JOURNAL OF MORPHOLOGY 247:172–195 (2001) Histological Study of the Development of the Embryo and Early Larva of Oreochromis niloticus (Pisces: Cichlidae) Carol M. Morrison,1* Tsutomu Miyake,2 and James R. Wright, Jr.3 1 Department of Pathology, IWK-Grace Health Center, Dalhousie University, Faculty of Medicine, Halifax, Nova Scotia, Canada 2 Center for Human Genetics, Boston University Medical School, Boston, Massachusetts 3 Department of Pathology, Surgery and Biomedical Engineering, IWK-Grace Health Center, Dalhousie University, Faculty of Medicine, Halifax, Nova Scotia, Canada ABSTRACT The developmental stages of Oreochromis niloticus are similar to those described in other mouthbreeding tilapias except that, as in zebrafish, no cavity was found in the blastula. Variation in the rate of development of the embryo and larva of O. niloticus was found within a clutch of eggs as well as between clutches. Hatching glands are described for the first time in tilapias. They are widely distributed within the ectoderm covering the head, body, tail, and surface of the yolk sac near its attachment to the embryo. Timing of larval development is similar to that in other mouthbrooding tilapias, but is slower than that found in substrate-spawning tilapias. A pneumatic duct connects the swimbladder to the digestive tract and swimbladder inflation and initiation of feeding Tilapias (genus Oreochromis) are among the most important fish commercially produced in aquaculture worldwide, with more than 75 countries raising the fish. Oreochromis niloticus is probably the most important of the tilapine species. Oreochromis niloticus is also currently a very useful species for divers studies in molecular genetics (Agnese et al., 1997; Kocher et al., 1998; Oliveira and Wright, 1998), endocrinology (Yang et al., 1997a; Wright et al., 1998b, 2000), and xenotransplantation (Yang et al., 1997b; Wright et al., 1998a; Yang and Wright, 1999; Yu and Wright, 1999; Wright and Pohajdak, in press). Like zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes), O. niloticus has become a very important species for transgenic research. Although these fish are members of the Class Actinopterygii, they belong to different orders and families (zebrafish: Order Cypriniformes, Family Cyprinidae; Japanese medaka: Order Beloniformes, Family Adrianichthyidae; O. niloticus: Order Perciformes, Family Cichlidae [www.fishbase.org]). All these fish are easy to rear in aquaria, but the larger size of O. niloticus makes it more suitable for some purposes, such as those involving physiological studies after transgenic manipulation (Chen et al., 1995; MacKenzie, 1996; Wright, 2001). Oreochromis nil© 2001 WILEY-LISS, INC. occurs at about the same time. The digestive tract of the larva 8 and 9 days after fertilization is similar to that found in the adult, except that there are no digestive glands. An endocrine pancreatic islet was first seen 76 h after fertilization. A prominent thymus gland is present at 100 h. Hematopoietic tissue develops in the vicinity of the pronephros during early larval development. A spleen develops later, 7 days after fertilization. J. Morphol. 247: 172–195, 2001. © 2001 Wiley-Liss, Inc. KEY WORDS: Oreochromis niloticus; tilapia; development; embryo; larva; histology; hatching glands; pancreatic islet oticus has a high fecundity (up to several thousand eggs in a clutch), year-round breeding capability, and a short generation period (i.e., fertilization to sexual maturity) of 5 to 6 months (Rahman et al., 1998; Wright, 2001). Nonetheless, O. niloticus has never really been exploited for developmental biology studies, even though its genome has been well characterized. The diploid chromosome number is 44 (Majunder and McAndrew, 1986) and a genetic linkage map with a length of ;1,000 –1,200 cM has been constructed (Kocher et al., 1998). The genome size is 1–2 3 109 basepairs (JM Wright, personal communication) and there is a Tilapia gene mapping project on the Internet (www.ri.bbsrc.ac.uk/ tilapia/) organized by the Roslin Institute, Edinburgh. A number of O. niloticus genes have been cloned (over 500 entries in NCBI Genebank Repository). Contract grant sponsor: I.W.K. Grace (to C.M.); Aquanet. *Correspondence to: Dr. Carol Morrison, Department of Pathology, IWK-Grace Health Center, Dalhousie University, Faculty of Medicine, Halifax, Nova Scotia, Canada B3J 3G9. E-mail: cmmorrison@iwkgrace.ns.ca DEVELOPMENT OF OREOCHROMIS NILOTICUS Figures 1–12 173 174 C.M. MORRISON ET AL. Recently, we initiated a study to investigate the development of insulin production in Oreochromis niloticus embryos (Lobsinger et al., unpublished observations; Mansour et al., 1998) and were unable to find any information on the development of their pancreatic islets. We also wanted to find the approximate time when the embryo reaches the 1,000 cell midblastula stage, because this has been identified as the optimal stage for obtaining embryonic stem cells in medaka (Hong and Schartl, 1996) and zebrafish (P. Collodi, Department of Animal Sciences, Purdue University, West Lafayette, IN, USA, personal communication). However, we found that estimates of timing of the blastula stage varied from 8 –12 h (Lingling and Qianru, 1981; Galman, 1980; Galman and Avtalion, 1989; Rana, 1990) and that no cell counts were available beyond the 64-cell stage. In fact, we were astounded at how little detailed information is currently available on embryonic and early larval development of O. niloticus and, therefore, initiated this study. We found that most accounts only described the features of Oreochromis niloticus embryology and early larval development that could be seen without histological sectioning, using dissecting microscopes (Galman, 1980; Lingling and Qianru, 1981; Rana, 1990). However, Galman and Avtalion (1989) described the surface features using scanning electron microscopy. Fishelson (1966) described the larval organogenesis of several systems in the substrate- Fig. 1 (Overleaf.) Oreochromis niloticus. Two-cell stage. The large cells are incompletely divided. No nuclei are visible. H&E. Bouin’s and formalin fixation. Bar 5 100 mm. Fig. 2. Blastodisc at 4 h. Two layers of cells are present in the center of the blastodisc. H&E. Bouin’s and formalin fixation. Bar 5 100 mm. Fig. 3. Blastodisc at 5 h. The EVL is darker than the other cells in the blastodisc. H&E. Bouin’s and formalin fixation. Bar 5 100 mm. Fig. 4. Same stage as Figure 3. Nuclei (N) and mitotic figures (MF) can be seen. H&E. Bouin’s and formalin fixation. Bar 5 100 mm. Fig. 5. Blastodisc at 6 h. There are about four layers of cells in the center of the blastodisc and the EVL is darker and more flattened than in Figure 3. H&E. Bouin’s and formalin fixation. Bar 5 100 mm. Fig. 6. Blastodisc at 721 h. There is a dome of cells about seven cells deep. H&E. Bouin’s and formalin fixation. Bar 5 100 mm. Fig. 7. Same stage as Figure 6. A peripheral rim of syncytial cells, the YSL, extends beneath the periphery of the blastodisc. H&E. Bouin’s and formalin fixation. Bar 5 100 mm. Fig. 8. Periphery of blastodisc at 11 h. The YSL extends over the yolk around the blastodisc. H&E. Bouin’s and formalin fixation. Bar 5 50 mm. Fig. 9. Transverse section of embryonic shield at 22 h. Laterally, Brachet’s cleft (B) separates the epiblast (E) from the hypoblast (H). Centrally the shield is thickened to form the embryonic axis. H&E. Bouin’s and formalin fixation. Bar 5 100 mm. Fig. 10. Periphery of embryonic shield at 22 h. The EVL is very thin and the YSL is continuous between the blastoderm and the yolk. H&E. Bouin’s and formalin fixation. Bar 5 50 mm. Figs. 11–12, 31-h embryos. Fig. 11. The anterior end of the neural keel is enlarged to form a brain primordium and an optic primordium (OpP) is visible. H&E. Bouin’s and formalin fixation. Bar 5 50 mm. Fig. 12. Transverse section near anterior end. There are otic primordia (OP) on either side of the neural keel (NK) in the region of the hindbrain. Ventrally is a cord of cells, the notochord (No). H&E. Formalin fixation. Bar 5 50 mm. spawner Tilapia tholloni from hatching to 30 days old and compared it with that of the mouthbrooder Oreochromis macrocephalus and, to a lesser extent, O. niloticus. Shaw (1954) included sectioned material in a description of the stages of embryonic development of O. macrocephalus, a mouthbrooder like O. niloticus. In the following account we provide light micrographs of sectioned material of the embryology and larval development up to swimbladder inflation and exogenous feeding of O. niloticus. We describe features such as the hatching glands, which have not previously been described in tilapine fish, and the rudimentary cement glands, which have previously only been described in drawings by Peters (1965). The numbering of the stages of embryonic development in Oreochromis niloticus differs in previous accounts and the timing also varies. In a study of the embryonic development of the zebrafish, Kimmel et al. (1995) subdivided embryogenesis into eight broad periods. In their system the stages were named, but not numbered to allow for flexibility in the staging series. This system is used in the following account. Age is recorded in hours after fertilization, later stages also in days, counting the day of fertilization as day 1. MATERIALS AND METHODS Eggs, most from 2-year-old Oreochromis niloticus niloticus (Linnaeus, 1758) (Dalhousie University Marine Gene Probe Laboratory Hatchery, Halifax, Canada), were incubated at 27–29°C. About 600 eggs and larvae from 10 different clutches at various developmental stages were studied. In most instances, larvae were examined under a dissecting microscope prior to fixation. Most were fixed in Bouin’s fixative overnight, then transferred to 10% buffered formalin, but some were fixed in formalin only. About 80 of these samples were embedded in paraffin and either sectioned at multiple levels or serially sectioned. Since the yolk was large, most of it was cut off hatched larvae, so that they could be oriented more accurately. The sections were stained with hematoxylin and eosin, Masson’s trichrome stain (Drury et al., 1967), periodic-acid Schiff, and Alcian blue at pH 2.5 (Vacca, 1985) or an immunohistochemical technique for tilapia insulin (Yang et al., 1999). Cell counts at the beginning of development (2– 8 cells) were made from direct observation of the eggs, which have a transparent envelope. The egg of Oreochromis niloticus is large (2.06 –2.40 mm 3 1.35– 1.80 mm; Lingling and Qianru, 1981) compared to zebrafish (ca. 0.7 mm; Kimmel et al., 1995) and medaka (ca. 1.2 mm; Iwamatsu, 1994). Therefore, to obtain cell counts later in development the embryo had to be separated from the yolk. The egg envelope was removed using microdissecting scissors and forceps. The embryo was then teased away from the Drawings 1–5 176 C.M. MORRISON ET AL. yolk and a squash preparation was made so that the cells could be counted. The number of cells at 91⁄2 h was counted from a photograph of a squash preparation of the embryo. Lengths of hatched larvae were obtained from fixed larvae using a dissecting microscope and are given as the mean and standard deviation. Ten larvae were measured from each clutch and two or three clutches were measured for each day. Drawings were made from photographs of the sectioned material used for the photographic plates. Organs from different sections through each larva have been included, so that the reader can orient these in the micrographs. The outline of each larva was taken from whole, fixed specimens. RESULTS We noticed some variation in speed of development among embryos in the same clutch, as well as among different clutches. The timing of developmental stages given in this account therefore applies to the particular embryos and larvae that we studied and could be different in other specimens. Zygote Period (0 –11⁄2 h) After fertilization, cytoplasm accumulates at the animal pole. After about 11⁄2 h this cytoplasm bulges † Drawing 1 (Overleaf.) Oreochromis niloticus. 31-h embryo. An optic primordium (OpP) is present on either side of the brain primordium. There are about 11 somites (S), a notochord (No) is present, and Kuppfer’s vesicle (KV) can be seen ventral to the tail bud. Drawing 2. 46-h embryo. There is a lens primordium (LP) in the center of the eye cup, an olfactory vesicle (OlfV) is present, and there are dorsal and ventral cement glands (CG). The brain is subdivided, and there is a vertically oriented cerebellum (Ce) and a hindbrain divided into rhombomeres (R). A liver (Li) is beginning to form and a digestive tract (DT) and tubular heart (He) are present. Drawing 3. 76-h embryo. Pharyngeal arches (PA) have formed and there is an otic vesicle (Ot). An endocrine pancreatic islet (EP) has formed and the glomerulus (G) of the pronephros can be seen. Drawing 4. 100-h embryo. An oropharyngeal membrane (OM) covers the mouth, a pseudobranch (Ps) is forming, and there are gill arches (GA) around the pharynx. A prominent thymus gland (TG) is present and convoluted pronephric tubules (PT) have formed. The swimbladder primordium (Sw) is attached to the stomach (St) by a pneumatic duct, and the digestive tract forms a loop around the endocrine pancreas (EP). The otocyst (Ot) is partially divided into chambers. Drawing 5. 124-h larva. The mouth is still covered by an oropharyngeal membrane, and the pectoral fins (PF) face dorsally. A branchiostegal membrane (BM) is beginning to form. A thymus gland (TG), uninflated swimbladder (Sw), endocrine pancreas (EP) and gallbladder (Ga) are present. The stomach (St) forms a small cecum to one side of the gut. The pronephric tubules (PT) lead to a urinary bladder (UB). The notochord turns up posteriorly, forming the urostyle (U). up to form a disc and a distinct perivitelline space is apparent. Cleavage Period (11⁄2–5 h) The first cleavage, at 11⁄2–2 h, results in two large cells (Fig. 1). By 2–21⁄2 h, most eggs are at the 4-cell stage and by 3 h eggs have 4 – 8 cells. By 4 h there are 16 cells in a 4 3 4 array, or 32 cells, in many cases forming a 4 3 8 array, as described by Kimmel et al. (1995). The arrangement of cells in the 32-cell stage is variable and cleavages become more irregular in the following stages. A horizontal cleavage forms the 64-cell stage. In some eggs this starts to takes place at 4 h. The central cells divide before the marginal cells, so that two layers of cells form in the center of the blastodisc (Fig. 2). Blastula Period (5–22 h) Five hours after fertilization the cells are variable in size and about 72 cells were counted from a squash preparation of an embryo. These cells form approximately three layers. An outer enveloping layer (EVL) of cells is somewhat flattened and has darker cytoplasm than the other cells in the blastodisc (Fig. 3). Mitotic spindles could be seen in many cells (Fig. 4). The blastodisc begins to look ball-like at about 6 h. There are about 150 cells by 51⁄2 h and 270 cells by 6 h. The latter are arranged in about four layers in the center of the blastodisc (Fig. 5). The EVL becomes more distinct and consists of dark, flattened cells. There are about 400 cells by 61⁄2 h. At 71⁄2 h there is a smooth dome of cells approximately seven cells deep (Fig. 6). The peripheral blastomeres (the first-tier EVL cells) join together to form the “yolk syncytial layer” (YSL, Kimmel et al., 1995). As in the zebrafish, the YSL forms a ring around the blastodisc (Fig. 7). Approximately 1,200 cells are present at 91⁄2 h and form a dome about seven cells deep in the center. There is no blastocoel. By 11 h the dome is seven to nine cells deep. The YSL extends under the rim of the blastodisc and is also beginning to form a thin flange over the yolk (Fig. 8). The blastodisc covers about 10% of the yolk. At 14 h a different clutch of eggs has a blastodisc that is about 13 cells deep. The YSL extends under about one-third of the edge of the blastodisc, but has not extended over the yolk to the same extent as the 11-h sample. In another clutch the blastodisc is further flattened by 20 h and covers about 20% of the yolk. Gastrula Period (22–26 h) † These drawings have been prepared by overlaying photographs of slides used for the micrographs in this account. It is not possible to show the full extent of all features, since some obscure others. Only some sections of such features as myotomes and neural and hemal arches and spines are therefore shown. Bar 5 1 mm. In three different clutches epiboly is about 30% complete in most eggs at approximately 22, 24, and 26 h, respectively. The germ ring is beginning to thicken and a thickened triangular region, the em- DEVELOPMENT OF OREOCHROMIS NILOTICUS Figure 13–22 177 178 C.M. MORRISON ET AL. bryonic shield, extends from the germ ring towards the animal pole. The YSL is now continuous between the embryonic cells and the yolk. Most of the blastoderm is so thin that, except for the embryonic shield and germ ring, it is difficult to distinguish the different layers of cells. Excluding the EVL and YSL, the embryonic shield consists of about six layers of cells separated into two layers and the germ ring is about two cell layers thick. In three other clutches at about the same time intervals the blastoderm covers about 40% of the surface of most eggs and the anterior–posterior axis of the embryo is visible as a thickening in the center of the embryonic shield, extending from the animal pole to the edge of the blastoderm. In the embryonic shield, Brachet’s cleft separates the epiblast from the hypoblast and the central embryonic axis extends down into the yolk (Fig. 9). The EVL is so thin in the embryonic shield that it is difficult to see and the YSL is continuous beneath the blastoderm (Fig. 10). Segmentation Period (26 – 48 h) 26 –30 h (day 2). Epiboly is 50 – 60% complete by 26 –30 h, depending on the clutch. The whole embryo becomes thickened, especially at the anterior end. Posteriorly, the tail bud extends ventrally from the germ ring. A 26-h embryo from one clutch has about Figs. 13–15 (Overleaf.) 31-h embryos. Oreochromis niloticus. Fig. 13. About 11 somites (S) are forming. H&E. Bouin’s and formalin fixation. Bar 5 100 mm. Fig. 14. Transverse section near posterior end. Mesoderm (M) is on either side of the neural keel (NK). H&E. Formalin fixation. Bar 5 50 mm. Fig. 15. Kupffer’s vesicle. This is found beneath the tail bud. Dorsally are columnar cells with cilia (C) and ventrally the elongate nuclei (N) of the YSL are visible. The cells of the notochord (No) are becoming vacuolated. Y, yolk. H&E. Bouin’s and formalin fixation. Bar 5 40 mm. Figs. 16 –22, 46-h embryos. Fig. 16. Brain showing the regions of the telencephalon (Te) and diencephalon (D) of the forebrain, dorsal midbrain (DM), ventral midbrain (VM), and the prominent transverse cerebellum (Ce) that separates the midbrain from the hindbrain rhombomeres (R). No, notochord. H&E. Bouin’s and formalin fixation. Bar 5 100 mm. Fig. 17. Eye with lens placode (LP) in center of optic cup with ventral choroid fissure (CF). An olfactory vesicle (OlfV) is present and erythrocytes (Er) can be seen in the thin-walled heart (He). H&E. Bouin’s and formalin fixation. Bar 5 50 mm. Fig. 18. There is a spinal cord floor plate (FP) between the neural tube (NT) and notochord (No). The digestive tract (DT) possesses a lumen and the liver (Li) is developing next to the yolk (Y). H&E. Bouin’s and formalin fixation. Bar 5 20 mm. Fig. 19. Primordial germ cells (PGC) are ventral to the pronephric tubule (PT) and somites (S). H&E. Bouin’s and formalin fixation. Bar 5 20 mm. Fig. 20. Anterior and posterior cement glands (CG) on the dorsal surface of head (anterior end to the right). H&E. Bouin’s and formalin fixation. Bar 5 20 mm. Fig. 21. Cement gland (CG) on the ventral side of the anterior end of the larva, ventral to the olfactory vesicle, OlfV. Op V, optic vesicle. H&E. Bouin’s and formalin fixation. Bar 5 20 mm. Fig. 22. Yolk periphery. Beneath the egg envelope (EE) is the periderm (Pe), then a blood space containing erythrocytes (Er) lined by endothelium (End). Beneath the endothelium is a nucleus (N) of the YSL. H&E. Bouin’s and formalin fixation. Bar 5 20 mm. four myomeres and a notochord and eye primordium are present. Kupffer’s vesicle is present as a small slit. Segmentation does not appear to have started in an embryo from another clutch at 30 h. 31 h (day 2). Epiboly is about 80% complete in embryos from one clutch, being more advanced on the side opposite the embryo than in the region of the embryonic axis, which is clearly visible. The neural keel is enlarged anteriorly to form the brain rudiment, which is not yet subdivided, and optic primordia are present on either side of this rudiment (Fig. 11; Drawing 1). In a cross section of the posterior part of the brain there are otic placodes and a notochord is ventral to the brain (Fig. 12). About 11 somites have formed (Fig. 13) and a cross section of this region shows mesoderm on either side of the neural keel (Fig. 14). The cells are beginning to elongate in the most anterior somites. Along most of the notochord the cells have a typical “stack of pennies” appearance (Kimmel et al., 1995). The posterior part of the notochord, which can be seen in the tail bud dorsal to Kupffer’s vesicle, does not yet have this appearance (Fig. 15). Kupffer’s vesicle is lined ventrally by a depression in the YSL and the roof is lined with ciliated columnar cells. 46 – 48 h (day 2). Epiboly is completed during this time. The eyes are visible but not pigmented and there is a patch of melanophores on the egg surface on each side of the embryo, which remains throughout the rest of embryonic and larval development. In 48-h embryos from one clutch, Kuppfer’s vesicle is still present. About 15 somites are visible in sections and the first one to four somites, depending on the embryo, are differentiated into myotomes with longitudinally oriented muscle fibers. Segmentation is not yet complete to the posterior end of the embryo. The cells of the notochord still have the “stack of pennies” appearance. In another clutch (Drawing 2), at 46 h myotomes have developed from the first 17–18 somites, which extend along most of the embryo. The brain is partitioned into the forebrain (diencephalon and telencephalon), the dorsal (optic tectum) and ventral midbrain, the cerebellum, and the hindbrain, which is divided into rhombomeres (Fig. 16). The cells of the anterior part of the notochord are vacuolated and beneath the notochord is the gut, which has a lumen. The posterior gut does not appear to be ciliated and Kupffer’s vesicle was not seen. The eye consists of an optic cup with a ventral choroid fissure and a central lens primordium (Fig. 17). The heart is a thin-walled tube containing erythrocytes. The liver is beginning to form between the yolk and gut (Fig. 18). Dorsal to the notochord is a row of cuboidal cells, the spinal cord floor plate. This plate is ventral to the neural tube, which possesses a lumen (Fig. 18). The floor-plate extends anteriorly to the hindbrain. Ventral to the somites are pronephric tubules and posteriorly there are also a few primordial germ cells, which could be recognized by their large size, Figures 23–32 180 C.M. MORRISON ET AL. round shape, and clear cytoplasm (Fig. 19). There are three pairs of cement glands on the head, one pair ventral to the anterior surface of the larva and two dorsal (Figs. 20, 21). Erythrocytes are present over large regions of the yolk, enclosed by thin endothelial layers that could be distinguished in some places between the periderm (formed from the EVL) and the YSL (Fig. 22). Pharyngula Period (48 –100 h) 76 h (day 4). Besides pigmentation on either side of the larva on the yolk, there are melanophores on the ventral side of the larva near the attachment to the yolk, as well as a few on the body dorsal to this region. The brain is enlarged and consists of five lobes and there is an ovoid otic vesicle with thickenings in the walls (Fig. 23). Another embryo of the same age from a different clutch had a more elongate, flattened head, with a longer distance between the otic vesicle and eye. Olfactory placodes are present and the notochord is well-developed and vacuolated. Several pharyngeal arches have formed (Drawing 3; the first two, the mandibular and hyoid arches, will form the jaws; the others will form the branchial arches). At this stage, the heart, which had been observed to be contracting rhythmically, is a simple tube with a multilayered wall, ventrolateral to the left side of the pharynx (Fig. 23). In one clutch the heart was beating at 50 h. The tail is free of the yolk. Figs. 23–27 (Overleaf.) 76-h embryos. Oreochromis niloticus. Fig. 23. The brain has enlarged and the ovoid otocyst (Ot) is closer to the eye than in younger larvae. The shape of the otocyst is slightly irregular, preparatory to the formation of chambers. Pharyngeal arches (PA) are present and there is a vesicular heart (He). The nuclei (N) of the photoreceptors are aligned in the central part of the optic cup. H&E. Bouin’s and formalin fixation. Bar 5 100 mm. Fig. 24. Part of eye. Small pigment granules (PG) are present in a layer of cells in the external part of the optic cup. L, lens. H&E. Bouin’s and formalin fixation. Bar 5 40 mm. Fig. 25. Segmental nerves (SN) are found ventral to the hollow nerve cord (NC). H&E. Bouin’s and formalin fixation. Bar 5 100 mm. Fig. 26. The pronephros consists of a pronephric tubule (PT) leading from a single glomerulus (G). H&E. Bouin’s and formalin fixation. Bar 5 50 mm. Fig. 27. An endocrine pancreatic gland (EP) is present. There are hatching glands (HG) in the ectoderm. S, somites. H&E. Bouin’s and formalin fixation. Bar 5 100 mm. Figs. 28 –32, unhatched 100-h embryos. Fig. 28. Embryo coiled around large yolk. H&E. Bouin’s and formalin fixation. Bar 5 1 mm. Fig. 29. Head of embryo with otocyst (Ot), pharyngeal arches (PA), several profiles of pronephric tubule (PT), and mouth covered by oropharyngeal membrane (OM). H&E. Bouin’s and formalin fixation. Bar 5 400 mm. Fig. 30. The esophagus has stratified epithelium (SE), and posterior to this are columnar cells with pale cytoplasm (PC) that appear to be ciliated. H&E. Bouin’s and formalin fixation. Bar 5 40 mm. Fig. 31. The uninflated swimbladder (Sw) is attached by a pneumatic duct (PD) to the digestive tract (DT). H&E. Bouin’s and formalin fixation. Bar 5 50 mm. Fig. 32. The liver (Li) is permeated by blood vessels containing erythrocytes (Er). Me, melanophore; S, somite; Y, yolk. H&E. Bouin’s and formalin fixation. Bar 5 50 mm. The central part of the eyecup is more developed than the peripheral regions. In this central region the layers of cells are connected by inner and outer plexiform layers and the photoreceptor nuclei are aligned (Fig. 23). Pigment granules are starting to form in the outer layer of the optic cup (Fig. 24). In an embryo from another clutch, eye pigmentation had not started by 80 h. The nerve cord is hollow. Segmented nerves extend to myotomes, which are present to the end of the tail (Fig. 25). Two pronephric tubules run from the glomeruli (Fig. 26) to the cloaca and an endocrine pancreatic islet with insulin immunopositive cells is present (Fig. 27). The ectoderm is two cell layers thick and unicellular hatching glands with prominent granules which stained positively with eosin and orange G are present in the ectoderm covering the head, the tail, and the surface of the yolk sac around the attachment to the embryo (Fig. 27). Hatching glands were also occasionally seen in the pharynx. Cement glands are present on the head. There is a lumen in the pharynx and esophagus, which are lined with cuboidal epithelium, and in the intestine, which is lined with columnar epithelium. The stomach is a diverticulum to one side of the intestine. Hatching Period (100 –124 h) 100 h (day 5). In one clutch, no embryos had hatched at 92 h (day 4), but they began hatching at about 100 h. In most clutches hatching started 5 days after fertilization and continued for about 24 h. Unhatched embryos are coiled around a large yolk sac (Fig. 28; Drawing 4). There is a patch of pigmentation on either side of the body posterior to the pectoral fin, where the embryo attaches to the yolk, and one or two pigment spots on the dorsal surface of the embryo. The brain is well-developed, several gill arches can be distinguished around the pharynx, and the pronephric tubules are coiled anteriorly (Fig. 29). There is a small amount of lymphoid and erythroid tissue around the pronephros. An oropharyngeal membrane (Fig. 29) covers the mouth. The epithelial lining of the anterior part of the esophagus is beginning to stratify and posterior to this is a region of cuboidal to columnar cells with pale cytoplasm, which appears to be ciliated (Fig. 30). A pneumatic duct with a narrow lumen attaches the primordial swimbladder to the digestive tract near the opening of the stomach (Fig. 31). There are no goblet cells in the esophagus or intestine and there is a small loop in the intestine around the pancreas and gallbladder (Drawing 4). A well-vascularized liver is present (Fig. 32) and the ovoid gallbladder is lined with cuboidal cells (Fig. 33). Some exocrine pancreatic cells are closely associated with an insulin-positive endocrine islet (Fig. 33). There are numerous melanophores in the gut cavity. Primor- Figs. 33– 41, unhatched 100-h embryos. Oreochromis niloticus. Fig. 33. The endocrine pancreatic islet (EnP) is associate with a small amount of exocrine pancreatic tissue (ExP). The gallbladder (Ga) is lined with cuboidal epithelium. Me, melanophore. H&E. Bouin’s and formalin fixation. Bar 5 40 mm. Fig. 34. Each gill arch contains a cartilaginous rod (CR) and a branchial artery (BrA). Ps, pseudobranch. H&E. Bouin’s and formalin fixation. Bar 5 100 mm. Fig. 35. In the central region of the optic cup the pigment layer (PL) is well-developed and the layers of cell nuclei are connected by plexiform layers (PlL). L, lens. H&E. Bouin’s and formalin fixation. Bar 5 100 mm. Fig. 36. The otocyst is divided into chambers containing maculae (Ma) and cartilage (Ca) is present ventrally. H&E. Bouin’s and formalin fixation. Bar 5 100 mm. Fig. 37. Anterior and posterior cement glands (CG) are present on the head of the embryo, dorsal to the eye. H&E. Bouin’s and formalin fixation. Bar 5 100 mm. Fig. 38. Neuromast (Ne) is present on the ventral surface of the head, dorsal to the yolk sac (Y). H&E. Bouin’s and formalin fixation. Bar 5 40 mm. Fig. 39. Squash preparation of larva, showing hatching glands (HG) in ectoderm. H&E. Bouin’s and formalin fixation. Bar 5 100 mm. Fig. 40. Hatching glands (HG) are seen in the ectoderm on the dorsal and ventral surface of tail. S, somite; BS, blood space around egg; Y, yolk; BV, caudal blood vessel. H&E. Bouin’s and formalin fixation. Bar 5 40 mm. Fig. 41. Oblique section through tail. The nerve cord (NC), notochord (No) and caudal blood vessels (BV) can be seen. H&E. Bouin’s and formalin fixation. Bar 5 200 mm. 182 C.M. MORRISON ET AL. dial germ cells are present near the pronephric tubules. Each gill arch contains a cartilaginous rod and branchial artery and the pseudobranch is beginning to form as a bulge on the anterior wall of the branchial cavity (Fig. 34). The trabeculum cranii and Meckel’s cartilages are present in the upper and lower jaws and there is a cartilaginous core in the pectoral fin. There are well-developed thymus glands on the dorsal wall of the pharyngeal cavity. There are trabeculae in the ventricle of the heart and there is a thick-walled bulbus venosus. In the central part of the eyecup the pigment cell layer is prominent and the nuclei of the rods and cones are becoming aligned into two layers (Fig. 35). The otocyst is partially divided into the utriculus, sacculus, and lagena, each containing a macula (Fig. 36). Olfactory organs are present. There are rudimentary cement glands on the head, which are not as prominent as in the 76-h-old embryo (Fig. 37). Neuromasts are also present on the head (Fig. 38). Numerous hatching glands are present in the epidermis of the head and tail, including the pectoral fins and the surface of the yolk sac near the attachment of the embryo (Figs. 39, 40). In a cross section of the tail, there are a hollow nerve cord, vacuolated notochord, and caudal blood vessels (Fig. 41). Posteriorly the notochord turns up, giving the tail a heterocercal appearance. Embryos usually hatch tail-first, leaving thin, ghost-like chorions behind. After hatching, the pectoral fins face dorsally. Early Larval Period 124 h (day 6); mean length 4.8 6 0.3 mm. Most larvae in most clutches have hatched, but still possess a large yolk sac. There are a few melanophores at the base of the dorsal and ventral fin folds and on the dorsal surface of the head, as well as the patch seen on either side of the body in the 100-h embryo. The pectoral fins face dorsally. The notochord is turned up posteriorly to form a urostyle, giving a heterocercal appearance. In some larvae, the mouth is covered by an oropharyngeal membrane, while in others it is open. Gill filaments are forming and the pigmented layer of the retina extends around the eye to the lens (Fig. 42). The photoreceptor nuclei are aligned on the inner side of the optic cup, forming a double row centrally (Fig. 43). The gill cover or branchiostegal membrane is starting to form, covering the first pair of gill arches (Fig. 42). The pronephric tubules are more convoluted anteriorly than in the 100-h embryo and there is a small amount of erythroid and lymphoid tissue associated with this region. The well-vascularized liver is prominent between the yolk sac and the intestine and the intestine is lined with columnar epithelium (Fig. 44). The stomach forms a small cecum to one side of the gut (Drawing 5). No goblet cells were found. The gall bladder is more expanded than in embryos in the egg, lined by a cuboidal to squamous epithelium, and an insulin-immunopositive pancreatic islet partially surrounded by exocrine tissue is present (Fig. 44). A spleen is present dorsal to the pancreas. The otic vesicle is partially surrounded by a cartilaginous capsule (Fig. 45). A prominent thymus gland is present. The urinary ducts open into a small bladder, which is closely associated with the posterior digestive tract (Fig. 46). Primordial germ cells are present in the dorsal part of the posterior body cavity. Dorsal pharyngeal tooth buds are forming in the jaw. There are still a few hatching glands in the epithelium on the tail and head of the larva and on the anterior surface of the yolk sac, but most are partially degranulated. 147–148 h (day 7); mean length 5.4 6 0.7 mm. The bases of the pectoral fins have rotated, so that they now face posteriorly. There are more melanophores on the dorsal and ventral fin folds than in the 124-h larva, as well as on the dorsal surface of the head. The myotomes are V-shaped, separated into dorsal and ventral bundles by a myoseptum. The mouth is open and jaw movements were observed prior to fixation. The yolk is still large and the heart has a well-developed thick-walled bulbus arteriosus, thin-walled atrium, and ventricle with trabeculae in the wall (Fig. 47). Thyroid follicles are present around the ventral aorta. The intestine runs posteriorly, then loops anteriorly before passing posteriorly to the cloaca (Drawing 6). There are no goblet cells in the intestine, the gallbladder is enlarged and lined by squamous epithelium, and segmented tubules of the mesonephros are forming (Fig. 48). There is a spleen consisting mainly of erythroid tissue dorsal to the pancreatic islet and the swimbladder is still not inflated (Fig. 49). The thymus is well-developed. Dorsal and ventral pharyngeal teeth have formed in the jaws (Fig. 50). Primordial germ cells are present in the dorsal part of the posterior body cavity (Fig. 51). Most of the ectoderm is only two cell layers thick. A cartilaginous neural arch encloses the nerve cord and small ventral cartilaginous processes surround the caudal artery and vein. Four hypural cartilages are present ventral to the urostyle and several primordial fin rays support the tail fin (Drawing 6). 169 –172 h (day 8); mean length 6.3 6 0.7 mm. In one clutch at 172 h pigmentation on the dorsal surface of the body has increased and the yolk is becoming reduced in size. The ciliated nasal epithelium is indented, with an opening to the exterior; lamellae have formed on the gill filaments and a branchiostegal membrane covers the branchial chamber (Fig. 52; Drawing 7). The layers of the retina are well-developed, with two layers of photoreceptor nuclei and elongate outer segments in the central region. The pseudobranch consists of several lamellae covered by an epithelial layer (Fig. 53). Several cartilages have formed in the head (Figs. 53, Figures 42– 47 184 C.M. MORRISON ET AL. 54) and a bony cleithrum is present. Flanges of the oropharyngeal membrane (Fig. 54) line the open mouth. Goblet cells are present in the epithelium lining the esophagus, oral and pharyngeal cavities, and gill arches (Fig. 55). In the esophagus, there are both large goblet cells that stained blue-purple with PAS/Alcian blue and small goblet cells that stained more red-purple (Fig. 56). The small goblet cells have a similar appearance to those found in the dorsal part of the oral cavity and on the dorsal surface of the gills. A muscle layer is present in the esophageal wall. Besides dorsal and ventral pharyngeal teeth (Fig. 55), there are teeth on the dorsal surface of the gill arches and on the jaws. There is a constriction forming a pyloric sphincter between the small stomach, which is lined with columnar cells with a PAS-positive apical border, and the intestine. The stomach extends as a caecum to the right side of the gut. There are small goblet cells in the intestine, part of which is closely associated with the liver, and the exocrine pancreas extends between the loops of the intestine (Fig. 57). The swimbladder is not yet inflated and is lined with columnar epithelium whose cells have a vacuolated appearance; capillaries form a primordial rete mirabile ventral to the swimbladder (Fig. 58). A pneumatic duct with a small lumen is present, running from the digestive tract near the junction of the esophagus and stomach to the posterior end of the swimbladder (Fig. 59). Hemopoietic tissue is developing around the pronephros (Fig. 60) and there is some lymphoid tissue in the spleen (Fig. 61). The mesonephros is becoming coiled. The gall bladder has a thinner squamous epithelial lining than in earlier stages (Fig. 61). The outer layer of the epidermis is becoming squamous, with flattened nuclei, and more than two layers of cells are present. Neuromasts are present on the head (Fig. 62). Four hypural cartilages and two less modified hemal arches support fin rays in the caudal fin (Fig. 63). Figs. 42– 46 (Overleaf.) 124-h larvae. Oreochromis niloticus. Fig. 42. The pigment layer (PL) of the eye extends to the lens and gill filaments (GF) are forming. The branchiostegal membrane (BM) covers the first pair of gills. H&E. Bouin’s and formalin fixation. Bar 5 200 mm. Fig. 43. There is a double row of photoreceptor nuclei (N) in the central part of the eye cup. L, lens; PlL, plexiform layer. H&E. Bouin’s and formalin fixation. Bar 5 100 mm. Fig. 44. The pancreatic islet contains insulin-immunopositive cells (In). Exocrine pancreatic cells (ExP) are associated with the islet. The intestinal epithelium (I) is columnar. The gallbladder (Ga) is lined mainly by cuboidal cells. Li, liver. Insulin. Bouin’s and formalin fixation. Bar 5 40 mm. Fig. 45. Much of the otocyst is surrounded by cartilage (Ca). The prominent thymus gland (TG) extends into the pharyngeal cavity. Ma, macula. H&E. Bouin’s and formalin fixation. Bar 5 200 mm. Fig. 46. The pronephric tubule (PT) leads to a urinary bladder (UB), which is close to the posterior intestine (I) near the cloaca. H&E. Bouin’s and formalin fixation. Bar 5 100 mm. Fig. 47. 148-h larvae. The mouth is open and the trabeculum cranii (TC) is well-developed. The heart ventricle (HV) is thick-walled with trabeculae. H&E. Bouin’s and formalin fixation. Bar 5 400 mm. Cartilaginous neural and hemal arches have spines extending between the myotomes (Fig. 64), which are S-shaped (Drawing 7). Between the spines the cartilage of the pterygiophores, as shown by slight Alcian blue staining, is being laid down at the base of the fin folds (Drawing 7; Fig. 64). Bone is forming around the notochord, forming centra (Fig. 64). The heart has a well-developed bulbus venosus, atrium, and ventricle separated by valves (Fig. 65). Thyroid follicles are present around the ventral aorta (Fig. 66). 193–196 h (day 9); mean length 7.3 6 0.6 mm. Melanophores line the dorsal surface of the body cavity and are also present on the dorsal surface of the larva from one clutch at 196 h. A few melanophores have appeared on the sides of the body and in the ventral fin-fold. The myotomes are W-shaped. The dorsal and anal fins are beginning to differentiate in the fin-fold, which is supported by actinotrichia, and pterygiophores can now be clearly seen in the base of the fin-fold (Drawing 8). In one clutch fin rays are forming in the dorsal and anal fins. Bone is forming around the neural and hemal arches and spines and the cartilages of the head. Thin dermal bones, the branchiostegal rays, have been laid down in the branchiostegal membrane and premaxillary bones are present. There are taste buds in the epithelial lining of the pharynx (Fig. 67) and goblet cells between the gill lamellae. The swimbladder is inflated and is lined by squamous epithelium and there is a prominent constriction, the pyloric sphincter, between the small stomach and the intestine (Fig. 68). The esophagus has a muscular layer in the wall and it is lined with stratified squamous epithelium, which is replaced by columnar epithelium in the stomach (Fig. 68). The intestine contains some food organisms (Fig. 69). Part of the digestive tract is closely associated with the well-developed liver, which lies between it and the yolk. Numerous mucous cells are present in the intestine, which is lined by columnar epithelium (Fig. 69). The gall bladder is expanded and contains flocculent material; the spleen contains more lymphoid material and there is exocrine pancreatic tissue between folds of the digestive tract (Fig. 69). The coils of the mesonephric tubules are becoming more extensive and the urinary bladder opens into a cloaca with the posterior digestive tract or rectum, which lacks goblet cells (Fig. 70). A narrow gonad containing primordial germ cells is present ventral to the mesonephros (Fig. 70). The lining of the proximal part of the pronephric tubule is PAS/Alcian blue-positive. The lateral part of the otocyst, where semicircular canals are developing, is more completely surrounded by cartilage than the medial region, where cartilage is only present ventrally. The remains of one otolith were visible (Fig. 71). Two layers of receptor cell nuclei extend to the peripheral part of the eye (Fig. 72), the nuclei of the sclerad layer being larger than those of the vitread layer. Drawings 6 – 8 186 C.M. MORRISON ET AL. The skin is stratified and the outer layer is cornified. Neuromasts and mucous cells are present on the head. DISCUSSION The times at which we found that various features developed are compared with those described by other authors in Table 1. Each author chose different features for staging, so we picked those used by more than one author. Each author also used a different numbering system for the stages. Most accounts of Oreochromis niloticus development describe only features seen macroscopically or using a dissecting microscope, or surface features seen with a scanning electron microscope (Galman and Avtalion, 1989). Larval organogenesis is described by Fishelson (1966) in his comparison of the development of O. macrocephalus and O. niloticus with that of Tilapia tholloni. However, he does not describe embryonic development. We therefore also give the staging system used by Shaw and Aronson (1954) for embryonic development in Table 1, although this is for O. macrocephalus, because this is the only histological account of a species closely related to O. niloticus with which we could compare our results. There is some variation in the timing of the developmental features given in each account. Some of this variation is probably caused by the different rearing temperatures used in each study. We also found that the rate of development depends on the age of the broodstock, being generally slower and Drawing 6 (Overleaf.) Oreochromis niloticus. 148-h larva. The small stomach (St) leads into an intestine which runs posteriorly and forms an anterior loop before leading posteriorly to the cloaca. The pronephros (PT) is present and a segmented mesonephros (Mes) is forming. A spleen (Sp) is dorsal to the endocrine pancreas (EP) and gallbladder (Ga). The swimbladder (Sw) is not inflated. Neural and hemal arches (NA and HA) are present. Cartilaginous hypurals (Hy) support primordial fin rays (FR) in the tail fin. Drawing 7. 172-h larva. The nasal epithelium (NE) is indented and the pseudobranch (Ps) consists of several lamellae. The branchiostegal membrane (BM) covers the gill filaments. Pronephric tubules (PT) and coiled mesonephric tubules (Mes) are present. The swimbladder (Sw) is not inflated. Posterior to the stomach (St) is a small constriction (Co) forming a pyloric sphincter. The spleen (Sp), gallbladder (Ga), and endocrine pancreas (EP) are close together. The heart consists of a thick-walled bulbus arteriosus (BA), atrium (A), and ventricle (V). Neural arches (NA) surround the nerve cord and hemal arches (HA) surround the caudal artery and vein (C). These arches possess spines (NS and HS), between which pterygiophores (Pt) are forming. S-shaped myotomes (My) are present. Four hypural cartilages (Hy) support the fin rays. Drawing 8. 196-h larva. The swimbladder (Sw) is inflated. Coiled pronephric tubules (PT) and mesonephric tubules (Mes) lead into a urinary bladder (UB). The stomach (St) leads into a digestive tract containing food organisms. There are well-developed pterygiophores (Pt) between the neural and hemal (HS) spines and there are actinotrichia (Ac) in the fin-fold. The myotomes (My) are W-shaped. The gallbladder (Ga) is thin-walled and expanded. more variable in eggs from older broodstock. We limited this variable by obtaining most eggs from broodstock that was 2 years old, but the broodstock age is not mentioned in other accounts. Kimmel et al. (1995) found, as we did, that there is asynchrony in development even within a single clutch that is fertilized simultaneously and reared under optimal conditions. These authors reported that this asynchrony becomes more pronounced as time progresses. This variation would be even more evident in different strains of Oreochromis niloticus, explaining many of the differences in timing found in different studies. Also, it is difficult to accurately correlate the timing of stages given by different authors, since development is a continuous process. A stage “is merely a device for approximately locating a part of the continuum of development” (Kimmel et al., 1995, p. 254). We found that generally the embryonic development of features seen in sectioned material is similar to that described in Oreochromis macrocephalus by Shaw and Aronson (1954). These authors found that the embryonic development of O. macrocephalus is not fundamentally different to that described in other cichlids, such as Hemichromis bimaculata (McEwen, 1930, 1940). The development of the cichlid Labeotropheus species (Balon, 1985) is also similar to that of tilapias, but Labeotropheus is a more specialized mouthbrooder than either O. macrocephalus or O. niloticus, having eliminated the defenseless larval period by producing fewer, larger eggs, which produce larger larvae and juveniles. These cichlids, unlike most teleosts, produce ovoid eggs in which the embryo develops at one end. We found some differences in the development of Oreochromis niloticus compared to that of Shaw and Aronson’s description of development in O. macrocephalus, such as the lack of a segmentation cavity in the blastula and lack of ciliation of the posterior part of the gut. Also, Shaw and Aronson (1954) found that the mouth of O. macrocephalus opened before hatching, whereas it did not open until after hatching in O. niloticus. The early cleavage stages are similar to those described in Oreochromis niloticus by Galman and Avtalion (1989) and in zebrafish by Kimmel et al. (1995). However, the cleavage period is completed 2 h after fertilization in zebrafish (Kimmel et al., 1995), but only after about 5 h in O. niloticus (Table 1). Heterogenous cell sizes are found at the 16-cell stage of O. macrocephalus and O. niloticus (Shaw and Aronson 1954; Galman and Avtalion, 1989) and we found variation in the arrangement of cells at this stage. We also found that the horizontal cleavage at the 64-cell stage divides the central cells at the animal pole before those at the periphery. This indicates that metasynchronous division is occurring, which is not found until the early blastula period of zebrafish (Kimmel et al., 1995). Metasynchronous division would result in cell counts which Figures 48 –57 188 C.M. MORRISON ET AL. do not show a geometric progression in numbers during the cleavage and early blastula period. This might explain the cell counts obtained by us and by Shaw and Aronson (1954) in the cleavage stages. A morula stage is followed by a blastula with a segmentation cavity in Oreochromis macrocephalus (Shaw and Aronson, 1954). However, we did not find a segmentation cavity in the blastula of O. niloticus, so it is not possible to distinguish between a morula and a blastula. Kimmel et al. (1995) also found no segmentation cavity in zebrafish and do not distinguish a morula stage. These authors state that a blastula without a cavity should be termed a “stereoblastula.” Blastomeres are loosely connected (Fishelson, 1995) and so may appear to be separated by a small cavity in some eggs. Iwamatsu (1994) described a morula and a blastula stage in the medaka and Galman (1980), Galman and Avtalion (1989), and Lingling and Qianru (1981) described morula formation at 5–7 h after fertilization in Oreochromis niloticus. However, these authors do not describe a segmentation cavity in the blastula and the timing corresponds with the timing of early blastula formation in our study. We found that a late blastula had formed by 91⁄2 h, which is close to the times given by the other authors in Table 1. Figs. 48 –52 (Overleaf.) 148-h larvae. Oreochromis niloticus. Fig. 48. The gallbladder (Ga) is enlarged and surrounded by squamous epithelium. Segmented tubules of the mesonephros (Mes) are forming. I, intestine. H&E. Bouin’s and formalin fixation. Bar 5 200 mm. Fig. 49. The spleen (Sp) is near the pancreas, which consists of an endocrine islet (EnP) and exocrine pancreas (ExP). The swimbladder (Sw) is not inflated. Y, yolk. H&E. Bouin’s and formalin fixation. Bar 5 200 mm. Fig. 50. Teeth (T) are developing on the dorsal and ventral pharyngeal plates. H&E. Bouin’s and formalin fixation. Bar 5 100 mm. Fig. 51. Primordial germ cells (PGC) are present dorsal to the intestine (I), which is lined with columnar cells. H&E. Bouin’s and formalin fixation. Bar 5 100 mm. Figs. 52–57, 172-h larvae. Fig. 52. The retina (Re) of the eye is well-developed and the nasal epithelium (NE) is indented. Gill lamellae (GL) have formed. The branchiostegal membrane (BM) covers the whole branchial cavity. Ot, otocyst; PT, pronephric tubules. H&E. Bouin’s and formalin fixation. Bar 5 400 mm. Fig. 53. Cartilages (Ca) and branchial arteries (BrA) are prominent in the gill arches and lamellae are present on the gills. The pseudobranch (Ps) is composed of several lamellae. Cartilage is present around the otocyst (Ot), as well as in the jaws. H&E. Bouin’s and formalin fixation. Bar 5 100 mm. Fig. 54. The open mouth is bordered by oropharyngeal membranes (OM). The trabeculum cranii (TC), ethmoid (Eth), and Meckel’s (Mec) cartilages are present. There is a basihyal cartilage (BC) supporting the tongue. H&E. Bouin’s and formalin fixation. Bar 5 200 mm. Fig. 55. Goblet cells (GC) are present in the epithelium lining the esophagus, pharyngeal cavity, and the dorsal part of the gill arches. T, tooth. PAS/Alcian blue. Bouin’s and formalin fixation. Bar 5 200 mm. Fig. 56. In the esophagus there are large goblet cells (LGC) and smaller goblet cells (SGC) which stain dark purple-red. There is a muscle layer (ML) in the wall. PAS/Alcian blue. Bouin’s and formalin fixation. Bar 5 100 mm. Fig. 57. Exocrine pancreas (ExP) is present between the loops of the digestive tract, part of which is close to the liver (Li). GC, goblet cell. PAS/Alcian blue. Bouin’s and formalin fixation. Bar 5 100 mm. In our study on sectioned material the YSL is beginning to spread over the yolk 11 and 14 h after fertilization. As described by Kimmel et al. (1995) in zebrafish, this region of the YSL is external to the blastodisc edge and advances across the yolk ahead of the cells of the blastodisc. Other authors (Table 1) also observed gastrulation at this stage. Kimmel et al. (1995) describe the yolk forming a dome at the animal pole at the beginning of gastrulation, which causes thinning of the blastodisc. We did not observe this in the Oreochromis niloticus egg, which has a much larger yolk. In Oreochromis niloticus there is 30 – 40% epiboly at 22 h, corresponding with about one-third epiboly in two other cichlid mouthbrooders by 18 –28 h (Fishelson, 1995). Before it is covered by the YSL during epiboly, the yolk sac is surrounded by the perivitelline pellicle, a noncellular membrane of lamellated gel (Fishelson, 1995). In the zebrafish the gastrula period is defined by Kimmel et al. (1995) as ending when epiboly is complete and the tail bud has formed just dorsal to the site of yolk plug closure. However, in Oreochromis niloticus and O. macrocephalus (Shaw and Aronson, 1954) the yolk is so large that the embryo does not extend to the vegetal pole. The blastoderm therefore migrates beyond the tail bud, delaying yolk plug closure so that segmentation of the embryo begins before epiboly is complete. A separate “neurula” stage is described by Shaw and Aronson (1954), but Kimmel et al. (1995) found that neurulation and segmentation overlapped extensively. We found Kupffer’s vesicle at an early segmentation stage (26 –31 h), as in Fundulus heteroclitus (Brummett and Dumont, 1978). The histology of this structure is similar to that described by these authors. Its function is unknown, although Brummett and Dumont (1978) summarize theories that it may play a nutritive function. Cilia in the roof of Kupffer’s vesicle may circulate yolk partly digested by the YSL, increasing absorption into the lengthening embryo. Shaw and Aronson (1954) reported that the posterior gut of Oreochromis macrocephalus is ciliated and that Kupffer’s vesicle is still present when rhythmical contraction of the heart starts, at the 20 –23 somites stage. However, in O. niloticus we were unable to find Kupffer’s vesicle at the 17somite stage (46 h) and the gut did not appear to be ciliated. Fishelson (1995) described similar timing of the development of the primordial blood capillary system to that found in Oreochromis niloticus in other mouthbrooding cichlids. He was able to clearly distinguish the fine endothelium surrounding the capillaries with transmission electron microscopy, which we could only see in some places using light microscopy. Kimmel et al. (1995) found large blood spaces over broad regions of the yolk ball in zebrafish, which later became restricted to the cardi- DEVELOPMENT OF OREOCHROMIS NILOTICUS 189 Figures 58 – 67 nal veins. This seems to be similar to the situation in O. niloticus. Optic bud formation and brain segmentation occur earlier in our embryos than in those of Galman and Avtalion (1989) and Lingling and Qianru (1981), which might be expected because ours were generally reared at a higher temperature. However, hatching occurred later in our embryos than in those reared by these authors. In the case of Lingling and Qianru (1981) this may be because the rearing tem- 190 C.M. MORRISON ET AL. perature was higher towards hatching. The differences may also have been partly due to different rearing conditions other than that of temperature and to different stocks of Oreochromis niloticus. Primordial germ cells were described in Oreochromis mossambicus just after hatching (Nakamura and Takahashi, 1973). We found them at this stage in O. niloticus, but they were also apparent as early as 46 h after fertilization. Larvae of tilapias bear three pairs of cement glands on the head, which enable substratespawning species to attach themselves to the substrate (Peters, 1965). However Peters (1965) showed that only rudiments of these glands are present in mouthbrooding tilapias such as Oreochromis niloticus 3 or 4 days after fertilization. According to Arnold et al. (1968) the cement glands of O. niloticus produce a small amount of polysaccharide substance, but this is not secreted. We found these glands at the same stage as Peters (1965), on day 4 of embryonic development. The glands were degenerating by day 5 and we did not find them in hatched larvae, presumably because they have no function in mouthbrooders. The term “pharyngula” (Ballard, 1981) is used for the stage when the basic bilateral anatomical pattern of the embryo is laid down. The term comes from the most conspicuous feature of this stage, the Figs. 58 – 65 (Overleaf.) 172-h larvae. Oreochromis niloticus. Fig. 58. The swimbladder (Sw) is lined with columnar epithelium. Ventrally there is a bundle of parallel capillaries, the primordium of the rete mirabile (RM). H&E. Bouin’s and formalin fixation. Bar 5 100 mm. Fig. 59. A pneumatic duct (PD) runs from the swimbladder (Sw) to the digestive tract. DA, dorsal artery. H&E. Bouin’s and formalin fixation. Bar 5 100 mm. Fig. 60. Lymphoid (Ly) and erythroid (Ery) tissue is present around the pronephric tubules (PT). S, somite; Y, yolk; Me, melanophore. H&E. Bouin’s and formalin fixation. Bar 5 100 mm. Fig. 61. The spleen (Sp) contains lymphoid and erythroid tissue. The gallbladder (Ga) is expanded and lined with squamous epithelium. An endocrine pancreatic islet (EnP) is partially surrounded by exocrine pancreas (ExP). Li, liver. H&E. Bouin’s and formalin fixation. Bar 5 100 mm. Fig. 62. A neuromast (Ne) is situated in the stratified epithelium on the dorsal surface of the head, near the anterior end of the larva. H&E. Bouin’s and formalin fixation. Bar 5 40 mm. Fig. 63. Four hypural cartilages (HyC) and two less modified hemal arches (HA) support fin rays (FR) in the caudal fin. The urostyle (U) is upturned posteriorly. S, somite. PAS/Alcian blue. Bouin’s and formalin fixation. Bar 5 200 mm. Fig. 64. Neural (NA) and hemal (HA) arches extend from the notochord (No). Pterygiophores (Pt) are beginning to form in the base of the ventral fin-fold, between the hemal spines (HS) which extend from the hemal arches. Bone (B) is being laid down at the periphery of the notochord. PAS/ Alcian blue. Bouin’s and formalin fixation. Bar 5 200 mm. Fig. 65. The sinus venosus (SV) of the heart leads to a thin-walled atrium (A), separated from the ventricle (V), which has trabeculae (Tr) in the wall, by an atrioventricular valve (AV). The thick-walled bulbus arteriosus (BA) leads to the ventral aorta and the branchial arteries. H&E. Bouin’s and formalin fixation. Bar 5 200 mm. Fig. 66. Thyroid follicles (TF) are present around the ventral aorta (VA). H&E. Bouin’s and formalin fixation. Bar 5 100 mm. Fig. 67. 196-h larvae. There are taste buds (TB) as well as large (LGC) and small (SGC) goblet cells in the pharyngeal epithelium. Teeth (T) are present in the pharyngeal plates. PAS/ Alcian blue. Bouin’s and formalin fixation. Bar 5 70 mm. pharyngeal segments. During this stage the head shortens rapidly as the distance between the otic capsule and eye decreases (Kimmel et al., 1995). This explains the difference found in embryos from two different clutches of eggs 76 h after fertilization. Unicellular hatching glands are a prominent feature of the pharyngula period in our study and also in zebrafish (Kimmel et al., 1995). Similar glands, with basal nuclei and prominent granules that stain with eosin and orange G, have been found in many species of teleost larvae. They are larger than other cells and can be differentiated from mucous glands that stain with mucicarmine (Yamagami, 1988). However, they differ in size in each species (Ishida, 1985) and their distribution in the larva also varies. Hatching glands have been shown to originate from the anterior end of the hypoblast, the polster, in zebrafish, salmon, and medaka (Inohaya et al., 1997). However, these cells migrate to different regions of the embryo, where they are covered by a single layer of peridermal or epithelial cells. According to the species, hatching glands may be found on the anterior part of the head and body, on the yolk sac, or lining the pharynx (Yanai et al., 1956; Willemse and Denuce, 1973; Rosenthal and Iwai, 1979; Schoots et al., 1982; Ishida, 1985; Luczynski et al., 1986; Inohaya et al., 1997). They were found both on the head and in the buccal cavity of Coregoninae and salmonid embryos (Yokoya and Ebina, 1976; Ishida, 1985; Luczynski et al., 1986; Luczynski and Ostaszewska, 1991; Inohaya et al., 1997). The distribution of hatching glands is more extensive in Oreochromis niloticus than in most of these species, since glands were found on the head, on the yolk sac around the attachment to the embryo, and on the dorsal and ventral surface of the tail. The hatching glands of the medaka Oryzias latipes are unusual in that they were found only in the wall of the mouth and pharynx (Ishida, 1944; Yamagami, 1988). In halibut larvae the hatching glands form a narrow collar and the egg envelope is digested only in this region, resulting in the envelope splitting into a rigid top and bottom half (Helvik et al., 1991). However, in most other larval species the effect on the egg envelope is more diffuse, resulting in the formation of thin egg envelope “ghosts,” as described in salmon (Helvik et al., 1991), and also in our study. According to Ishida (1985), hatching gland cell differentiation starts in the 10 –12 somite embryo of medaka and secretory granules form in these cells in the 15-somite embryo. However, we were unable to find hatching glands in Oreochromis niloticus at the 11-somite stage. They were not seen until 76 h, when the larva is fully segmented and pigment formation has just started in the eye. Most of the authors listed above found hatching glands at this time, with maximum development just before hatching. DEVELOPMENT OF OREOCHROMIS NILOTICUS 191 Figs. 68 –72, 196-h larvae. Oreochromis niloticus. Fig. 68. There is a constriction (Co), the pyloric sphincter, between the small stomach (St) and the intestine (I), both of which are lined with columnar epithelium. The esophagus (Es) is lined with stratified epithelium and the inflated swimbladder (Sw) with squamous epithelium. H&E. Bouin’s and formalin fixation. Bar 5 200 mm. Fig. 69. The digestive tract contains a food organism (FO), and the gallbladder (Ga) contains flocculent material. There are numerous goblet cells (GC) in the intestine (I). Sp, spleen; ExP, exocrine pancreas. PAS/Alcian blue. Bouin’s and formalin fixation. Bar 5 200 mm. Fig. 70. The mesonephric duct leads to a bladder (UB) which opens into the cloaca (Cl) near the opening of the posterior digestive tract (DT). There is a small gonad (Go) between the digestive tract and mesonephros (Mes). H&E. Bouin’s and formalin fixation. Bar 5 200 mm. Fig. 71. The otocyst consists of several chambers surrounded by cartilage (Ca). Near a macula (Ma) are the remains of an otolith (Oto). The thymus gland (TG) is prominent. H&E. Bouin’s and formalin fixation. Bar 5 200 mm. Fig. 72. The pigment layer (PL) and plexiform layers (PIL) are prominent and the external segments of the photoreceptors are elongate in the whole retina. L, lens. H&E. Bouin’s and formalin fixation. Bar 5 200 mm. Insulin-immunopositive cells were first found in a pancreatic islet 76 h after fertilization in Oreochromis niloticus, whereas these have not been reported until hatching in other teleosts (Berwert et al., 1995). Fishelson (1966) found that the alimentary tract and associated organs, sensory organs, fin, and pigmentation develop more rapidly in the substratespawner Tilapia tholloni than in the mouthbrooders Oreochromis macrocephalus and O. niloticus. The TABLE 1. Comparison of timing of some developmental features of Oreochromis niloticus Hours after fertilization (rearing temperature) Developmental features Zygote period Protoplasm bulges Cleavage period 2 cells 4–8 cells 16 cells 32–64 cells Blastula period Morula or early blastula Blastula Gastrula period Early gastrula 30–40% epiboly, embryonic shield Segmentation period Somite formation Optic primordia Brain segmentation Pharyngula period Otic vesicles Heart beat Retinal pigment Hatching period Hatching Early larval period Gill cover starts to form Mouth open, jaw movements Teeth Swimbladder inflates, swimming Active feeding Present study (27–29°C) 1.5 Galman, 1980 (26–27°C) Galman and Avtalion, 1989 (24–26°C) Rana, 1990 (28°C) Stage Lingling and Qianru, 1981 (25–29°C) Shaw and Aronson, 1954 Nussbaum and Chervinsky, 1968 Galman, 1980 Galman and Avtalion, 1989 0.5 1 1 2 1 2 2.5–3 4 4.5–5 2 3–4 5 6 2 3–4 5 6 3 2 3–4 5 6 5.5 7 7 4 6–8 Rana, 1990 Lingling and Qianru, 1981 1 0–1 5 1–5 2–3.5 4 5.5 5.5 7 5.5–10.5 5.5–14 8 10.5–12 10 8 8 8 5 8–9 7 2–8 22 22 11 15 24 24 10 14 11 28 9 10–11 9 10–11 6 7 10 10 8 9 2–9 3–2 26–30 31 46 30 30 30 40 52 58 30 30 37.5 38 40 12 13 14 12 13 14 9 9 9 11 12 13 10 9 4–2 5–1 5–2 76 50 76 48 60 72 65 72 65 40 52.5 72 15 15 18 15 15 19 11 12 13 14 15 14 11 100–125 100 72 90–120 79 24 20 15 15 12 7 124 147 150 156 72 98 127 127 20 21 17 22 15 18 17 prelarval " 148 196 228 164 180 223 20 21 " " 196 240 256 223 24 " 1.5–2 2–3 4 4–4.5 2–3 4 5 6 1–2 2 3–4 5 6 2–1 2–2, 2–3 2–4 2–5, 2–6 2–7 5–2 5–4 The staging systems of these authors are given as well as that of Oreochromis macrocephalus (Shaw and Aronson, 1954). The timing given by Lingling and Qianru (1981) has been rounded off to the nearest half-hour. Timing has not been given for Shaw and Aronson (1954) or Nussbaum and Chervinsky (1968), who both used the same system up to stage 15. Shaw and Aronson only gave times for the early cell divisions. Nussbaum and Chervinsky (1968) gave times for a few stages after Stage 15, but there was not enough detail to correlate these with other accounts. DEVELOPMENT OF OREOCHROMIS NILOTICUS musculature and axial skeleton also develop more rapidly up to 9 –11 days after fertilization, but then the mouthbrooders develop more quickly. We found that caudal fin rays first start to develop about 7 days after fertilization, whereas Fishelson (1966) found them in O. niloticus at 5– 6 days. Fishelson (1966) described the first fin rays in the dorsal and anal fins at 6 –7 days, but we did not find them in our study, except in one clutch 9 days after fertilization. This is more similar to Labeotropheus sp. (Balon 1985), where no fin rays are described at about 9 days, although the dorsal and anal fin are beginning to differentiate. Lingling and Qianru (1981) found an increase in pigmentation on the body of Oreochromis niloticus larvae as they develop, as described in our account. At hatching these authors found, as we did, that the branchiostegal membrane only covers the first pair of gill arches, but then grows posteriorly to cover the whole gill chamber. The pseudobranch of Tilapia tholloni has several lamellae at 8 days (Fishelson, 1966), which is similar to the situation in O. niloticus. We found that swimbladder inflation does not occur until day 8. Fishelson (1966) also found swimbladder inflation at 8 –9 days in both Oreochromis niloticus and O. mossambicus, and Lingling and Qianru (1981) found inflation in O. niloticus at 9 days. Swimbladder inflation occurs 4 –5 days after fertilization in Tilapia tholloni (Fishelson, 1966). The uninflated swimbladder is also lined by columnar epithelium in O. mossambicus (Doroshev and Cornacchia, 1979) and T. tholloni (Fishelson, 1966). As in our study, swimbladder inflation occurs at about the same time as the start of feeding activity in other tilapias (Doroshev and Cornacchia, 1979; Lingling and Qianru 1981; Takemura, 1996). There is some controversy concerning swimbladder inflation in physoclistous larvae. In some the swimbladder is connected to the digestive tract by a pneumatic duct during larval development and swimbladder inflation may be initiated by gulping air that passes via the pneumatic duct to the swimbladder. The duct degenerates after swimbladder inflation (Doroshev et al., 1981). However, Doroshev et al. (1981) suggest that inflation may be by gas secretion into the swimbladder in other species, such as Oreochromis mossambicus, in which they found no pneumatic duct. We found a patent pneumatic duct just before swimbladder inflation in O. niloticus, and a pneumatic duct was also reported by Shaw and Aronson (1954) in O. macrocephalus, so there may be no fundamental difference in the mechanism of swimbladder inflation between tilapias and other species of fish. Lymphoid tissue is found in the thymus, headkidney, and spleen of adult Oreochromis mossambicus (Sailendri and Muthukkaruppan, 1975). We found that the thymus had developed in the same location as in the adult O. mossambicus in the 100-h embryo of O. niloticus. The superficial location of the 193 thymus in the pharyngeal cavity, in contact with the water current entering through the branchial cleft, is well shown in Galman and Avtalion (1989, fig. 8). This location may be associated with defense against pathogenic organisms (Tatner and Manning, 1982). The pronephros lies close to the thymus and continuity between them has been noted in some fish (Manning, 1981). A distinct spleen is found close to the pancreas, as in the adult O. mossambicus, in the day 7 larva of O. niloticus. As in O. niloticus, the spleen was found to be predominantly erythroid and slow in becoming lymphocytic in larval rainbow trout, Salmo gairdneri (Grace and Manning, 1980). Head kidney lymphoid and erythroid tissue have developed around the pronephros of O. niloticus by day 8. The head kidney is an important site of hemopoiesis in adult teleosts, where the pronephros degenerates and is replaced by hemopoietic tissue (Al-Adami and Kuntz, 1977). In Oreochromis mossambicus, Jirge (1971) noted positive PAS and Alcian blue (pH 5 2.5) staining in the pronephric tubules of 5 mm larvae, which become restricted to the luminal lining of 8 –12 mm larvae. This was similar to our results in day 9 larvae, which were about 7 mm long after fixation. A PAS-positive lining was also reported in the proximal segment of the pronephros of newly hatched larvae of O. mossambicus by Hwang and Su-Mei (1989). Lingling and Qianru (1981) described a periodically contracting urinary bladder 3 days after hatching, which would correspond with the enlarged urinary bladder found 9 days after fertilization in our study. The ciliated region we found posterior to the esophagus of Oreochromis niloticus in the hatching period, before the mouth and anus open at the beginning of the early larval period, has been described in larvae of other teleosts and may help to circulate the gut contents (Morrison et al., 1997). The digestive tract of mouthbrooding cichlids develops more slowly than in bottom spawners (Fishelson, 1966). For example, goblet cells were found in the esophagus of Tilapia tholloni at 4 days, whereas we did not find them in O. niloticus until 8 days. We found that the digestive tract of the latter is similar to that of the adult (Morrison and Wright, 1999) in that two goblet cell types were found in the esophagus, there was a small sac-like stomach with apical PAS-positive staining, and goblet cells were found in the intestine. However, the stomach does not contain the multicellular gastric glands found in the adult and the region from the esophagus to the intestine “bypassing” the stomach is not present. Development of goblet cells occurs the day before we observed food organisms in the digestive tract of O. niloticus. Lingling and Qianru (1981) found that the mouth and anus are open at this stage (3 days after hatching), and that the intestine is discharging its contents. We found that the liver develops between the YSL surrounding the yolk and the digestive tract 5 days 194 C.M. MORRISON ET AL. after fertilization, which is comparable to the 4 –5 days described by Fishelson (1995). He found yolk granules in the hepatocytes and suggested that the liver may aid in yolk digestion. We found no yolk granules in the hepatocytes, but the close association of the liver with the YSL indicates that it is important in processing yolk partially broken down by the YSL. Since the liver is well-vascularized, the products of yolk digestion could then be circulated to the gut and other organs. We found elements of the exocrine pancreas near the digestive tract of the early larval stages of Oreochromis niloticus but none in the liver, although most of it is associated with the liver in the adult (Yang et al., 1999). Eye development in Oreochromis niloticus is similar to that of Tilapia leucosticta (Grün, 1975), which is also a mouthbrooding tilapia, except that the photoreceptor nuclei start to form a double layer earlier in O. niloticus, at 76 h. As in other teleosts (Ali and Anctil, 1976), this double row consists of an outer layer of cone nuclei and an inner layer of smaller vitread rod nuclei. Shaw and Aronson (1954) found two otoliths in the otocyst before hatching, whereas we did not find remains of otoliths until day 9. However, the sensory maculae, which would support the otoliths, are present before hatching, so the otoliths probably dissolved in the fixative that we used. We found that, as in other fish, the ectoderm of the hatching larva consists of only two layers. According to Bouvet (1976) there is an outer peridermal layer and an inner layer that will give rise to the epidermis. The peridermal layer disintegrates as the epidermis develops. The epidermis becomes multilayered in the larva, which is completely covered by a stratified epidermis by day 9. ACKNOWLEDGMENTS The authors thank C. Isenor, K. Lovett, B. Conrad, and J. Tam for expert assistance in preparing the stained paraffin sections used in this study; M. 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