Introduction
The species Zungaro jahu belongs to the order Siluriformes. It is the largest fish in the Paraná-Paraguay and Uruguay River basins, reaching up to 144 cm in length and 150 kg in weight (Agostinho et al., Reference Agostinho, Gomes, Suzuki, Júlio, Carolsfield, Harvey, Ross and Baer2004). First maturation occurs when adults are 70 cm long and spawning takes place between December and February, with animals migrating upriver during the reproductive season (Vaz et al., Reference Vaz, Torquato and Barbosa2000).
Induced reproduction and incubation techniques are well understood in different fish species (Shardo, Reference Shardo1995). However, few studies have analysed the basic aspects of the embryonic period from fertilization to hatching. A good understanding of the initial phases of the life cycle is of extreme importance in taxonomy and species ecology studies (Sanches et al., Reference Sanches, Nakatani and Bialetzki1999).
Embryonic development is a complex process that begins when the oocyte is fertilized by the spermatozoon in the micropyle and consists in the reorganization of the egg components (Depêche & Billard, Reference Depêche and Billard1994). Embryo studies are of great importance in fish reproductive biology (Coward et al., Reference Coward, Bromage, Hibbitt and Parrington2002) and are essential in a great variety of research, including species ontogeny and biotechnology, where they can be used as bioindicators of environmental quality, and to determine the effects of toxic substances on aquatic fauna (Flores et al., Reference Flores, Araiza and Valle2002; Botero et al., Reference Botero, Fresneda, Montoya and Ángel2004; Ninhaus-Silveira et al., Reference Ninhaus-Silveira, Foresti and Azevedo2006). However, in order to be fully understood, embryonic studies must be combined with topological analysis of fertilized eggs (Depêche & Billard, Reference Depêche and Billard1994).
Embryonic development has been studied in several fish species and is often classified into stages. Currently, the following native Siluriformes species have been studied: Rhamdia hilarii (Godinho et al., Reference Godinho, Fenerich and Narahara1978), Rhamdia sapo (Cussac et al., Reference Cussac, Matkovic and Maggese1985; Matkovic et al., Reference Matkovic, Cussac and Cukier1985); Pseudoplatystoma coruscans (Cardoso et al., Reference Cardoso, Alves, Ferreira and Godinho1995; Landines et al., Reference Landines, Senhorini, Sanabria and Urbinati2003), and Pimelodus maculatus (Luz et al., Reference Luz, Reynalte-Tataje, Ferreira and Zaniboni-Filho2001). However, in the majority of studies, embryonic development was analysed only by stereomicroscopy, with only a few studies using light microscopy.
Data on the biology and ecology of Z. jahu are scarce, with most data being related to nutrition or parasitology (Agostinho et al., Reference Agostinho, Gomes, Suzuki, Júlio, Carolsfield, Harvey, Ross and Baer2004). Successful rearing of fish depends on the understanding of the species’ initial biology, including fertilization and embryonic development characteristics, which directly influence fertilization and hatching rates (Matkovic et al., Reference Matkovic, Cussac and Cukier1985).
The Z. jahu is a vulnerable species with a reduced natural stock and is in danger of extinction due to changes and destruction of its habitat. Thus, it has been included in the List of Fauna Species in Danger of Extinction in the State of Minas Gerais under the category ‘Critically Endangered’ (Minas Gerais, 2008) and in the List of Vertebrate and Invertebrate Species of the Wild Fauna in Danger of Extinction in the State of São Paulo (São Paulo, 2014). The Z. jahu has not been included in the national list of endangered species in Brazil because it can also be found in the Paraguay River basin where, in spite of overfishing, it cannot be considered endangered due to the relatively intact environmental state of the Pantanal ecosystem (Machado et al., Reference Machado, Drummond and Paglia2008).
The aim of this study was to structurally and ultra-structurally identify the events that take place during the embryonic development of Z. jahu in order to better understand its biology and reproduction, and thus improve husbandry practices in this species.
Materials and methods
Collection and fertilization
Broodstock of the species Z. jahu Ihering, 1898 (= Paulicea luetkeni Steindachner, 1875) from the fishery Usina Hidrelétrica Engenheiro Souza Dias (Companhia Energética do Estado de São Paulo, Castilho, SP, Brazil) were subjected to induced reproduction and dry fertilization during the reproductive period of the species, from December to February, according to the techniques by Woynarovich & Horváth (Reference Woynarovich and Horváth1983).
The females (n = 5) received two injections of common carp (Cyprinus carpio) pituitary extract (0.8 and 5.0 mg/kg body weight) in the coelomic cavity at 12-h intervals. The males (n = 8) received a single dose (2.5 mg/kg body weight) concomitantly to the females’ second dose.
Approximately 12 h after the effect of the hormone, the gametes were obtained by manual craniocaudal massage of the abdomen and subsequently mixed and hydrated.
After extrusion, semen and oocytes were gently homogenized and water was added to the mixture for gamete activation and egg hydration. The eggs were washed under running water to remove excess semen and transferred to 60-litre incubators with a continuous water flow, where they remained for the whole experimental procedure. The following physical–chemical characteristics of the water were recorded: temperature and dissolved oxygen (mg/L), by YSI 550 A equipment, pH, conductivity (μS/cm), by YSI 63 equipment, and alkalinity (mg/L) ammonia (μg/L) by the Goltermann et al. (Reference Goltermann, Clymo and Ohnstad1978) method.
Samples were collected at oocyte extrusion; when oocytes and semen were mixed together (fertilization–time zero); at 10 and 30 seconds post-fertilization; at 1, 2, 5, 7, 10, 15, 20, 30, and 45 min post-fertilization (mpf), then every 15 min up to 2 h post-fertilization, and at every hour until hatching. Fertilization rate (developing eggs/total eggs collected in 1 g) ×100, was determined 5 h after the male and female gametes were mixed (fertilization time).
Twenty-five oocytes/eggs/larvae were collected at each sampling time. These samples were fixed in modified Karnovsky solution (2.5% glutaraldehyde + 2.5% paraformaldehyde) for 24 h, washed in 0.1 M sodium cacodylate buffer solution (pH 7.4) and stored in 70% alcohol for further analysis at the School of Agrarian Sciences and Veterinary Medicine (FCAV), UNESP – Univ Estadual Paulista, Campus Jaboticabal, Department of Morphology and Animal Physiology, Laboratory of Histology and Animal Embryology.
Stereomicroscopy and morphometry
Samples were analysed and photographed using LEICA DFC 280 photomicrography equipment attached to a LEICA MZ8 stereomicroscope. Linear measures (oocyte/egg/embryo diameter and larval total length) were obtained using the programme IM 50-LEICA. Twenty oocytes/eggs/larvae were measured for each collection time.
Light microscopy (LM)
Samples were prepared and embedded in historesin (kit Leica Historesin®) according to the manufacture's recommendations and the histological sections (3 μm) were stained with haematoxylin-phloxine (Tolosa et al., Reference Tolosa, Behmer and Freitas-Neto2003). Analysis of slides and photodocumentation were performed using Leica DFC 280 photomicrography equipment attached to a LEICA DM 2500 photomicroscope.
Scanning electron microscopy (SEM)
Samples were post-fixed in 1% osmium tetroxide solution for 2 h, washed in 0.1 M sodium cacodylate buffer, and dehydrated in a graded series of ethanol (30, 50, 70, 80, 90, and 95%) for 5 min each. Subsequently, the slides were dried to critical point in liquid CO2 (BAL-TEC dryer), mounted onto a copper grid, coated in palladium/gold (Desk II Denton Vacuum equipment), and analysed under a scanning electron microscope (JEOL-JSM 5410).
Transmission electron microscopy (TEM)
Oocytes and eggs collected during the first hour of development were analysed by TEM. These samples were post-fixed in 2% osmium tetroxide for 2 h and dehydrated in a graded series of ethanol (similarly to SEM). Samples were infiltrated with a mixture of araldite and acetone and subsequently embedded in araldite. Semi-fine sections (0.5 µm) were obtained and stained with 1% toluidine blue in saturated boric acid for selection and analysis of the areas of interest. The best sections were selected and ultra-fine sections (60 nm) obtained using a diamond blade. These sections were contrasted, analysed, and electron micrographed with a transmission electron microscope (JEOL – JEM 1010).
Results
The embryonic period of Z. jahu comprised the period from fertilization to hatching and lasted for 780 mpf (13 h) at 29.4 ± 1.5°C. Fertilization rate was 95%. The physical–chemical characteristics of the water from the incubators were: ammonia = 11.13 μg/l, pH = 8.14, conductivity = 34 μS/cm, dissolved oxygen = 7.26 mg/l, and alkalinity = 21 mg/l.
The embryonic development of Z. jahu was divided into seven stages and classified according to Faustino et al. (Reference Faustino, Nakaghi and Neumann2011 and Reference Faustino, Makino, Neumann and Nakaghi2015) into: zygote, cleavage, morula, blastula, gastrula, organogenesis, and hatching. The different stages and their corresponding characteristics are detailed in Table 1. The main characteristics of each stage are illustrated in Figures 1 b–j and 2 a–f.
Table 1 Characteristics of the different stages of embryonic development in Zungaro jahu at 29.4°C
mpf: minutes post-fertilization.
Figure 1 Photomicrography of oocytes (a) and eggs (b–j) of Z. jahu. (a) Oocytes (extrusion). (b) Zygote phase (0–15 mpf), development of the egg cell or zygote (*). (c–h) Cleavage phase: (c) presence of two blastomeres (20 mpf); (d) four blastomeres (30 mpf); (e) eight blastomeres (45 mpf); (f) 16 blastomeres (60 mpf); (g) 32 blastomeres (60 mpf); (h) 64 blastomeres (75 mpf). (i) Morula phase (90 mpf). (j) Blastula phase (120 mpf). mpf, minutes post-fertilization; ps, perivitelline space; y, yolk. Bars represent 100 μm.
Figure 2 Photomicrography of eggs (a–c), embryos (d, e) and hatched larvae (f) of Z. jahu. (a–c) Gastrula phase: (a) 25% epiboly (180 mpf); (b) 50% epiboly (240 mpf); (c) formation of yolk plug (360 mpf). (d, e) Organogenesis phase: (d) differentiation of cephalic and caudal regions (420 mpf); (e) caudal elongation of the embryo, presence of somites, optic (arrowhead) and Kupffer vesicles (arrow), and start of pigmentation (600 mpf). (f) Hatching phase (780 mpf), detail of primitive gut (circle) and embryonic fin (broken arrow). ca, caudal region; ce, cephalic region; yp, yolk plug. Bars represent 100 μm.
The term ‘oocyte’ refered to the female gamete before fertilization while ‘egg’ comprised the stages from zygote (fertilization time) to the end of the gastrula stage, when the embryonic axis was formed (organogenesis stage) and the term ‘embryo’ used forthwith. The denomination ‘larvae’ was used from the time of chorion rupture.
Oocytes and zygotes (extrusion, 0–15 mpf)
At the time of extrusion, oocytes showed a mean diameter of 1.38 ± 0.06 mm, which remained virtually unaltered until organogenesis. The oocytes were spherical, yellowish, with a large perivitelline space (Figures 1 a and 3 b), and a gelatinous layer over the chorion.
Figure 3 Photomicrography of oocytes (extrusion; a–c) and eggs (d–f) of Z. jahu. (a) Detail of the micropyle. (b) Oocyte during fertilization. (c) Cortical cytoplasm with cortical alveoli distributed in the periphery, chorion with a thin gelatinous layer (arrow). (d) Development of the egg cell (0–15 mpf). (e) Two blastomeres (20 mpf). (f) Eight blastomeres (45 mpf). ap, animal pole; ca, cortical alveolus; cc, cortical cytoplasm; m, micropyle; ps, perivitelline space; vp, vegetative pole; y, yolk. Bars: (a) represent 20 μm; (b, d–f) represent 100 μm; (c) represent 30 μm.
Each oocyte showed a single micropyle, a small opening where the spermatozoon penetrated during fertilization (Figure 4 a). The micropylar apparatus consisted of two vestibules and a short micropylar canal (Figures 3 a and 4 a).
Figure 4 Scanning (a, d–f) and transmission electron micrography (b, c) of oocytes (extrusion, a–d) and eggs (0–15 mpf; e, f) of Z. jahu. (a) Oocyte, presence of a single micropyle (arrow and inset). (b) Cortical cytoplasm with cortical alveoli distributed in the periphery, chorion with pores. (c) Cortical cytoplasm with mitochondria (arrowhead) and ruptured cortical alveoli. (d) Presence of multiple spermatozoa at the entrance of the oocyte micropyle. (e) Two blastomeres (*). (f) Four blastomeres. ca, cortical alveolus; cc, cortical cytoplasm; ch, chorion. Bars: (a) represent 100 μm; (b, e, f) represent 10 μm; inset, (d) represent 2 μm.
Transversal sectioning (TEM) revealed a perforated chorion containing several pores responsible for water intake and exchange of substances necessary for embryonic development (Figure 4 b), as well as the presence of a thin gelatinous layer (Figure 3 c).
Under LM and TEM, it was possible to observe several cortical alveoli of various sizes distributed throughout the whole periphery of the oocyte, forming a thin basophilic layer (ML) that constituted the cortical cytoplasm (Figures 3 c and 4 c).
The zygote stage comprised the period from fertilization to the development of the animal pole, also known as the egg cell.
Under LM, at the time of fertilization, it was possible to observe a cytoplasmic movement towards the egg's micropyle region, characterizing the animal pole; which was formed by the union between the male and female pronucleus and the dislodged cytoplasm. The animal pole was basophilic and homogeneous (Figure 3 d), while the vegetative pole consisted of a large amount of yolk.
The alveoli initially seen in the oocytes ruptured after fertilization, contributing to the elevation of the chorion and consequent increase in the perivitelline space. By 5 mpf, the cortical alveoli were absent. The development of the perivitelline space and its increase resulted in the separation of the chorion from the egg membrane. It was also observed that the cortical cytoplasm contained mitochondria, ribosomes, and secretion vesicles (Figure 4 c).
Under SEM, several spermatozoa could be seen at the entrance of the micropyle from the time of fertilization up to 1 mpf (Figure 4 d). By 30 s post-fertilization, some eggs had been fertilized and were protected from polyspermia by the development of a fertilization cone, a round structure that projected from the micropyle and obstructed its opening.
Cleavage stage (20–75 mpf)
This stage began with the first cleavage, on the vertical plan of the animal pole, which divided the blastodisc into two cells (blastomeres) of equal size (Figures 1 c, 3 e and 4 e). Subsequently, another division took place in the vertical plane, perpendicular to the first, generating four blastomeres (Figures 1 d and 3 f). The third division was vertical and parallel to the first, resulting in eight blastomeres arranged 4 × 2 (Figures 1 e, 4 f and 5 a). The fourth division was vertical and parallel to the second, forming 16 blastomeres arranged 4 × 4 (Figures 1 f and 5 b). The fifth division was vertical and parallel to the first, originating 32 blastomeres arranged 4 × 8 (Figures 1 g, 5 c and 6 a) and the sixth cleavage was on the horizontal plane, resulting in two layers of cells and 64 blastomeres (Figures 1 h and 6 b).
Figure 5 Scanning electron micrography of eggs (a–d), embryo (e) and larva (f) of Z. jahu. (a–c) Presence of eight, 16, 32 blastomeres (45, 60, 75 mpf), respectively. (d) Morula phase (90 mpf). (e) Differentiation of the cephalic and caudal regions (420 mpf). (f) Hatched larva with embryonic fins (780 mpf) (broken arrow); inset highlighting primordial barbels (*), oral cavity (white open arrow), respiratory cavity (arrow) and nostril region (white arrowhead). ca, caudal region; ce, cephalic region. Bars: a–e, detail represent 100 μm, f represent 500 μm.
Figure 6 Photomicrography of the eggs of Z. jahu. (a) Thirty-two blastomeres (60 mpf). (b) Sixty-four blastomeres (75 mpf). (c) Morula phase (90 mpf), with yolk syncytial layer (arrow). (d) Large number of mitosis (90 mpf) (arrowhead). (e, f) Gastrula phase: (e) highlighting the epiboly movement with the development of epiblast and hypoblast (300 mpf); (f) presence of yolk plug (360 mpf). bla, blastocele; e, epiblast; h, hypoblast; yp, yolk plug. Bars: (a–c, e, f) represent 100 μm, (d) represent 20 μm.
As the egg developed, the number of blastomeres increased while their size decreased. It was also noted that small globules of yolk penetrated the area of the blastomeres in a fragmented way (Figure 6 a).
Morula stage (90–105 mpf)
This stage was characterized by the presence of more than 64 blastomeres in the eggs. The final phase of the morula stage was determined when the blastomeres formed a cellular mass shaped like a half berry (Figures 1 i, 5 d and 6 c). At this stage it was possible to observe the development of the yolk syncytial layer, known as a periblast (Figure 6 c).
Blastula stage (120 mpf)
This stage was characterized by intense cellular division, with no distinction between the boundaries of the blastomeres. There was the formation of the blastoderm, a dome-shaped structure over the yolk, and the appearance of the blastocele, characterized as a large space between the yolk and the blastomeres (Figures 1 j and 6 d).
Gastrula stage (180–360 mpf)
The gastrula stage was defined by the start of the epiboly movement in the eggs, the migration of the embryonic cells from the animal to the vegetative pole (Figure 2 a). Once 50% of the yolk was covered, the involution movement began. It projected below and in the opposite direction to the epiboly movement of the blastoderm, generating the epiblast and hypoblast (Figures 2 b and 6 e). This stage ended with the end of the epiboly, characterized by the complete coverage of the yolk by the embryonic cells, forming the vitelline plug or blastopore (Figures 2 c and 6 f).
Organogenesis stage (420–720 mpf)
This stage began with the differentiation of the cephalic and caudal regions of the embryo (Figures 2 d, 5 e and 7 a), followed by the appearance of somites (somitogenesis), notochord, otic vesicle, and Kupffer vesicle (Figures 2 e and 7 b). Subsequently, there was elongation of the embryo by the cephalo-caudal axis, differentiation of large round nuclei myoblasts into flat nuclei myomeres (Figure 7 c), and pigmentation of the body and yolk. The anterior region of the neural tube expanded, forming the prosencephalon and mesencephalon, and the optic vesicle was evident (Figure 7 d). The embryo tail became free and began to move, and would later cause the rupture of the chorion.
Figure 7 Photomicrography of embryos (a–d) and larva (e) of Z. jahu. (a) Differentiation of the cephalic and caudal regions (420 mpf). (b) Elongation of the embryo by the cephalo-caudal axis (600 mpf). (c) Differentiation of myomeres (arrowhead). (d) Development of the nervous system and optical vesicle (720 mpf). (e) Hatched larva (780 mpf. ca, caudal region; ce, cephalic region; me, mesencephalus; no, notochord; op, optic vesicle; ot, otic vesicle; pr, prosencephalon; so, somites; y, yolk. Bars: (a, b, e) represent 100 μm; (c) represent 20 μm; (d) represent 50 μm.
Hatching stage (780 mpf)
This stage was characterized by the total rupture of the chorion and free swimming of the larvae (Figures 2 f, 5 f and 7 e). Under SEM, contrast revealed the primordium of the first pair of maxillary barbels, oral cavity, developing branchiae, olfactory organ with few cilia, embryonic membrane covering the whole caudal region, and large yolk sack (Figure 5 f). Under a stereomicroscope, it was possible to observe a rudimentary digestive system in the mid-posterior region of the larvae (Figure 2 f), and LM revealed the outline of the heart in the region anterior to the yolk sac (Figure 7 e).
Discussion
The physical–chemical parameters of the water from the incubators, where the embryonic development of Z. jahu took place, were considered good for the development of fresh water fish (Sipaúba-Tavares, Reference Sipaúba-Tavares1995).
According to Vazzoler (Reference Vazzoler1996), the fecundity and diameter of fish oocytes can vary and show inter- and intra-specific variations between reproductive periods. Furthermore, these characteristics are associated with reproductive behaviour; in which migratory species with complete spawning, external fertilization, and no parental care produce smaller oocytes and have greater fecundity rates. This is the case for Z. jahu, whose mean diameter of the oocytes, eggs and embryos are considered small (1.38 ± 0.06 mm) when compared with species of no migratory fish. Oocyte diameter also affects incubation time, with species that produce smaller oocytes having shorter incubation periods than those that have large oocytes, such as the majority of sedentary species (Sargent et al., Reference Sargent, Taylor and Gross1987).
Z. jahu produced free non-adhesive oocytes with a chorion surrounded by a thin gelatinous layer. According to Rizzo et al. (Reference Rizzo, Sato, Barreto and Godinho2002), this layer is made of an entanglement of delicate fibrils and is often found in Siluriformes. It does not seem to be related to the adhesiveness of the eggs, as it has been reported in both adhesive and non-adhesive fish eggs. Furthermore, in species that do not provide parental care, this gelatinous layer enables oocytes and eggs to adhere to substrates, thus protecting them from water currents (Rizzo et al., Reference Rizzo, Sato, Barreto and Godinho2002). In species with parental care, this facilitates parental behaviour (Melo et al., Reference Melo, Arantes, Sato, Santos, Rizzo and Bazzoli2011). This layer has been reported by Faustino et al. (Reference Faustino, Nakaghi, Marques, Makino and Senhorini2007) and Perini et al. (Reference Perini, Sato, Rizzo and Bazzoli2009) in P. corruscans × P. fasciatum hybrid and in Rhinelepis aspera, respectively.
The micropylar apparatus of the oocytes of Z. jahu had two vestibules, a smooth surface, and a short and narrow micropylar canal. According to Rizzo et al. (Reference Rizzo, Sato, Barreto and Godinho2002) and Ganeco & Nakaghi (Reference Ganeco and Nakaghi2003), the micropyle is a concave area located in the chorion, consisting of a continuous vestibule with an internal canal that narrows towards the plasmatic membrane of the egg. Rizzo & Bazzoli (Reference Rizzo and Bazzoli1993) reported that the funnel-shaped micropyle is present in the majority of teleostei and allows the passage of a single spermatozoon. Studies suggest that differences in the micropyle can be found among species of the same genus or family, as a mechanism to prevent inter-specific hybridization (Chen et al., Reference Chen, Shao and Yang1999). Conversely, some studies have reported similarities between the micropylar apparatus in different species, suggesting a possible relationship between systematic groups (Rizzo et al., Reference Rizzo, Sato, Barreto and Godinho2002).
At the time of fertilization, a cytoplasmic movement towards the micropyle region begun in the eggs of Z. jahu, characterizing the animal pole. Similar findings have been reported by Kimmel et al. (Reference Kimmel, Ballard, Kimmel and Ullmann1995) in Danio rerio, by Ninhaus-Silveira et al. (Reference Ninhaus-Silveira, Foresti and Azevedo2006) in Prochilodus lineatus, by Marques et al. (Reference Marques, Nakaghi, Faustino, Ganeco and Senhorini2008) in P. corruscans, and by Faustino et al. (Reference Faustino, Nakaghi and Neumann2011) in Brycon gouldingi, who have reported stimulation of this cytoplasmic movement after fertilization. However, Ganeco (Reference Ganeco2003) observed this cytoplasmic movement in Brycon orbignyanus oocytes at extrusion, before fertilization.
Oocytes and eggs of Z. jahu showed a large perivitelline space throughout the embryonic development, which according to Matsumura (Reference Matsumura1972) would protect the embryos from environmental damages and contribute to greater survival in running water.
The eggs from Z. jahu are classified as telolecithal due to the large amount and distribution of the yolk, showing partial or meroblastic segmentation restricted to the animal pole; which is characteristic of fish eggs (Lagler et al., Reference Lagler, Bardach, Miller and Passino1977; Leme dos Santos & Azoubel, Reference Leme dos Santos and Azoubel1996).
At the cleavage stage, it was observed that the cell divisions began from the centre towards the edge of the blastodisc, as also reported by Matkovic et al. (Reference Matkovic, Cussac and Cukier1985) in Rhamdia sapo and by Shardo (Reference Shardo1995) in Alosa sapidissima. Successive cleavage occurred up to the 64 blastomeres phase, when the number of blastomeres was inversely proportional to their size, as also reported by Wourms & Evans (Reference Wourms and Evans1974) in Xiphister atropurpureus and by Faustino et al. (Reference Faustino, Nakaghi and Neumann2011) in B. gouldingi. According to Gilbert (Reference Gilbert2003), these cleavages (mitotic divisions) are essential for a new balance to be established between the nucleus and cytoplasm of the animal pole. The yolk globules observed entering the blastomeres in Z. jahu probably aid their absorption by the cells, a fact also noted by Ninhaus-Silveira et al. (Reference Ninhaus-Silveira, Foresti and Azevedo2006).
At the morula stage, the yolk syncytial layer or periblast was formed, a continuous cytoplasmic layer located between the blastoderm disc and the yolk that resulted from the incomplete division of the blastomeres (Ninhaus-Silveira et al., Reference Ninhaus-Silveira, Foresti, Azevedo, Agostinho and Veríssimo-Silveira2007). According to Kimmel et al. (Reference Kimmel, Ballard, Kimmel and Ullmann1995), this layer constitutes an organ and can only be found in teleostei, positioned extra-embryonically and thus not contributing to the development of the embryo. According to Balinsky (Reference Balinsky1970), this layer is important in the breakdown of the yolk, converting it into available energy for embryo development. The development of the periblast during the morula stage has also been observed by Long & Ballard (Reference Long and Ballard1976) in Catostomus commersoni and by Matkovic et al. (Reference Matkovic, Cussac and Cukier1985) in Rhamdia sapo. However, in Oryzias latipes (González-Doncel et al., Reference González-Doncel, Okihiro, Villalobos, Hinton and Tarazona2005), Prochilodus lineatus (Ninhaus-Silveira et al., Reference Ninhaus-Silveira, Foresti and Azevedo2006), and B. gouldingi (Faustino et al., Reference Faustino, Nakaghi and Neumann2011) this layer was observed at the blastula stage.
In Z. jahu, the morula stage was followed by the blastula stage, which was characterized by the formation of the blastocele, a single and significant space between the blastoderm and periblast; which was also observed in Oreochromis niloticus by Galman & Avtalion (Reference Galman and Avtalion1989). Conversely, Kimmel et al. (Reference Kimmel, Ballard, Kimmel and Ullmann1995), in a study in B. rerio, have suggested that the majority of teleostei only shows irregular extracellular spaces between the blastomeres without a well defined blastocele as observed by Faustino et al. (Reference Faustino, Nakaghi and Neumann2011) in B. gouldingi.
The start of the gastrula stage in Z. jahu was characterized by the epiboly movement, when the embryonic cells moved from the animal to the vegetative pole (Leme dos Santos & Azoubel, Reference Leme dos Santos and Azoubel1996). The phases of the epiboly movement are often characterized according to the percentage of yolk coverage, as previously reported by Buzzolo et al. (Reference Buzollo, Veríssimo-Silveira, Oliveira-Almeida, Okuda and Ninhaus-Silveira2011) in Pimelodus maculatus and Faustino et al. (Reference Faustino, Makino, Neumann and Nakaghi2015) in B. gouldingi. In Z. jahu eggs, when approximately 50% of the yolk was covered, the most superficial cells began to move contrary to the epiboly movement (involution), originating two embryonic layers: epiblast and hypoblast. The end of the gastrula stage was characterized by the formation of the blastopore or vitelline plug, in agreement with the findings by Buzzolo et al. (2011) in Pimelodus maculatus and Faustino et al. (Reference Faustino, Nakaghi and Neumann2011) in B. gouldingi.
Organogenesis was defined by tissue and organ development. It began with the appearance of the embryonic axis, formed by the differentiation of the cephalic and caudal regions (Faustino et al., Reference Faustino, Nakaghi and Neumann2011, Reference Faustino, Makino, Neumann and Nakaghi2015). This was followed by the appearance of somites, which are muscle precursors that develop in blocks of transient cells and are necessary for segmental organisation of embryos (Gilbert, Reference Gilbert2003). The notochord was also present, a transitory organ related to the development of the neural tube (future central nervous system, CNS) and the establishment of the antero-posterior body axis (Gilbert, Reference Gilbert2003). The expansion of the neural tube culminated in the development of the three primary vesicles of the CNS, similarly to that observed in R. sapo by Cussac et al. (Reference Cussac, Matkovic and Maggese1985), D. rerio by Kimmel et al. (Reference Kimmel, Ballard, Kimmel and Ullmann1995), P. lineatus by Ninhaus-Silveira et al. (Reference Ninhaus-Silveira, Foresti and Azevedo2006) and B. gouldingi by Faustino et al. (Reference Faustino, Nakaghi and Neumann2011, Reference Faustino, Makino, Neumann and Nakaghi2015). According to Kimmel et al. (Reference Kimmel, Ballard, Kimmel and Ullmann1995), the optical vesicles form at the start of prosencephalon development and the otic vesicles originate from the posterior region of the rhombencephalon.
The organogenesis stage was followed by larval hatching, the final stage in the embryonic development of Z. jahu. Hatching occurs after vigorous muscle contractions in the tail and body of the larvae (Nakatani et al., Reference Nakatani, Agostinho, Baumgartner, Bialetzki, Sanches and Cavicchioli2001), leading to the softening of the chorion by enzymatic activity (Blaxter, Reference Blaxter1988). At this stage in Z. jahu, the primordial heart was observed in the anterior region of the yolk, which according to Hu et al. (Reference Hu, Sedmera, Post and Clark2000) is the first definite organ to develop and become functional during embryogenesis, signalling the formation of the circulatory system.
Conversely, the presence of a rudimentary digestive system, an embryonic fin covering the whole body, primordial branchiae and barbels, oral cavity with small fissures hinting at the separation of the lips, olfactory organ with few cilia, outline of the optic and otic vesicles, and large yolk sac represent a group of morphological characteristics that demonstrate that Z. jahu larvae have low swimming ability and little perception of the environment at hatching. This situation makes the search for food and scape from predators difficult, thus making the larvae dependent on endogenous energy reserves.
These findings corroborate with Nakatani et al. (Reference Nakatani, Agostinho, Baumgartner, Bialetzki, Sanches and Cavicchioli2001), who reported that the majority of fresh water fish larvae are not fully developed at hatching and often follow this pattern until the first stage of development after hatching (vitelline larvae). However, it is important to note that, especially in migratory species, the development of structures that enable the adaptation to adverse environment conditions occurs within a short time (Vandewalle et al., Reference Vandewalle, Germeau, Besancenet, Parmentier and Baras2005).
The data presented in this study are the first of its kind to be reported in Z. jahu and could be used to improve production in captivity as well as to aid conservation studies in this species.
Acknowledgements
The authors would like to thank Mr Orandi Mateus for his help in processing the material, the Companhia Energética de São Paulo (CESP) and Mr Roberto Soares Ferreira for sample collection, and to Comissão de Aperfeiçoamento de Pessoal do Nível Superior [Coordination for the Improvement of Higher Level] (CAPES) and Conselho Nacional de Pesquisa [National Research Council] (CNPq) for the financial support provided.
