Introduction
The study of embryological development is an important tool that provides knowledge on a species life history (De Alexandre et al., Reference De Alexandre, Ninhaus-Silveira, Veríssimo-Silveira, Buzollo, Senhorini and Chaguri2009). This phase of development comprises fish formation events from fertilization of the oocyte by spermatozoa to larval hatching (Solnica-Krezel, Reference Solnica-Krezel2005). At this phase, the animal is more vulnerable to any environmental disturbance, which can change its morphology, cause deformities, or even result in death. Therefore, to investigate the effects of changes in climatic variables on the embryonic development of teleosts, many studies have described this phase and associated its development with abiotic factors such as temperature (Hansen and Falk-Peterson, Reference Hansen and Falk-Petersen2001; Rodrigues-Galdino et al., Reference Rodrigues-Galdino, Maiolino, Forgati, Donatti, Mikos, Carneiro and Rios2010; Arashiro et al., Reference Arashiro, Yasui, Calado, Nascimento, Santos, Alves do Santos and Senhorini2018), water acidification (Villanueva et al., Reference Villanueva, Quintana, Petroni and Bozzano2011), and water dissolved oxygen content (Keckeis et al., Reference Keckeis, Bauer-Nemeschkal and Kamler1996), among others.
Studies on embryonic development are also important to subsidize research on phylogeny and taxonomy of species, revealing knowledge about evolutionary history and phylogenetic relationships (Godinho et al., Reference Godinho, Lamas and Godinho2009; Weber et al., Reference Weber, Arantes, Sato, Rizzo and Bazzoli2012; Dos Santos et al., Reference Dos Santos, Yasui, Xavier, de Macedo Adamov, do Nascimento, Fujimoto and Nakaghi2016). In addition, Godinho and Lamas (Reference Godinho, Lamas and Godinho2009) showed that the characteristics of eggs, when fertilized, help in the development of reproductive strategies for teleosts.
In Brazil, embryological studies focus mainly on species with an established commercial value such as Siluriformes Pseudoplatystoma coruscans (Cardoso et al., Reference Cardoso, Alves, Ferreira and Godinho1995; Marques et al., Reference Marques, Okada Nakaghi, Faustino, Ganeco and Senhorini2008), the Characiformes Colossoma macropomum (Leite et al., Reference Leite, Melo, Oliveira, Pinheiro, Campello, Nunes and Salmito-Vanderley2013), Brycon insignis (Isaú et al., Reference Isaú, Rizzo, Amaral, Mourad and Viveiros2011), and Brycon cephalus (Romagosa et al., Reference Romagosa, Narahara and Fenerich-Verani2001; De Alexandre et al., Reference De Alexandre, Ninhaus-Silveira, Veríssimo-Silveira, Buzollo, Senhorini and Chaguri2009), among many other large-sized animals. However, those works do not cover the diversity of species considering the abundance of described species, especially of freshwater fish (3148 species described until 2018; ICMBio, 2018).
The genus Moenkhausia (Eigenmann, 1903), for example, includes about 90 species of freshwater fish distributed across South America in Venezuela, Guyana, and Amazonia (Froese and Pauly, Reference Froese and Pauly2018), and all Brazilian watersheds (Lima and Toledo-Piza, Reference Lima and Toledo-Piza2001). This genus belongs to the Characiformes order, and it is currently allocated to Incertæ sedis in the Characidae family, due to lack of detailed research about its phylogeny. Although some taxonomic studies have already been carried out (Hojo et al., Reference Hojo, Santos and Bazzoli2004; Benine et al., Reference Benine, Castro and Santos2007, Reference Benine, Mariguela and Oliveira2009; Carvalho et al., Reference Carvalho, Sarmento-Soares and Martins-Pinheiro2014), its current classification is still unclear, as most studies are limited to the description of species of the genus.
This misclassification of the species Moenkhausia oligolepis (Gunther, 1864) is currently under discussion due to the wide distribution of Moenkhausia species coexisting and exhibiting similarities of colours and patterns. For this reason, Costa (Reference Costa1994) and Benine and colleagues (Reference Benine, Mariguela and Oliveira2009) proposed M. oligolepis to be a complex of species. However, according to Domingos et al. (Reference Domingos, Moraes, Moresco, Margarido and Venere2014), the coexistence and similarity between species usually results in an incorrect definition of their conservation status. While it is called black tail tetra in some areas (Matos et al., Reference Matos, Matos, Corral and Azevedo2003), this species reaches around 10 cm total length when mature (Froese and Pauly, Reference Froese and Pauly2018). It also presents a reticulated body colour and reddish pigmentation on the dorsal margin of the eye, giving it the popular name (fire-eye tetra), and it also has a dark spot on the stalks of the caudal fin.
Therefore, this study aimed to describe the embryonic development of M. oligolepis under laboratory conditions to contribute knowledge on the biology and species conservation, in addition to furthering its identification and classification. The study describes the timing of typical stages after fertilization, based on external morphology, in captive individuals of M. oligolepis. It was found that the embryonic development lasted 14 h at 25ºC, and these stages occurred similarly to that of closely related species (e.g. Brycon gouldingi: Faustino et al., Reference Faustino, Nakaghi and Neumann2011; Astyanax bimaculatus: Weber et al., Reference Weber, Arantes, Sato, Rizzo and Bazzoli2012; Astyanax altiparanae: Dos Santos et al., Reference Dos Santos, Yasui, Xavier, de Macedo Adamov, do Nascimento, Fujimoto and Nakaghi2016).
Materials and methods
Sampling of animals
Sexually mature individuals of M. oligolepis were collected from streams in the Tocantins Basin, located in the interior of the Amazon Forest, in the ‘Fundação Zoobotânica de Marabá’ – PA (collection authorization ICMBio no. 62027-1). Nylon nets (1.10 mm, 4.75 × 1 mesh, 10 cm) were used to sample the fish, which were transported to the laboratory in 30-l plastic bags filled with water and equipped with portable aerators. The species was identified in the Laboratory of Biology and Fish Genetics at the Institute of Biosciences of the Universidade Estadual Paulista (UNESP), in Botucatu of the state of São Paulo, Brazil (voucher: 25622).
Fish acclimatization lasted 4 months in glass tanks (23 × 21 cm, capacity of 13 l of water) with aeration pumps and internal bacteriological filter. The animals were fed three times a day with commercial food (4200 kcal/kg and 28% crude protein), and the tank water was partially exchanged daily.
Breeding preparation
Four males and three females were separated in a tank that had the same dimensions of the acclimatization tanks, also designed with constant circulation of water. Those animals were submitted to a monitored photoperiod cycle of 12 h of light/dark, for 45 days. During this period, the water parameters (dissolved ammonia, nitrite, dissolved O2, pH, and temperature) were analyzed daily. The same commercial food was offered throughout the day in three rations of 0.100 g each, totalling 0.300 g of food per day.
Induction to spawning and fertilization
On the 45th day, the animals were anaesthetised with 1 ml of Eugenol solution (20 ml of Biodynamic Eugenol in 100 ml of absolute alcohol) diluted in 500 ml of water. Both males and females were injected with carp crude pituitary extract that was macerated and diluted in 0.9% saline solution. The solution was applied in the coelomic cavity at the base of the pectoral fin using an insulin syringe (1 ml) with a needle. This step was based on the protocol of Ninhaus-Silveira et al. (Reference Ninhaus-Silveira, Foresti and De Azevedo2006), in which females received two hormonal doses: the first dose was 0.5 mg/kg body weight; and after a 12-h interval, the second dose was 5.0 mg/kg body weight. Males received a single dose of 1.0 mg/kg body weight at the same time as the second dose of females.
Embryo collection and analysis
Samples were collected at the following time intervals after fertilization: every 10 min up to 2 h post fertilization (hpf); then every 20 min up to 4 hpf: and afterwards, every 30 min until hatching. The collected embryos were fixed in a solution of 2.5% glutaraldehyde sodium phosphate buffer 0.1 M, pH 7.3, and they were observed using a trinocular stereoscope (TNE-10TN Opton). The images were captured using the TC Capture program and a digital camera (Samsung A3, 2015, 8 MP) and then processed using the CorelDRAW program (version 2018).
The embryonic development of M. oligolepis was classified in the standard phases (zygote, cleavage, blastula, gastrula, segmentation, and hatching) based on previous studies (Arashiro et al., Reference Arashiro, Yasui, Calado, Nascimento, Santos, Alves do Santos and Senhorini2018). The temperature and parameters of the water were monitored and documented during the development of the embryos.
Results and Discussion
In this study, the embryonic development of M. oligolepis, a Characidae of disputed taxonomic position from the Amazon, was described up to hatching. It was found that embryonic development lasted 14 h at 25ºC with the described development stage occurring at similar times as that of closely related species. The ontogenetic development in fish is sensitive to changes in temperature, as its metabolic activities can be accelerated or retarded and, therefore, can alter the rhythm of the embryonic development (Santos et al., Reference Santos, Padilha, Bomcompagni-Júnior, Santos, Rizzo and Bazzoli2006; Faustino et al., Reference Faustino, Nakaghi and Neumann2011). This period is variable among species, being as short as observed in M. oligolepis, or it may be even shorter as observed in M. sanctaefilomenae, whose embryonic development lasted 13 h (Walter, Reference Walter2011). Conversely, Prochilodus lineatus presented embryo development time similar to that of the present study at higher temperatures (28ºC) (Ninhaus-Silveira et al., Reference Ninhaus-Silveira, Foresti and De Azevedo2006), which made clear that each species has its own relationship with abiotic factors, this observation reflects the life history and evolutionary strategy of each species.
Egg sampling and morphology
The spawning occurred seminaturally about 2 h after the application of the last hormonal doses, and the fertilized eggs measured 0.85 ± 0.5 mm (mean ± SD) in diameter. They were demersal, spherical, and translucent after fertilization, and did not present any oil droplets. The chorion had an adhesive surface, and the perivitelline spaces measured 0.1 ± 0.02 mm (mean ± SD) (Fig. 1). The diameter of the eggs was also directly related to the reproductive strategy, as small eggs are usually found in migratory species with total spawning, while the largest eggs are observed in non-migratory species (Godinho et al., Reference Godinho, Lamas and Godinho2009). The diameter of M. oligolepis eggs is similar to those observed by Sato et al. (Reference Sato, Sampaio, Fenerich-Verani and Verani2006) and Weber et al. (Reference Weber, Arantes, Sato, Rizzo and Bazzoli2012) in other small Characiformes, Astyanax bimaculatus and Tetragonopterus chalceus, respectively, and these are both rheophilic species. Astyanax bimaculatus also reproduces in lentic waters (Webber et al., Reference Weber, Arantes, Sato, Rizzo and Bazzoli2012).
Figure 1. Morphological characteristics of M. oligolepis eggs. Presence of chorion (Co), perivitelline space (EsP), and initial cleavage, blastomeres (Bl). Scale bar, 0.25 mm.
It was also observed that the eggs of M. oligolepsis showed characteristics of adhesiveness. According to Kolm and Ahnesjö (Reference Kolm and Ahnesjö2005), adhesive eggs are a characteristic of the species with partial spawning and parental care. Godinho and colleagues (2010) also observed higher adhesiveness in eggs of lentic species with multiple spawning, whereas lotic species presented free eggs and total spawning. Judging the characteristics of the environment in which the matrices of this study were collected, it can be suggested that M. oligolepis is a species that spawns in lentic waters, however it is different from other species with adhesive eggs as there was no evidence of parental care in this M. oligolepis.
Egg adhesion to the substrate contributes to the viability and protection of the offspring in the natural environment but, in captivity, it may cause high mortality of the embryos, as egg and embryo agglomeration impairs gas exchange between the developing embryo and the external environment. Moreover, egg adhesion can contribute to proliferation of fungi and bacteria, causing death or malformation in the embryos. Many techniques have been developed to mitigate such damage (Siddique et al., Reference Siddique, Psenicka, Cosson, Dzyuba, Rodina, Golpour and Linhart2014) such as incubators equipped with a closed water recirculation system to promote the circulation of water and embryos, therefore preventing their deposit and agglomeration at the bottom of the tank (Luz et al., Reference Luz, Reynalte-tataje, Ferreira and Zaniboni-Filho2001). In the studied species, it was observed that, although the eggs fixed to each other or the aquarium walls presented strong adhesiveness to forming embryo clusters, the aerator was sufficient to keep them suspended in the water, eliminating the need for more elaborate techniques.
Another important structure in the embryological staging of fish is the chorion, as hydration of the egg causes it to expand to form the perivitelline space (Siddique et al., Reference Siddique, Psenicka, Cosson, Dzyuba, Rodina, Golpour and Linhart2014), and this will aid in embryo development, protecting it from external injuries often caused by water flow. This vulnerability makes eggs with large perivitelline spaces characteristic of species that reproduce in agitated waters. Conversely, smaller perivitelline spaces suggest eggs that spawn in calm waters, an aspect that reflects different species adaptations to their environment (Yamagami et al., Reference Yamagami, Hamazaki, Yasumasut and Masuda1992; De Alexandre et al., Reference De Alexandre, Ninhaus-Silveira, Veríssimo-Silveira, Buzollo, Senhorini and Chaguri2009; Ribeiro and Guimarães, Reference Ribeiro and Guimarães2012; Yang et al., Reference Yang, Zhang, Liu, Hu, Wang, Du and Yan2014). M. oligolepis presents pelagic eggs similar to other Characiformes such as Acestrorhynchus spp., Hoplias lacerdae, Prochilodus spp., and Leporinus sp. that were observed by Rizzo et al. (Reference Rizzo, Sato, Barreto and Godinho2002).
Embryogenesis
Phases, stages, and time of development of M. oligolepis embryogenesis are listed in Table 1.
Table 1. Phases and time of embryonic development in M. oligolepis at the temperature of 25°C
Zygote phase
An increase of the perivitelline space and formation of the blastodisc was observed, defining the animal and vegetal poles and evidencing a great quantity of yolk (Fig. 2A).
Figure 2. Embryonic development of Moenkhausia oligolepis after fertilization. Zygote, cleavage, blastula, gastrula, epiboly, and neurula phases. Scale bar, 0.25 mm. See Table 1 for key.
Cleavage phase
Cleavage followed the pattern of discoidal meroblastic division, noting the presence of 2, 4, 8, 16, 32, and 64 consecutive blastomeres (Fig. 2B–G). This phase took approximately 30 min.
Blastula phase
This phase was initiated at the sixth cleavage, doubling the number of cells in the sequences of 128, 256, and 512 blastomeres, which was achieved at 1 h 30 min after fertilization (AF). The dome phase was reached at 1 h 40 min AF, and was characterized by the organization of thousands of blastomeres in several layers at the top of the yolk (Fig. 2H–K).
Gastrula phase
This phase began approximately 2 h AF. The blastoderm cells of started the epiboly movement, moving toward the yolk and gradually evolving. At 2 h 40 min, a germinative ring was observed (Fig. 2L). At 4 h AF, 90% of the yolk was surrounded by the blastula, and the blastopore was observed (Fig. 2M–P).
Neurula
This stage occurred at 5 h and 30 min AF. It was characterized mainly by 100% epiboly, whose blastoderm completely envelops the yolk (Fig. 3A).
Figure 3. Embryonic development of Moenkhausia oligolepis after fertilization. Segmentation and hatching phases. s: somites, op: optic vesicle, ot: otic vesicle; arrow: Kupffer vesicle. Scale bar, 0.25 mm. See Table 1 for key.
Segmentation
The segmentation phase is the last phase of embryonic development. It represents the differentiation of the cephalic and caudal poles, and it also includes the appearance of somites, vesicles, and some external and internal organs of the embryo, extending until the moment of hatching. Segmentation lasted about 8 h 10 min. The embryo presented the first somite around 5 h 50 min AF, eight somites at 6 h 30 min, and at 7 h 30 min, it was possible to visualize the optical vesicle. At 7 h 40 min AF, there were 17 somites, and 8 h 10 min AF, the appearance of the Kupffer vesicle was observed, followed by the appearance of the otic vesicle at 9 h AF. At 11 h 30 min AF, there were 27 somites, and after that, there were about 30 somites just before hatching (Fig. 3B–F).
Hatching phase
The embryo presented a free tail at 12 h 30 min AF, followed by larvae hatching at 14 h AF with about 30 somites (Fig. 3F).
Considering that this is only the second embryological study of the genus Moenkhausia, this work brings important data about the embryology of M. oligolepis. It is noted that although much information has been revealed and supported, some of the data needed more detailed and elaborate assessments. The use of such data to clarify the incomplete picture in species and genus classification is encouraged. As suggested by Webber et al. (Reference Weber, Arantes, Sato, Rizzo and Bazzoli2012), studies like this one are important to support future studies on reproduction, phylogeny, and taxonomy.
Acknowledgements
We thank the Laboratory of Neurosciences and Behaviour ‘Frederico Guilherme Graeff’ (LaNec) for the structure used. The authors also thank Professor Claudio de Oliveira from the Laboratory of Biology and Fish Genetics of the Institute of Biosciences of the State University of São Paulo (UNESP) for identification of fish. Additional thanks are given to the Laboratory of Science and Technology of Madeira (UEPA) and Professor Luiz Eduardo de Lima Melo for facilities support. Lastly, we appreciate all students from the Research Group of Studies in Reproduction of Amazonian Fish (GERPA) and the Fundação Zoobotânica of Marabá – PA for allowing collection of fish in its facilities.
Financial support
This work was funded by ‘Conselho Nacional de Desenvolvimento Científico e Tecnológico’ CNPq, which awarded a PIBIC grant (PIBIC-2017660905051).
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical standards
The authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional guidelines on the care and use of laboratory animals.
