Chinese Journal of Oceanology and Limnology   2016, Vol. 34 Issue(1): 136-152     PDF       
http://dx.doi.org/10.1007/s00343-016-5008-2
Institute of Oceanology, Chinese Academy of Sciences
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Article Information

GAO Xiaoqiang(高小强), HONG Lei(洪磊), LIU Zhifeng(刘志峰), GUO Zhenglong(郭正龙), WANG Yaohui(王耀辉), LEI Jilin(雷霁霖)_L
An integrative study of larval organogenesis of American shad Alosa sapidissima in histological aspects
Chinese Journal of Oceanology and Limnology, 2016, 34(1): 136-152
http://dx.doi.org/10.1007/s00343-016-5008-2

Article History

Received Jan. 9, 2015
accepted in principle Mar. 3, 2015;
accepted for publication Mar. 16, 2015
An integrative study of larval organogenesis of American shad Alosa sapidissima in histological aspects
GAO Xiaoqiang(高小强)1, 2, HONG Lei(洪磊)2, LIU Zhifeng(刘志峰)1, 2, GUO Zhenglong(郭正龙)3, WANG Yaohui(王耀辉)3, LEI Jilin(雷霁霖)2        
1 College of Fisheries, Ocean University of China, Qingdao 266003, China;
2 Key Laboratory for Sustainable Development of Marine Fisheries, Ministry of Agriculture, Qingdao Key Laboratory for Marine Fish Breeding and Biotechnology, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China;
3 Jiangsu Zhongyang Group, Nantong 226600, China
ABSTRACT:We describe organogenesis at a histological level in American shad (Alosa sapidissima) larvae from 0 until 45 days after hatching (DAH). Larval development was divided into four stages based on the feeding mode, external morphological features, and structural changes in the organs: stage 1 (0-2 DAH), stage 2 (3-5 DAH), stage 3 (6-26 DAH) and stage 4 (27-45 DAH). At early stage 2 (3 DAH), American shad larvae developed the initial digestive and absorptive tissues, including the mouth and anal opening, buccopharyngeal cavity, oesophagus, incipient stomach, anterior and posterior intestine, diff erentiated hepatocytes, and exocrine pancreas. The digestive and absorptive capacity developed further in stages 2 to 3, at which time the pharyngeal teeth, taste buds, gut mucosa folds, diff erentiated stomach, and gastric glands could be observed. Four defined compartments were discernible in the heart at 4 DAH. From 3 to 13 DAH, the excretory systems started to develop, accompanied by urinary bladder opening, the appearance and development of primordial pronephros, and the proliferation and convolution of renal tubules. Primordial gills were detected at 2 DAH, the pseudobranch was visible at 6 DAH, and the filaments and lamellae proliferated rapidly during stage 3. The primordial swim bladder was first observed at 2 DAH and started to inflate at 9 DAH; from then on, it expanded constantly. The spleen was first observed at 8 DAH and the thymus was evident at 12 DAH. From stage 4 onwards, most organs essentially manifested an increase in size, number, and complexity of tissue structure.
KeywordsAlosa sapidissima     larval development     ontogeny     histology     organ diff erentiation    
1 INTRODUCTION

Greater underst and ing about the major ontogenic events during the early stages of fish is needed. During this stage, ontogeny involves important changes in the function and structure of larval tissues, organs, and systems(Zambonino-Infante and Cahu, 2001). Species level research into organogenesis is essential to both optimize rearing conditions, develop feeding protocols and larval-rearing techniques, and ensure the healthy development and optimal growth of larvae in culture(Yúfera et al., 2000; Padrós et al., 2011). A number of studies have documented the histological changes in diff erent tissues and organs during larval development in a range of species, including flatfish brill Scophthalmus rhombus(Hachero-Cruzado et al., 2009), dentex Dentex dentex L.(Santamaría et al., 2004), Atlantic blue-fintuna Thunnus thynnus(Yúfera et al., 2014), Senegal sole Solea senegalensis(Padrós et al., 2011), common P and ora Pagellus erythrinus(Micale et al., 2006), and redb and ed seabream Pagrus auriga(Sánchez-Amaya et al., 2007).Taken together, these studies suggest the mechanism of organogenesis is similar between species, but the relative timing of ontogeny is species-specific. Thus, comparative studies of the ontogeny of each species at diff erentdevelopmental stages can lead to a better underst and ing of their functional systemic capabilities and increase the survival rate of larvae.

The American shad(Alosa sapidissima)is an economically and ecologically valuable anadromous species native to the Atlantic coast of North America from southern Labrador in Canada to the St. Johns River in northern Florida(Walburg and Nichols, 1967; Scott and Crossman, 1973; Limburg et al., 2003). The American shad spends most of its life at sea, but swims up rivers to spawn. Northern populations are iteroparous whereas southern populations exhibit semelparity(Leonard and McCormick, 1999; Leonard et al., 1999). Because of its size, consumer dem and , and high economic value, the abundance of American shad has increased rapidly following its introduction into China in the early 21st century. As a result, the species is an important c and idate for commercial freshwater aquaculture. The scale of American shad culture has increased gradually during the past decade to meet the Chinese market dem and. However, there is a very high mortality rate in culture, particularly during the incubation period and the lecithoexo trophic stage. This mortality is the most significant obstacle to sustainable industrial farming.

Recent studies into American shad early larval development have focused on factors such as morphogenesis, salinity tolerance, feeding characteristics, allometric growth, and artificial propagation(Shardo, 1995; Zydlewski and McCormick, 1997; Wu et al., 2004; Zhang et al., 2010; Hong, 2011; Xu et al., 2012; Gao et al., 2015a, b). However, there has been no comprehensive study describing the histology of organogenesis during larval development in American shad. Our objective was to describe the primary histological changes in the digestive system and other organs(gills, liver, pancreas, heart, kidney, spleen and thymus)during the ontogeny of American shad.

2 MATERIAL AND METHOD

The brood-stock American shad were derived from the Zhongyang group, Jiangsu Province, China. Sexually mature individuals were selected and transferred to a spawning pond(700 m 3) and held under a natural photoperiod. Spawning occurred spontaneously at a water temperature of 19-20°C. The fertilized eggs were collected in nylon mesh and incubated in a 400-L fiberglass hatch cylinder with a flow-through fresh water system(egg density: 20-30 eggs/L). The water exchange rate was 15-25 L/min during the incubation period. Water quality conditions were monitored daily: dissolved oxygen 6.8- 8.2 mg/L, pH 7.2-7.9, water temperature 20.5-21.5 ° C(Leach and Houde 1999).

After hatching(71±1 h post-fertilization), the larvae were placed in three cement tanks(3.5 m×2.5 m×1.0 m)at an initial density of 1 100-1 500 larvae/m 3 and reared in deep well water(freshwater). Larvae were fed on enriched rotifers(Brachionus calyci florus)at a rate of 12-15 individuals/mL, four times per day from 3 to 15 d after hatching(15 DAH). Cladoceran and copepod nauplii were collected from outside ponds and added to the larval diet at a rate of 15-20 individuals/mL, three times per day from 12 to 20 DAH. From 20 to 35 DAH, the primary food source was large cladocerans and copepods, fed three times per day. From 30 DAH, the larvae were gradually switched to a microbound diet(produced by Shengsuo Fishery Culture Feed Research Centre of Sh and ong Province, China; diameter 100-200 μm; nutrient composition: crude protein ≥50%, crude fat ≥8%, crude fiber ≤3%, crude ash ≥16.5%, lysine ≥2%, phosphorus ≥1%, calcium ≥5%, water content ≤ 12%)before switching to zooplankton. During the larval culture period, the water temperature was 19.5- 21.5°C, dissolved oxygen was 6.0-7.5 mg/L, and NH 3 levels were <0.05 mg/L(Klauda, 1994). The residue and feces were cleaned from the ponds and 30% of the gross deep well water was replenished every day.

A r and om sample of forty-five larvae was collected from the rearing tanks daily from hatch(0 DAH)to 15 DAH, every second day from 16 to 42 DAH, and a final sample was taken at 45 DAH. A sub-sample of 15 larvae was collected daily from tanks to measure total length using an ocular micrometer with the aid of a dissecting microscope and electronic digital caliper. Each sample was fixed in Bouin’s solution for 24 h at room temperature, dehydrated in a graded series of ethanol and embedded in paraffin. Serial transverse and sagittal sections(5-10 μm)were cut and then stained with haematoxylin-eosin(H&E)for histological study(Comabella et al., 2013).

3 RESULT 3.1 Primary developmental stages and growth of American shad larvae

The larval development of American shad was divided into four stages based upon the diet, external morphological features, and structural changes of the organs during ontogeny(Sánchez-Amaya et al., 2007; Yúfera et al., 2014)( Tables 1-2): stage 1 or lecithotrophic(0-2 DAH), stage 2 or lecithoexotrophic(3-5 DAH), stage 3 or exotrophic I(6-26 DAH), and stage 4 or exotrophic II(27-45 DAH).

Table 1 Schematic synthesis of the main ontogenetic events occurring in American shad larvae during lecithotrophic (stage 1) and lecithoexotrophic (stage 2)

Table 2 Schematic synthesis of the main ontogenetic events occurring in American shad larvae during exotrophic I (stage 3) and exotrophic II (stage 4)

The total length of American shad larvae is described in Fig. 1. The growth of larvae was represented by an exponential curve from hatching until the end of the experiment(45 DAH) and described by: Total length=7.978e0.035×DAH(R2=0.993).

Fig. 1 Growth in total length (mm) of American shad larvae from 1 to 45 days after hatching (DAH)
The feeding protocol is also included (mean±SD, n =15).
3.2 Histological characteristics of American shad larval development 3.2.1 Endogenous reserves

At hatching, American shad larvae had a large and homogenous acidophilic yolk sac without an elliptic oil globule that occupied most of the abdominal cavity(Fig. 2a, b). The yolk sac was surrounded by a simple epithelium or syncytial layer of squamous cells. The yolk sac reserves were largely consumed between 2 and 3 DAH. During stage 2, as yolk sac-absorption proceeded, the yolk appeared granular and formed heterogeneous droplets and was strongly acidophilic until its complete depletion by 5 DAH.

Fig. 2 Microsections of American shad larvae during stage 1
a. an acidophilic and homogeneous yolksac, undiff erentiated digestive tract, primordial pronephric duct, and cord-like cells at hatching; b. yolksac and gill anlage at hatching; c. heart and gill anlages at hatching; d. at 1 DAH, yolksac, incipient digestive tract, primordial gill arches; e. closed urinary bladder at 1 DAH; f. posterior portion of the digestive tract bending slightly at 1 DAH; g. at 1 DAH, the cord-like cells proliferated; h. exocrine pancreatic region with zymogen granules, gall bladder, and liver at 2 DAH; i. the primordial swim bladder, digestive tract, and liver at 2 DAH; j. first thyroid follicle at 2 DAH; k. at 2 DAH, discernible heart diff erentiated into three compartments. A: atrium; An: anus; BA: bulbus arteriosus; Cl: cord-like cells; DT: digestive tract; ExP: exocrine pancreas; GA: gill arches; Ga: gill anlage; GB: gall bladder; H: heart; L: liver; PC: pericardial cavity; PD: pronephric duct; PS: perivitelline space; SB: swim bladder; TF: thyroid follicle; UB: urinary bladder; V: ventricle; YS: yolksac.
3.2.2 Digestive tract

At hatching, the digestive tract was a straight and undiff erentiated tube attached dorsally to the yolk sac, and the mouth and anus were closed(Fig. 2a). The epithelium of the undiff erentiated digestive tract consisted of a monostratified layer of cuboidal and columnar cells, which had basal nuclei and obviously apical microvilli. The posterior portion of the digestive tract became slightly bent at 1 DAH(Fig. 2f). At early stage 2, the mouth and anus were opened and the digestive tract diff erentiated into the buccopharyngeal cavity, oesophagus, incipient stomach, and intestine.

3.2.2.1 Buccopharyngeal cavity

At stage 2, the buccopharyngeal cavity was covered with a monostratified squamous epithelium and surrounded by connective tissue and a layer of circular musculature(Fig. 3b). Taste buds appeared in the buccopharyngeal cavity at 3 DAH and were clearly developed at 26 DAH(Figs. 3b, 4x). The goblet cells first appeared in the buccopharyngeal cavity epithelium at 5 DAH and were clearly visible at 8 DAH(Figs.3k, 4h). The pharyngeal teeth first appeared at 8 DAH, and the inner layer of circular and longitudinal muscle was thickened(Fig. 4h). From 8 to 26 DAH, the pharyngeal teeth underwent progressive proliferation and were completely developed at 39 DAH(Figs.4u, 5f).

Fig. 3 Microsections of American shad larvae during stage 2
a. ileo-rectal valve, folds in the anterior and posterior intestine at 3 DAH; b. at 3 DAH, the buccopharyngeal cavity was surrounded by connective tissue and a layer of circular musculature, and the taste buds were visible; c. yolk resorption, buccopharyngeal cavity, heart, goblet cells in the oesophagus at 3 DAH; d. gill arches with a cartilaginous framework, primordial gill filaments, heart, buccopharyngeal cavity at 3 DAH; e. the primordial swim bladder, liver, incipient stomach, oesophagus and anterior intestine at 3 DAH; f. the gas gland and pneumatic duct anlage at 3 DAH; g. goblet cells and folds in the oesophagus, an external circular layer of striated muscle at 3 DAH; h. endocrine (islet of Langerhans) and exocrine pancreas evident, several renal tubules and the hepatic sinusoids appeared at 4 DAH; i. buccopharyngeal cavity, four cardiac cavities, valves between ventricle and atrium and trabecular proliferation visible in the heart, goblet cells in the oesophagus at 4 DAH; j. liver, uninflated swim bladder with gas gland, extended pneumatic duct at 4 DAH; k. the goblet cells in the buccopharyngeal cavity at 5 DAH; l. exocrine pancreas with strongly acidophilic zymogen granules, hepatocytes containing large lipid vacuoles at 5 DAH; m. ileo-rectal valve in the anterior and posterior intestine, goblet cells in the posterior intestine at 5 DAH; n. primordial renal pronephros evident at 5 DAH; o. primordial gill filaments and lamellae in the gill arches, cartilaginous framework, chloride cells, and blood cells in the internal vascular system at 5 DAH; A: atrium; AI: anterior intestine; BA: bulbus arteriosus; BC: blood cells; Bc: Bowman capsule; BphC: buccopharyngeal cavity; Ca: cardia; CC: chloride cells; CM: circular muscle; Ct: connective tissue; ExP: exocrine pancreas; F: fold; GA: gill arches; GC: goblet cells; GF: gill filaments; gg: gas gland; Gl: glomerulus; H: heart; IL: islet of Langerhans; IV: ileo-rectal valve; L: liver; La, lamellae; MI, middle intestine; OE: oesophagus; PB: pseudobranch; Pc: pyloric caecum; Pd: pneumatic duct; Ps: pyloric sphincter; PI: posterior intestine; RT: renal tubules; SB: swim bladder; St: stomach; Si: sinusoids; Sv: sinus venosus; TB, taste bud; Tr: trabeculae; V: ventricle; Va: valve; YS: yolksac.

Fig. 4 Microsections of American shad larvae during stage 3
a. the developed stomach, anterior intestine, liver, oesophagus, and swim bladder at 6 DAH; b. primordial pseudobranch at 6 DAH; c. a developed swim bladder with gas gland and rete mirabile, stomach, liver, and oesophagus at 6 DAH; d. enterocytes with supranuclear inclusions in the anterior intestine at 6 DAH; e. the goblet cells and anus sphincter in the anus at 6 DAH; f. goblet cells and infranuclear vesicles in the medium intestine at 7 DAH; g. the spleen anlage, exocrine pancreas, and islet of Langerhans at 8 DAH; h. goblet cells and taste buds in the buccopharyngeal cavity surrounded by circular muscle and longitudinal muscle evident, first pharyngeal teeth at 8 DAH; i. islet of Langerhans and exocrine pancreas, and spleen surrounded by exocrine pancreas at 11 DAH; j. thymus anlage at 12 DAH; k. at 13 DAH, the trabeculae appeared in the atrium; l. a diff erentiated stomach with cardia, fundus, pylorus, and pyloric sphincter, anterior intestine, cardiac region with several mucosal folds, and oesophagus at 14 DAH; m. increase in kidney size; convolution and proliferation of renal tubules, lymphocytes, and sinusoid and renal capsule at 18 DAH; n. at 18 DAH, the lymphoblasts and reticular cells were present in the thymus; o. pseudobranch exhibiting gill filaments with lamellae at 20 DAH; p. the gas gland increased considerably in size and the gas chamber expanded consistently at 20 DAH; q. the gastric submucosa exhibiting some gastric pits, mucosal folds at 20 DAH; r. the lymphoblasts and macrophages were seen in the spleen at 20 DAH; s. at 22 DAH, the lymphoblasts developed into small lymphocytes in the thymus; t. heart valves were completely formed, four heart compartments, and trabecular proliferation at 22 DAH; u. the pharyngeal teeth underwent a progressive proliferation at 26 DAH; v. an increase in hepatic sinusoids with blood cells at 22 DAH; w. gastric gland proliferation, pyloric sphincter, and exocrine pancreas at 26 DAH; x. developed taste buds at 26 DAH. A: atrium; AI: anterior intestine; An: anus; AS: anus sphincter; BA: bulbus arteriosus; BphC: buccopharyngeal cavity; Ca: cardia; CM: circular muscle; Ct: connective tissue; ExP: exocrine pancreas; F: fold; Fu: fundus; GA: gill arches; GC: goblet cells; Gc: gas chamber; GF: gill filaments; gg: gas gland; GG: gastric glands; Gl: glomerulus; GP: gastric pits; HS: hematopoietic stem cells; IL: islet of Langerhans; INV: infranuclear vesicles; L: liver; La: lamellae; LM: longitudinal muscle; Ly: lymphocytes; Lyb: lymphoblasts; Mac: macrophages; OE: oesophagus; PB: pseudobranch; Pc: pyloric caecum; Pd: pneumatic duct; Pht: pharyngeal teeth; Ps: pyloric sphincter; Py: pylorus; RC: reticular cells; Rc: renal pronephros; RM: rete mirabile; RT: renal tubules; S: spleen; SB: swim bladder; Si: sinusoids; SNI: supranuclear inclusions; St: stomach; Sv: sinus venosus; T: thymus; TB: taste bud; Tr: trabeculae; V: ventricle; Va: valve.
3.2.2.2 Oesophagus

The oesophagus, a short and rather narrow lumen, connected the buccopharynx and incipient stomach. It was made up of a pseudostratified epithelium and surrounded by an external circular layer of striated muscle cells(Fig. 3c, g). During the lecitoexoteophic period(stage 2), the oesophagus was stretched and exhibited visible longitudinal folds(Fig. 3g). The goblet cells were first observed in the oesophagus at 3 DAH(Fig. 3g), and continued to proliferate during larvae growth. The number of folds also increased continuously. At stage 4, until the oesophagus was completely developed, the longitudinal folds were further increased and the muscular circular layer surrounded oesophagus mucosa thickened(Fig. 5c).

Fig. 5 Microsections of American shad larvae during stage 4
a. pronephric and mesonephric kidney at 28 DAH; b. spleen showing the splenic ellipsoids, the primitive splenic sinusoids, lymphoblasts, macrophages, and lymphocytes at 28 DAH; c. oesophagus completely developed at 30 DAH; d. at 36 DAH, the liver and pancreas increased in size and the gall bladder was completely developed; e. pseudobranch exhibiting gill filaments and lamellae proliferation at 38 DAH; f. the pharyngeal teeth were completely developed at 39 DAH; g. filaments and lamellae at the end of stage 4 (40 DAH); h. spleen with white pulp and red pulp and numerous lymphocytes, splenic ellipsoids and macrophages at 42 DAH; i. thyroid follicle proliferation at 45 DAH; j. a completely developed thymus at 45 DAH; k. numerous gastric glands and completely developed pyloric caeca at 45 DAH. CM: circular muscle; Co: cortex; De: dentine; ExP: exocrine pancreas; GA: gill arches; GB: gall bladder; GC: goblet cells; GF: gill filaments; GG: gastric glands; L: liver; La: lamellae; Ly: lymphocyte; Lyb: lymphoblasts; Mac: macrophages; Mn: mesonephros; Me: medulla; OE: oesophagus; Od: odontoblasts; PB: pseudobranch; Pc: pyloric caeca; Pn: pronephros; RP: red pulp; SE: splenic ellipsoids; Si: sinusoids; St: stomach; TF: thyroid follicles; Tr: trabeculae; WP: white pulp.
3.2.2.3 Stomach

The incipient stomach appeared at 3 DAH as a spindly and short tube at a posterior dilatation of the oesophagus, and was lined by a simple cubic epithelium(Fig. 3e). From 6 DAH, loose connective tissue and an external muscular circular layer were observed surrounding the stomach mucosa(Fig. 4a). The primordial pyloric sphincter, which separates the stomach from the anterior part of intestine, was visible at 3 DAH and was completely developed at 14 DAH. The pyloric caecum was detected in the anterior intestine wall at 6 DAH(Fig. 4a), after which the number and size of pyloric caecae increased, and development was complete at 45 DAH(Fig. 5k). During the exotrophic I period(stage 3), the stomach developed at a constant rate accompanied by a rapid thickening of the mucosal layer surrounding the stomach. At 14 DAH, three anatomically and histologically diff erentiated regions were clearly distinguishable: cardia, fundus, and pylorus, (Fig. 4l). The gastric submucosa exhibited some gastric pits, which constituted the primordial gastric gl and s at 20 DAH(Fig. 4q). Gastric gl and s that were lined by a simple cuboidal epithelium were first observed in the fundus region of the stomach at 26 DAH(Fig. 4w). From 28 DAH onwards, the stomach increased in size and complexity by virtue of an increase in the size and number of mucosal folds in both the cardiac and fundic regions, as well as proliferation of gastric gl and s. Development of the stomach was complete at 45 DAH(Fig. 5k).

3.2.2.4 Intestine

At hatching, larvae possessed an incipient intestine lined by a simple layer of columnar epithelium with a basal nucleus. At 1 DAH, the posterior region of the intestine was slightly bent and the anus was closed(Fig. 2f). Intestine loop formation was evident at 3 DAH. The intestinal valve(ileo-rectal valve)appeared and separated the anterior intestine and the posterior intestine(Fig. 3a). At 5 DAH, the goblet cells first appeared in the posterior intestine(Fig. 3m), and then in the middle intestine at 7 DAH(Fig. 4f). After this, the number of goblet cells tended to increase throughout larval development. At 6 DAH, the goblet cells were visible in the anus and the anal sphincter was clearly visible(Fig. 4e). The first signs of lipid absorption within enterocytes of the medium intestine were observed at 7 DAH(Fig. 4f). The supranuclear vacuoles were visible in the rectum at 5 DAH, and increased in size and number as larvae grew. At 6 DAH, the supranuclear inclusions were also visible in the anterior intestine(Fig. 4d). During exotrophic I(stage 3, from 10 DAH onwards) and stage 4, we did not observe any histological changes in the intestine, with the exception of an increase in the length of the tract and the size and number of mucosal folds.

3.2.3 Accessory gl and s

At hatching, the primordial liver and exocrine pancreas appeared as undiff erentiated deep-stained basophilic cells(cord-like structure), lying between the developing digestive tract and the posterior yolk sac(Fig. 2a). The cord-like cells proliferated and became rounded at 1 DAH(Fig. 2g), after which the rounded cells started to diff erentiate into the hepatocytes and pancreatic cells at 2 DAH(Fig. 2h). The hepatocytes were tightly packed between sinusoids and exhibited an obvious concentric nucleus; the pancreatic acini were formed by pyramidal cells with a basophilic cytoplasm containing strongly acidophilic zymogen granules(Fig. 3h). At 2 DAH, the gall bladder anlage was visible and consisted of a monostratified epithelium surrounded by connective tissue(Fig. 2h). A single islet of Langerhans(endocrine pancreas)was also present. The pancreas then divided into two regions: exocrine and endocrine pancreas(Fig. 3h). From 3 to 7 DAH, the number of large fat vacuoles increased in the hepatocytes, occupying the majority of the cytoplasm and displacing the nucleus to the periphery of the cell(Fig. 3l). After this, the volume of hepatic lipid deposits gradually decreased. The blood cells were clearly visible in the hepatic sinusoids at 9 DAH. From this point on, both the blood cells and sinusoids proliferated throughout stage 3(Fig. 4v). At the end of stage 3 and stage 4, both the hepatic and pancreatic tissues increased in size without any substantial morphological change relative to the previous stage(Fig. 5d).

3.2.4 Heart

The heart was visible at hatching as an undiff erentiated tubular structure, located cephalically to the coelomic cavity and ventrally to the gill primordium, and opening directly into the perivitelline space(Fig. 2c). At 2 DAH, the three compartments(ventricle, atrium, and bulbus arteriosus)in the heart were distinguishable(Fig. 2k). At the beginning of stage 2(4 DAH), four defined compartments were discernible in the heart: atrium, ventricle, bulbus arteriosus, and sinus venosus. Additionally, the ventricle trabeculae and the valve between the atrioventricle were also detected(Fig. 3i). During stage 3, the first trabeculae appeared in the atrium at 13 DAH, after which the trabeculae proliferated at a constant rate(Fig. 4k, t). The valves between the ventricle-bulbus arteriosus and sinus venosus-atrium were first observed at 8 and 22 DAH, respectively. At this time, the heart valves were completely formed at 22 DAH(Fig. 4t). The heart was fully developed at the end of stage 3.

3.2.5 Swim bladder

The primordial swim bladder was first seen at 2 DAH. The swim bladder diff erentiated from the dorsal wall of the digestive tube and its epithelium was lined by a layer of columnar cells, like those of the gut(Fig. 2i). At 3 DAH, the gas gl and and the pneumatic duct anlage were visible(Fig. 3f). At 4 DAH the pneumatic duct had increased in length without connecting to the digestive tube(Fig. 3j). At the end of stage 2(at 6 DAH), the swim bladder was more diff erentiated, the gas gl and was located at the anterior region, the rete mirabile was in the posterior region, and the swim bladder connected the gas chamber to the cardiac region of the stomach through a pneumatic duct(Fig. 4c). From 9 DAH onwards, the swim bladder started to inflate. The gas gl and increased considerably in size and the gas chamber exp and ed at a constant rate(Fig. 4p). During stage 4, the entire bladder organ exp and ed further as the larvae grew and the pneumatic duct was visible at all times.

3.2.6 Gills and pseudobranch

At hatching, the gill anlage was visible in the pharyngeal region(Fig. 2b). At 1 DAH, the four pairs of primordial gill arches appeared, formed by cores of chondroblasts carpeted with an undiff erentiated epithelium, at this time, the branchial cavity was still closed(Fig. 2d). At 3 DAH, the undiff erentiated epithelial cells proliferated continually, forming the primordial gill filaments(Fig. 3d). During stage 2, these structures further diff erentiated and proliferated in all the arches. At 5 DAH, the internal vascular system of the gill filaments, with blood cells and gill lamellae, were clearly visible. Concurrently, the first chloride cells appeared at the base of the gill filaments(Fig. 3o). During stages 3 and 4, the number and length of the gill filaments and lamellae increased significantly(Fig. 5g).

The pseudobranch was first visible at the end of stage 2 as a paired structure of undiff erentiated cells located in the opercular cavity, very close to the posterior region of the eye(Fig. 4b). During stage 3, the pseudobranch continued to develop and proliferate. The filaments and lamellae were visible(Fig. 4o). At the end of stage 3 and during stage 4, there was a constant increased in the length and number of filaments and lamellae in the pseudobranch. Following this period of development, the structure of the pseudobranch was similar to that of juveniles(Fig. 5e).

3.2.7 Kidney and urinary bladder

At hatching, the kidney was already visible and consisted of a pair of primordial pronephric straight ducts running just below the notochord axis(Fig. 2a). Additionally, the urinary bladder, a closed wall sac lined by cuboidal epithelium, was present in the vicinity of the anus(Fig. 2e). At 2 DAH, the glomerulus and a few haemocytopoietic cells appeared. At 3 DAH, the pronephric tubules started to convolute. The first renal corpuscle was visible at 5 DAH, consisted of a glomerulus and incipient capsule(Fig. 3n). As the vascular and hemopoietic elements developed, sinusoids containing blood cells developed by 9 DAH. The lymphoblasts first appeared as large and thick-stained cells at 11 DAH. At 18 DAH, the pronephic kidney exhibited primary tubules that were more convoluted, the hemopoietic elements and sinusoids proliferated, and a mass of lymphocytes were visible(Fig. 4m). At the end of stage 3, we observed the first evidence of the pronephros, containing large numbers of lymphocytes and hemopoietic tissue(20 DAH), and the development of the mesonephros with renal tubules(22 DAH). At 28 DAH, two zones were identified in the kidney. The former was occupied by hematopoietic tissue and the latter contained the developing mesonephric elements(Fig. 5a). From this point on, the kidney progressively increased in size.

3.2.8 Spleen and thymus

The spleen anlage was first seen at 8 DAH and was located in the ventral zone of the swim bladder adjacent to the wall of the intestine. It consisted of a loose small spherical cluster of mesenchymal cells(Fig. 4g). At 11 DAH, the spleen adopted its elliptic shape. As development proceeded, the hematopoietic stem cells and reticular cells became visible, and the spleen was completely surrounded by the pancreas tissue(Fig. 4i). At stage 3, the spleen substantially increased in size, particularly from 14 DAH onwards. The lymphoblasts and macrophages were first seen at 20 DAH(Fig. 4r). At 28 DAH, the splenic ellipsoids, the primitive splenic sinusoids and lymphocytes were visible(Fig. 5b). At the end of stage 4, the white pulp and the red pulp were visible in the spleen, and the reticuloendothelial system of the spleen was completely developed. At this time, the structure of the splenic organ was similar to that of juvenile fish(Fig. 5h).

At 12 DAH, the thymus anlage was visible as a paired structure, located at the dorso-superior cornu of the gill cavity and close to the dorso-joint of the operculum(Fig. 4j). It consisted of basophilic blast cells with large pale nuclei. At 18 DAH, the lymphoblasts and reticular cells were present(Fig. 4n). At 22 DAH, the lymphoblasts developed into small lymphocytes(Fig. 4s). From then on, the thymus increased substantially in size. During stage 4, the thymic tissue was diff erentiated into two regions: the cortex and the medulla, which were packed with lymphoid cells and epithelioid, reticular cells, respectively. The thymus was completely developed during this stage(Fig. 5j).

3.2.9 Endocrine elements

At 2 DAH, the thyroid gl and appeared as a single follicle located behind the eye and formed by a layer of cubic epithelium that surrounded a small lumen with a stained acidophilic colloid material(Fig. 2j). By 12 DAH, the number of the follicles had increased to 12. From this time until the end of the study, the number and size of the thyroid follicles significantly increased(Fig. 5i).

The endocrine pancreas appeared at 4 DAH as a single Langerhans islet within the exocrine pancreas(Fig. 3h). During stages 2, 3, and 4, it consisted of a single islet.

4 DISCUSSION

We successfully documented changes in histology associated with organogenesis during early larval development in American shad(from hatching to 45 DAH). Development was divided into four ontogenetic stages: lecithotrophic, lecithoexotrophic, exotrophic I, and exotrophic II. The general pattern of tissue, organ, and system development was similar to that in other teleosts(Falk-Petersen 2005; Yang et al., 2010; Papadakis et al., 2013), but the timing of organ diff erentiation and development diff ered. This is likely a result of genetic and environmental factors, including egg quality and composition, rearing temperature, and food composition(Falk-Petersen, 2005).

During stage 1, or the lecithotrophic period, the American shad larvae possessed an undiff erentiated and straight digestive tract that was attached dorsally to the yolk sac but without an exterior connection, as previously reported in other teleosts, such as rock bream Oplegnathus fasciatus(He et al., 2012) and Cuban gar Atractosteus tristoechus(Comabella et al., 2013). Additionally, undiff erentiated cord-like cell groups(liver and pancreas) and incipient structures(gills, heart, kidney and urinary bladder)were visible. Our observations were similar to those in redb and ed seabream Pagrus auriga(Sánchez-Amaya et al., 2007), Atlantic blue-fintuna Thunnus thynnus(Yúfera et al., 2014). Interestingly though, Hong et al.(2013)noted that the mouth and anus of American shad larvae opened, the liver appeared, and the buccopharynx and intestine began to diff erentiate at 1 DAH when held in water temperatures of 24.0-25.0  C. The diff erences in developmental rate are likely a function of rearing temperature conditions. At 2 DAH, the three compartments(ventricle, atrium, and bulbus arteriosus)in the heart were distinguishable, which is earlier than in common dentex Dentex dentex L(Santamaría et al., 2004) and Atlantic cod Gadus morhua(Hall et al., 2004). Because the heart plays a major role in transporting oxygen and carbon dioxide, Yúfera et al.(2014)speculated that the early diff erentiation of the heart provides an advantage for predatory larvae by facilitating increased swimming speeds.

During stages 2 and 3, the endogenous reserves are depleted(3 DAH) and the digestive tract divides into five segments: the buccopharynx, oesphagus, primordial stomach, intestine, and rectum. Additionally, the mouth opens coincident with the time of first feeding, which is consistent with observations in other fishes(Kato et al., 2004; Mai et al., 2005; Zaiss et al., 2006). The functional liver, gall bladder, and pancreas were clearly distinguishable at this time, suggesting that the larvae acquired the ability to ingest, digest, and absorb exogenous food before endogenous reserves were completely depleted(Govoni et al., 1986; Segner et al., 1989). Unstained vacuoles were first observed in the liver at 3 DAH, coincident with first feeding, and indicating that lipids were being accumulated(Hachero-Cruzadoa et al., 2009). From 4 to 7 DAH, hepatic lipid accumulation increased progressively, but then gradually decreased thereafter. This pattern of hepatic lipid storage is consistent with observations in other species(Kozarić et al., 2008; Yang et al., 2010; Pradhan et al., 2012). Deposition of lipids in the liver ensures is important for development of the digestive system and proper functioning of the pancreas and intestine(Zambonino-Infante et al., 2008). The rate of change of lipid deposits in American shad suggests that during stage 2 and 3, the digestive system developed rapidly and acquired the ability to digest and store lipid.

Taste buds are chemosensory endorgans consisting of modified epithelial cells, which play a critical role in gustation, including sampling of potential food and selecting or rejecting substances according to their edibility(Sánchez-Amaya et al., 2007). In American shad larvae, the taste buds were first observed in the buccopharyngeal clavity at the onset of exogenous feeding, which suggest that first-feeding larvae had the ability to select for food. This is consistent with observations in butter catfish Ompok bimaculatus(Pradhan et al., 2012), Cuban gar Atractosteus tristoechus(Comabella et al., 2013), and Takifugu rubripes(Wan et al., 2006). The first functional goblet cells were observed in the oesophagus and buccopharyngeal cavity(5 DAH) and intestine(5-8 DAH). The abundance of these mucous cells increase gradually throughout larval development. Goblet cells protect the digestive mucosa from viral and bacterial attacks and lubricate the digestive mucosa by producing mucosubstances(Hirji and Courtney, 1983; Murray et al., 1994; Gisbert et al., 1999; Ribeiro et al., 1999; Arellano et al., 2002; Gisbert et al., 2004).

During early ontogeny, gas exchange occurs primarily through the skin and the primary function of the larval gills is related to osmoregulation rather than respiration(Rombough, 2004; Santamaría et al., 2004; Padrós et al., 2011). During stages 1 and 2, the American shad larvae had an incipient gill that had gill arches with primordial lamellae and filaments. At this point, the immature gills play a minor role in respiration. During stage 3, the gill completed full morphological development and the larvae underwent a functional transition from cutaneous to branchial gas exchange. The endocrine pancreas(islet of Langerhans)first appeared at 4 DAH, 1 day after first feeding. The first thyroid follicle was observed at 2 DAH, before mouth opening. The number of thyroid follicles increased rapidly thereafter. Although our observations do not confirmed the functionality of these tissues, endocrine tissues are known to become functional at hatching or before complete absorption of yolk in most teleosts(Tanaka et al., 1995). The early appearance of endocrine tissues is critical to larval metabolism and further development and metamorphosis(Zambonino-Infante et al., 2008; Padrós et al., 2011; Yúfera et al., 2014).

During stage 3, larvae were fed primarily with cladocerans and copepods nauplli. These have an excellent nutritional composition, being very rich in proteins, n-3 fatty acids, polarlipids, and essential vitamins and microminerals(Conceição et al., 2010; Hamre et al., 2013; Yúfera et al., 2014). At this time, the tissue and organ development could be clearly observed in the larvae. We observed an increase in the folding of the mucosa in the oesophagus, stomach, and intestine, the appearance and proliferation of pharyngeal teeth and taste buds, appearance of supranuclear vacuoles in the rectum enterocytes, and development of lipid infranuclear vesicles in the enterocytes of the anterior and middle intestine. The changes in the structures and organs suggested the digestive tract was gradually maturing, resulting in more efficient digestive and absorptive ability(Hachero-Cruzadoa et al., 2009; Comabella et al., 2013). Thus, the initial addition of cladocerans and copepods is most appropriate during stage 3.

The incipient stomach was first observed in American shad larvae at 3 DAH. Three anatomically and histologically diff erent regions were clearly distinguishable by 14 DAH: cardia, fundus, and pylorus. The gastric gl and s first appeared at 26 DAH, marking the end of stage 3, after which there was a significant proliferation of the gastric gl and s indicating the formation of the functional stomach and improvement in gastric digestive ability(Stroband and Kroon, 1981; Moyano et al., 2005; Sánchez-Amaya et al., 2007). The formulation of a functional stomach with the ability to secrete HCl and pepsin from the gastric gl and s is a vital event for young fish as it ensures they have the ability to digest compounds in their diets(Zambonino-Infante et al., 2008). Thus, the development of these traits may be used as external criteria for the start of weaning(Verreth et al., 1992; Segner et al., 1993). The American shad larvae were reared on microdiets from 30 DAH. The rearing protocols for American shad larvae are designed to change with the functional development of stomach. Therefore, we suggest that the weaning process for American shad larvae should be initiated at 26 DAH. The first observation of gastric gl and s in the stomach occurred earlier than in gilthead sea bream Sparus aurata L.(Elbal et al., 2004), and later than in flatfish brill Scophthalmus rhombus L.(Hachero-Cruzadoa et al., 2009), redb and ed seabream Pagrus auriga(Sánchez-Amaya et al., 2007), Atlantic blue-fintuna Thunnus thynnus(Yúfera et al., 2014), and rock bream Oplegnathus fasciatus(He et al., 2012). The earlier development of a functional stomach allows for earlier weaning, and a reduction in the costs associated with cultivation of live foods for feeding larvae.

During stages 1 and 2, the circulatory and excretory systems became functional in the American shad larvae. This process included compartmentalization of the heart and the development of renal corpuscles and the collecting duct connected to the urinary bladder through the ureter. These developments facilitate an increase in swimming speed and predatory efficiency of American shad larvae. Such functionalities have also been described in flatfish brill Scophthalmus rhombus(Hachero-Cruzado et al., 2009)at 4 DAH, the redb and ed seabream Pagrus auriga(Sánchez-Amaya et al., 2007)from 3 to 7 DAH, and in Atlantic cod Gadus morhua(Morrison, 1993)at 8-9 DAH, 3-4 d after first feeding.

The immune organs of American shad larvae develop in the sequence: pronephric kidney(visible at 5 DAH), spleen(first seen at 8 DAH), and thymus(first seen at 12 DAH). This is consistent with the pattern of development observed in other marine teleosts(Chantanachookh et al., 1991; Padrós and Crespo, 1996; Watts et al., 2003; Kato et al., 2004; Santamaría et al., 2004). The timing at which immune organs become lymphoid is variable in teleosts(Zapata et al., 2006). In our study, the pronephric kidney was lymphoid by 18 DAH and the thymus and spleen began to develop lymphoid capacity at 22 and 28 DAH, respectively. This timing is similar to that observed in rock bream Oplegnathus fasciatus(Xiao et al., 2013) and barfin flounder Verasper moseri(Xiao et al., 2008). However, in flounder Paralichthys olivaceus(Liu et al., 2004), the time sequence of immune organs becoming lymphoid was thymus, kidney, then spleen. The diff erence in the timing of lymphoid development is likely influenced by species, environmental factors, and immune system evolution in fish(Schrøder et al., 1998; Watts et al., 2003; Zapata et al., 2006). During early larval development in dentex, the lymphocytes were only observed in the thymus(Santamaría et al., 2004), which was thought to explain the high mortality rates during the larval rearing process. Thus, development of lymphoid capacity in the immune organs is important for the health of larvae.

(Liu et al., 2004), the time sequence of immune organs becoming lymphoid was thymus, kidney, then spleen. The diff erence in the timing of lymphoid development is likely influenced by species, environmental factors, and immune system evolution in fish(Schrøder et al., 1998; Watts et al., 2003; Zapata et al., 2006). During early larval development in dentex, the lymphocytes were only observed in the thymus(Santamaría et al., 2004), which was thought to explain the high mortality rates during the larval rearing process. Thus, development of lymphoid capacity in the immune organs is important for the health of larvae.

In conclusion, the organogenesis of American shad larvae conformed to the general pattern observed in other teleost fish species, although the timing of organ and system development exhibited interspecific diff erences. These ontogenic diff erences should be taken account during artificial culture to ensure the healthy development and growth of larvae. By studying and analyzing the tissue structures and organ status during ontogenic development, we can better underst and the physiological characteristics and functional systemic capabilities of the American shad larvae. Our observation can be used to formulate feeding strategies, improve culture techniques, and increase the survival of larvae during the early life stages.

5 ACKNOWLEDGEMENT

Thank the Jiangsu Zhongyang Group to provide the larvae used in the present study, and thank Yellow Sea Fisheries Research Institute for their excellent technical assistance.

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