in still water at 19 21 C, with moderate aeration in 0.5 m³ tanks (density: 200 eggs L 1 ). The water volume replacement accounted for 10% per day, water temperature, dissolved oxygen and ph were maintained at 19 21 C, 7 9 mgl 1 and 6.8 7.6 respectively. Natural photoperiod was applied throughout the trial. Rotifers (mainly Brachionus, 15 20 individuals ml 1 ) were offered to larvae from 3 to 20 DAH. From 13 DAH Cladocerans (mainly Moina, 8 15 individuals ml 1 ) were also added to the larval fish tanks. Fish was weaned on commercial pellet feed (0.8 1.0 mm diameter; protein 40%, fat 9%, ash < 10%; from Haid Co., LTD, Guangzhou, China) using a transition time between 18 and 25 DAH. After 25 DAH, only commercial feed was given. All experimental procedures involving fish were approved by the institution s animal care and use committee of the Huazhong Agricultural University, China. Fish larvae were randomly sampled daily from hatching to 35 DAH, then once every 5 days until the end of the experiment. The fish were anaesthetized using tricaine-methanesulfonate (MS-222, 100 mgl 1 ) before measuring and fixation. Total length (L T ) of 30 specimens was individually measured to the nearest 0.1 mm using an ocular micrometer of a dissection microscope and a vernier caliper once the larvae reached 10 mm. Another 10 specimens (digestive tract was picked out after total length over 15 mm) were fixed in Bouin s solution, dehydrated in graded alcohol, and embedded in paraffin wax. A series of sagittal and cross-sections (5 7 lm) were cut from each paraffin block, mounted on glass slides (six to nine serial sections per slide), air dried, and stained with haematoxylin-eosin for general histological features, or periodic acid-schiff (PAS) for the neutral mucins or alcian blue (AB) at ph 1.0 and 2.5 for acid mucins, in addition the AB (ph 2.5) was employed in combination with PAS technique for neutral and acid mucins (Ben & Li 2001). AB (ph 2.5) followed by PAS which was used for classifying epithelial mucous cells in the digestive tract. All stained slides were mounted permanently in neutral balsam and observed under an Olympus light microscope. Photographs were taken with a motic digital photomicrographic attachment (Motic, Decatur, GA, USA). Results Morphology Total length growth of mud loach larvae followed a linear function curve (Fig. 1). At hatching (L T = 2.35 mm), the alimentary canal appeared as an undifferentiated straight tube lying dorsally to the yolk sac. At 1 DAH (L T = 3.87 mm), the external gills formed and their number and size reached a maximum value until 3 DAH (L T = 5.37 mm), and branchiostegal membrane extended backward. At 3 DAH, the mouth and anus opened, rotifers could be clearly seen in the digestive tract due to the transparency of the body. Furthermore, most of the yolk sac was absorbed. At 5 DAH, (L T = 6.89 mm), the yolk sac was completely absorbed, the digestive tract elongated and the gut lumen increased in size. At 15 DAH (L T = 20.01 mm), the external gills were no longer visible and remained covered by the operculum, and the branchiostegal membrane reached enough length to cover the branchial cavity. Histology and histochemistry At hatching, the digestive tract appeared as a straight tube. Within 3 days from hatching, rapid developmental changes took place in the digestive tract of loach larvae, as the digestive tract differentiated into three segments including buccophyarynx, oesophagus and intestine. Based on the observations of sections stained with AB-PAS (AB ph 2.5) staining, the epithelial 2014 The Authors. Aquaculture Research Published by John Wiley & Sons Ltd., Aquaculture Research, 47, 1180 1190 1181

Ontogeny of digestive tract in mud loach larvae J Zhang et al. Aquaculture Research, 2016, 47, 1180 1190 Figure 1 Growth in size of Misgernus anguillicaudatus larvae from hatching to 45 DAH. mucous cells in the digestive tract were classified into three types. Type I stained with red secreted neutral mucins. Type II stained with blue secreted acid mucins. Type III stained with bluish purple secreted mixed mucins, more acid than neutral. Buccopharynx At 1 DAH, the buccopharyngeal cavity was lined by a single layer of squamous epithelium (Fig. 2a). The mouth opened and the oral valve appeared at 3 DAH, when the buccopharyngeal mucosa was formed by a stratified squamous epithelium. Rotifers could be found in the larvae s rudimentary gut which implied that the larvae had started feeding. Simultaneously, taste buds and a few acidophilic goblet cells appeared in the buccopharyngeal epithelium (Fig. 2b). Two or three mucous cells of type II were detected in the buccopharynx at 3 DAH (Fig. 2c). The taste buds and goblet cells in the buccopharynx became obvious and numerically increased continuously with age. No noticeable morphological and histological changes of the buccopharynx were observed until the end of the study (Fig. 2d). opened at 3 DAH. Two different regions could be distinguished in the oesophagus at 3 DAH: an anterior region lined by a stratified squamous epithelium which was lack of goblet cells in mucosa, and a posterior region lined by a simple columnar epithelium which has abundant goblet cells in mucosa (Fig. 3a). The oesophagus was surrounded by a layer of muscles consisting of circular and longitudinal muscle fibres. As larvae grew, the muscle layer thickened (Fig. 3c). A number of mucous cells of oval and round shape were detected and rapidly increased in number showing the presence of acid mucins and neutral mucins (Fig. 3b). At 5 DAH, longitudinal mucosal folds appeared in the posterior region of the oesophagus. More longitudinal folds formed between the oesophagus and intestine, while single columnar epithelium was lined at the junction of the oesophagus and intestine (Fig. 3c). At 13 DAH, the mucosa was almost full of mucous cells of type II and type III indicating a large number of acid glycoproteins or muco-substances. As larvae grew, the size of mucous cells increased, and different mucous cells with round, rodlike, goblet or pyriform shapes were observed (Fig. 3d). Oesophagus At 1 DAH, the oesophagus appeared as a narrow lumen similar to the incipient gut which was lined by a single layer of squamous epithelium (Fig. 2a). No mucous cells could be detected in the oesophagus at this time. Goblet cells appeared in mucosa of the oesophagus when the mouth Intestine At hatching, the incipient intestine appeared as a straight tube laying dorsally to the yolk sac. The lumen of intestine appeared at 1 DAH lined by a single layer of columnar cells (Fig. 4a). At 3 DAH, the anterior region of intestine expanded and thickened more than the rest of the digestive tract. Intes- 1182 2014 The Authors. Aquaculture Research Published by John Wiley & Sons Ltd., Aquaculture Research, 47, 1180 1190

Ontogeny of digestive tract in mud loach larvae J Zhang et al. (a) (b) Figure 2 Sagittal section of the buccopharyngeal cavity during the development of loach larvae. (a) 1 DAH (HE), show the initial buccopharyngeal cavity, oesophagus and intestine. (b). 3 DAH, note the presence of taste buds and goblet cells (HE). (c). 3 DAHnote the presence of mucous cells in buccopharynx cavity and oesophagus. (AB- PAS). (d) 11 DAH (HE), show the structure of buccopharynx cavity. Abbreviations: AI, anterior intestine; BC, buccopharynx; ES, oesophagus; GA, gill arch; GC, goblet cell; L, liver; M, muscle; MC, mucous cells; N, notochord; OV, oral valve; PC, pharynx cavity; SSE stratified squamous epithelium; TB, taste bud; YS, yolk sac. (c) (d) (a) (b) (c) (d) Figure 3 Sagittal section of the oesophagus during the development of loach larvae. (a) 3 DAH, note the presence of goblet cells and two different regions (HE). (b) 13 DAH, note the presence of mucous cells (AB-PAS). (c) 18 DAH, show the differences of two regions in oesophagus and the junction of oesophagus and intestine (HE). (d) 30 DAH, show the abundant mucous cells of different types in oesophagus (AB-PAS). Abbreviations: AI, anterior intestine; AES: anterior oesophagus; BC, buccopharynx; ES, oesophagus; GC, goblet cell; IN, intestine; L, liver; LF, longitudinal fold; M, muscle; PES, posterior oesophagus; SCE, single columnar epithelium; SSE, stratified squamous epithelium; YS, yolk sac; II and III, mucous cells type II and III. 2014 The Authors. Aquaculture Research Published by John Wiley & Sons Ltd., Aquaculture Research, 47, 1180 1190 1183

Ontogeny of digestive tract in mud loach larvae J Zhang et al. Aquaculture Research, 2016, 47, 1180 1190 (a) (b) (c) (d) (e) (g) (i) (k) (f) (h) (j) (l) Figure 4 Sagittal section of the intestine of loach larvae. (a) 1 DAH (HE), show the initial intestine. (b) 4 DAH, show the presence of lipid vacuoles in the enterocytes of the enterocytes of the anterior region of intestine (HE). (c) 5 DAH, note the presence of acidophilic supranuclear vacuoles in posterior part of intestine and the brush border (HE). (d) Anterior part of intestine of 10 DAH larvae, show the presence of goblet cells (HE). (e) Posterior part of intestine of 15 DAH larvae (HE). (f) Intestine of 20 DAH larvae, show the appearance of intestine valve in the anterior region of intestine (HE). (g) Show the anterior intestine of 20 DAH larvae (HE). (h) Show the mid-intestine of 20 DAH larvae (HE). (i) Show the posterior intestine of 20 DAH larvae (HE). (j) Show the anterior intestine of 45 DAH larvae (HE). (k) Show the mid-intestine of 45 DAH larvae (HE). (l) Show the posterior intestine of 45 DAH larvae (HE). Abbreviations: AI, anterior part of intestine; B, intestine valve; BB, brush border; BC, buccopharynx; CM, circular muscle; ES, oesophagus; GC, goblet cell; I, intestine; L, liver; LM, longitudinal muscle; LV, lipid vacuole; M, muscle; m, mucosa; P, pancreas; PI, posterior part of intestine; R, rectum; S, serosa; SM, submucosa; SNV, supranuclear vacuole; YS, yolk sac; white arrows: capillaries with red blood cells. 1184 2014 The Authors. Aquaculture Research Published by John Wiley & Sons Ltd., Aquaculture Research, 47, 1180 1190

Ontogeny of digestive tract in mud loach larvae J Zhang et al. tine folds appeared at the same time. The intestine possessed a mucosa epithelium of simple columnar cells with nucleus located basally (Fig. 3a). At 4 DAH, lipid vacuoles appeared in the enterocytes of the anterior region of the intestine (Fig. 4b). Subsequently, the mucosal folds showed different views of the intestine at 5 DAH, with their height decreasing gradually from the anterior to posterior part of the intestine. During this period, numerous acidophilic supranuclear vacuoles appeared in the posterior part of the intestine. A PAS-positive reaction was observed in the brush border in the intestine (Fig. 4c). At 10 DAH the wall of the intestine thickened and the internal surface was folded more heavily. Meanwhile, the first goblet cells appeared in the anterior part of the intestine. Their size and number increased as larvae grew (Fig. 4d). At 15 DAH, blood capillaries with several red blood cells were visible in the submucosa of the posterior part of the intestine, while supranuclear vacuoles became more abundant and visible.(fig. 4e). At 20 DAH, an intestine valve appeared in the anterior region of the intestine (Fig. 4f) dividing it into two parts. The anterior part was the anterior intestine. According to the different height of mucosal folds, the posterior part of the intestine was further divided into two regions: the midintestine with higher mucosal folds and the posterior intestine with almost no mucosal folds. There were many visual differences between the three parts of the intestine. The height of the epithelial cell layer was progressively reduced from the anterior to the posterior intestine, as well as the number and depth of the mucosal folds. In the anterior intestine there were abundant goblet cells, which were not found in the posterior intestine. More capillaries with red blood cells were found just beneath the epithelial layer of the mid-intestine and the posterior intestine. The mucosal folds of the anterior intestine appeared very prominent and the mucosa showed shallow folds in the midintestine, while the intestinal mucosal folds of the posterior intestine were almost smooth giving this area a very thin wall. Supranuclear vacuoles disappeared in the posterior part of the intestine at 20 DAH (Fig. 4g,h,i). As larvae grew, the differences became more obvious. There were enhanced density blood capillaries with vast blood cells and the mucosa thinned in the posterior intestine (Fig. 4j,k,l). The characteristics of mucins secreted by the epithelial mucous cells of the intestine in the larvae were summarized in Table 1. The reaction with AB at ph 2.5 and 1.0 was first detected on 3 DAH at buccopharynx and oesophagus and the reaction with PAS was first detected on 5 DAH. As larvae grew, the epithelial mucous cells of the digestive tract were PAS-positive and they secreted neutral mucins. The mucous cells appeared blue with AB at ph 2.5 and 1.0 indicating the presence of carbonic and sulphated mucins respectively. From 10 DAH the sections stained more strongly with PAS, AB ph 2.5 and AB ph 1.0 indicating the presence of more neutral and acid mucins in the buccopharynx, oesophagus and the posterior part of the intestine. The mucous cells were highly vacuolated using haematoxylin and eosin staining, and gave a strongly positive reaction by PAS. In the intestine, the mucous cells were rodlike, goblet or round. Mucous cells of type II were first detected in the intestine at 5 DAH. At 10 DAH, mucous cells of type I and type II were scattered in the whole intestine, with slightly more in the anterior and posterior parts. As larvae grew, both the size and number of mucous cells increased. From 20 DAH, the mucous cells were abundant in the intestine, with type I and type III rich in the anterior intestine while type II and type III were rich in the posterior intestine. Table 1 Carbohydrate histochemistry of digestive tract during development of loach larvae Method DAH BC ES AI MI PI PAS 1 3 5 + 10 + ++ + 20 ++ ++ + + ++ 45 ++ +++ ++ ++ ++ AB (ph 2.5) 1 3 + + 5 + + 10 + ++ + 20 ++ ++ + + ++ 45 ++ +++ ++ + ++ AB (ph 1.0) 1 3 + + 5 + + + 10 + ++ + + + 20 ++ ++ + + ++ 45 ++ +++ ++ + ++ The intensity of the reaction is expressed in grades:, negative;, weak; +, moderate; ++, intense; +++, very intense. 2014 The Authors. Aquaculture Research Published by John Wiley & Sons Ltd., Aquaculture Research, 47, 1180 1190 1185

Ontogeny of digestive tract in mud loach larvae J Zhang et al. Aquaculture Research, 2016, 47, 1180 1190 Liver and pancreas The liver and pancreas were not formed at hatching. At 2 DAH, the rudimentary liver and pancreas appeared between the intestine and the yolk sac (Fig. 5a). Liver cells substantially increased in number from 3 DAH. Numerous lipid vacuoles was visible in the liver at 11 DAH (Fig. 5b). At 18 DAH, the hepatocytes became more contiguous and were polyhedral in appearance, with nucleus central and larger size vacuoles in cytoplasm (Fig. 5c). As larvae grew, the arteries and veins also grew in number, and the cytoplasm of hepatocytes filled with vacuoles (Fig. 5d). The pancreas of loach, which appeared on 2 DAH and appeared strongly basophilic subsequently at 5 DAH (Figs 5a, 4b) was diffused and mainly located around the anterior region of the intestine, close to the liver and extended in the posterior region of the abdominal cavity. At 7 DAH, the acini composed of exocrine pancreatic cells formed, and acidophilic zymogen granules were visible in the centre of the acini (Fig. 5e). The pancreas increased in size and complexity with larval ontogenetic development (Fig. 5f). Discussion The loach is an air-breathing fish that makes use of its intestine as an accessory respiratory organ. In addition to its digestive function, the intestine of loach also affords to share a respiratory function (Moitra et al. 1989). The ontogeny of the digestive tract of M. anguillicaudatus follows the general pattern of most freshwater fish species (Yang, Xie, Fan, Gao & Fang 2010; Pradhan et al. 2012). The peculiar developmental characteristics of M. anguillicaudatus larvae were mainly on the dual function development of the intestine. The initial digestive tract was a narrow straight tube with a simple structure like other species of stomachless teleosts (Wu 1994; Sun & Zhang 1997). It was also similar to those of Cichlids and catfishes (Trevino, Alvarez-Gonzalez, Perales-Garcia, Arevalo-Galan, Uscanga-Martinez, Marquez- Couturier, Fernandez & Gisbert 2011; Pradhan (a) (b) (c) (e) (d) (f) Figure 5 Sagittal section of the liver and pancreas of loach larvae (HE). (a) 2 DAH, show the appearance of rudimentary liver and pancreas (HE); (b) Liver of 11 DAH. (c) Liver of 18 DAH. (d) Liver of 45 DAH. (e) 7 DAH, note the presence of acidophilic zymogen granules in pancreas. (f) Cross section of the liver and pancreas of 20 DAH, show the pancreas. Abbreviations: AI, anterior intestine; BD, bile duct; CV, central vein; ES, oesophagus; IN, intestine; L, liver; LV, lipid vacuole; N, notochord; P, pancreas; YS, yolk sac; ZG, zymogen granules; black arrowhead: nucleus of hepatocytes; white arrowhead: cytoplasm of hepatocytes with larger size vacuoles. 1186 2014 The Authors. Aquaculture Research Published by John Wiley & Sons Ltd., Aquaculture Research, 47, 1180 1190

Ontogeny of digestive tract in mud loach larvae J Zhang et al. et al. 2012). The appearance of taste buds and acidophilic goblet cells in the buccopharynx at 3 DAH indicated the start of exogenous feeding as demonstrated in Oxyeleotris marmoratus larvae (Abol-Munafi, Liem, Van & Ambak 2006). The absence of teeth during the study indicated that the preferred feeding habit of early loach larvae was suction of whole prey, rather than biting, seizing or cutting. Goblet cells in the buccopharynx played an important role in lubrication and protection in swallowing food like other freshwater fish species (Xu, Wang, Xiao, Zhong, Wen, Liu & Hu 2011). During larvae rearing, although the oral valve appeared when the mouth opened at 3 DAH, the operculum could not close until the external gills disappeared at 15 DAH, leading to the fact that the efficiency of gas exchange in internal gills was restricted. Therefore, the external gills played a crucial role in breathing during this period. The development of the oesophagus was similar to the pattern of Pelteobagrus fulvidraco (Yang et al. 2010). Coinciding with the opening of the mouth, two different regions were distinguished in the oesophagus. The anterior region lack of goblet cells was associated with food transporting and the posterior oesophagus had an additional function of mucosa protection supported by the abundant goblet cells as demonstrated in P. fulvidraco (Yang et al. 2010). Increased epithelial stratification in correspondence with the larger number of mucous cells containing acid and neutral glycoproteins was related to a supportive function for the oesophageal mucosa. In the early stage, the development of the intestine of loach was similar to other species. Three different regions are distinguished along the intestine according to their histological organization (Gisbert, Ortiz-Delgado & Sarasquete 2008). The intestinal valve divided the loach intestine into two parts at 20 DAH. It was different from the other ten kinds of minnow species which lack any type of valves or ceca (German, Nagle, Villeda, Ruiz, Thomson, Balderas & Evans 2009). The specialized digestive tract might be connected with different functions in different regions of the intestine. The first intestinal absorptive cells were the lipid vacuoles of the mucosal epithelial cells in the anterior region of the intestine at 4 DAH. This was a sign of the onset of active lipid digestion from 4 DAH. The presence of lipid inclusions in the enterocytes of fish is a common phenomenon in fish larvae and juveniles (Gisbert et al. 2008). Lipid vacuoles were also found in the anterior intestinal segment of adult stomachless fish (Stroband & Debets 1978). The presence of the large lipid vacuoles has been described as temporary fat stores within the gut epithelial cells. With the appearance of a PAS-positive brush border at 5 DAH the function of carbohydrates absorption in the intestine improved. The appearance of numerous acidophilic supranuclear vacuoles at 5 DAH in the posterior part of the intestine indicated the start of pinocytosis and intracellular digestion of protein as described in other fish larvae as their enzymatic system was poorly developed (Govoni, Boehlert & Watanabe 1986; Umur, Chris & _Ihsan 2008). Changes in the accumulation of these inclusions may be indicative of changes in the nutritional physiology of the larvae (Gisbert et al. 2008). These supranuclear vacuoles may last into adulthood in some stomachless teleosts as Carassius auratus (Gauthier & Landis 1972). In the present study, the number and size of supranuclear vacuoles in the posterior part of the intestine reached the maximum at 15 DAH and disappeared at 20 DAH. This indicated the changes in the nutritional physiology of the loach larvae. Different types of mucous cells can reflect different histological features during the development process of larvae. In most teleost, mucous cells of type I, which contain only neutral mucins and mucous cells of type II which only contain acid mucins were considered to be immature mucous cells, and mucous cells of type III which contain both neutral and acid mucins was considered to be mature mucous cells (An, Meng, Yang, Yin & Wang 2001). The variation of mucous cells in different types in the intestine of loach larvae was in step with the ontogenetic process of the digestive tract. According to the dates in the present study, mature mucous cells played a dominant role in the intestine of loach larvae at 20 DAH, indicating that the digestive function of the intestine is stable and mature. Large numbers of mucous of type I in the anterior intestine at 20 DAH was similar to many other fish such as Cyprinus carpio (An et al. 2001) and Lateolabrax japonicas (Xie, Lin & Lin 2007), which indicates that the dominating physiological function of the anterior intestine was digestion. However, in the posterior part types of mucous cells were different from those in other 2014 The Authors. Aquaculture Research Published by John Wiley & Sons Ltd., Aquaculture Research, 47, 1180 1190 1187

Ontogeny of digestive tract in mud loach larvae J Zhang et al. Aquaculture Research, 2016, 47, 1180 1190 fish larvae. In loach larvae, there were large numbers of mucous cells of type II in the posterior part of the intestine. This may be caused by the auxiliary respiratory function of the posterior part of the intestine of loach larvae. Supranuclear vacuoles, demonstrated above, contain different mucosubstances in the enterocytes of other teleost larvae (Govoni et al. 1986). The enterocytes of the posterior part of the intestine of loach larvae appeared Alcian blue (ph 1.0)-positive content at 5 DAH, which contained acidic sulphated mucosubstances. The PAS-positive and Alcian blue (ph 2.5)-positive content appeared at 10 DAH, indicating the presence of neutral mucosubstances and carbonic mucosubstances. Different distribution of the mucous cells at 20 DAH showed that there were more neutral mucins in the anterior intestine than acid mucins, while in the posterior intestine more acid mucins were present. The mucus histochemistry of the larval intestinal epithelium was stained positive for combinations of acid and neutral mucins at 20 DAH, which was similar to that described in the adult (Park & Kim 2001). Such a mucus lining also indicated the development of the dual functions in the intestine at 20 DAH. Large numbers of acid muco-substances in the posterior intestine confirmed to be characteristic of the gastro-respiratory tube of the loach (Moitra et al. 1989). Sulphated and carbonic mucins in the posterior intestine may play another role in cleaning the respiratory zone and protecting against bacterial attack. According to the present study, loach larvae began to modify various elements of the intestinalrespiratory tube to suit its dual role of digestion and respiration from 15 DAH. Large numbers of neutral mucins and goblet cells with absorption functions of short chain fatty acids presented in the complex mucosa folds indicated that the anterior part of the intestine serves a digestive function but not a respiratory one, while the posterior part of the intestine was confirmed to have a respiratory function similar to the result of the adult (Park & Kim 2001). The existence of abundant blood capillaries scattered into the submucosa of the posterior part of the intestine made air-breathing possible. Similarly to most air-breathing fish like Anabas testudineus (Hughes, Dube & Datta Munshi 1973) and Pangasius sutchi (Pan, Liu, Zheng, Liu & Fan 1988), respiratory epithelium of loach was highly vascularized and had a short diffused distance for gas exchange. At 20 DAH, the relatively low mucosal folds in the posterior intestine gave this zone a very thin wall, which made the gaseous exchange easily possible and gave this zone a short diffuse distance for air exchange. The existence of large numbers of capillaries suggests that the posterior intestine can service gas exchange between the circulating blood and the swallowed air by mouth (Park & Kim 2001). As larvae grew, more and more blood cells were found in the high-density blood capillaries in the posterior intestine, indicating a higher capacity of gas exchange. In the present study, digestive function and intestinal respiration have developed well from 20 DAH based on the structural characteristics. Like other stomachless fish, intestinal mucosal folded at a rather early stage in loach larvae, then appeared very complicated and pronounced in the anterior intestine at 20 DAH, indicating a better function as the increase in intestinal length and digestion surface (Micale, Garaffo, Genovese, Spedicato & Muglia 2006). The complicated mucosal folds also played an important role in food transportation. In physiological trials on the intestinal respiration of loach, it has been reported that undigested food reached the posterior part of the intestine by the movements of the anterior part of the intestine directly, and was pushed out of the anus by air used for intestinal respiration (Koyama 1958). The liver and pancreas were believed to be the main sources of enzymes for digestion and absorption in the intestine of the larvae due to the lack of stomach (Abol-Munafi et al. 2006). The liver and pancreas in the loach differentiate early through development, as with other species such as Paralabrax maculatofasciatus and Pelteobagrus falvidraco (Pena, Dumas, Villalejo-Fuerte & Ortiz-Galindo 2003; Yang et al. 2010). Both liver and pancreas had improved function by the appearance of zymogen granules in pancreatic acini at 7 DAH and the hepatocyte vacuoles in liver cells at 11 DAH. In conclusion, the ontogeny of the digestive tract of loach larvae has its own characteristics due to the dual role of the intestine which has not been described for other species. The air-breathing region is mainly in the posterior intestine, and the anterior intestine plays an important role in digestion. According to the date in this study, the airbreathing function of the intestine developed from 15 DAH, and a critical period for loach larvae is from 15 to 20 DAH. The structure and function 1188 2014 The Authors. 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Ontogeny of digestive tract in mud loach larvae J Zhang et al. was similar to adults from 20 DAH as the gasblood barrier formed and large numbers of mucous cells in the intestine at 20 DAH indicated a better development of the digestive tract. This information provides fundamental knowledge for larvae rearing management for this species. Acknowledgments Authors thank two anonymous reviewers for their constructive and helpful advice to the manuscript. We thank Professor Miklos Bercsenyi and Ms. Claudia Molnar for their helpful revision. This work was financially supported by the Special Fund for Agro-scientific Research in the Public Interest (Grant No. 201403012), National Natural Foundation of China (Program No. 31372180) and CSC (Grant No. 201308420422). References Abol-Munafi A.B., Liem P.T., Van M.V. & Ambak M.A. (2006) Histological ontogeny of the digestive system of Marble Goby (Oxyeleotris marmorats) larvae. Journal of Sustainability Science and Management 1, 79 86. 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