Introduction
Fish are the most diverse group of vertebrates with over 34,700 known species (Fricke et al., Reference Fricke, Eschmeyer and Van der Laan2018). More than 6000 of these species inhabit Neotropical freshwater environments. These Neotropical species are highly abundant, making up 20–25% of the global fish population (Reis et al., Reference Reis, Albert, Di Dario, Mincarone, Petry and Rocha2016). However, several factors including pollution, the introduction of invasive species, and riparian forest degradation (Vitule et al., Reference Vitule, Freire and Simberloff2009; Barletta et al., Reference Barletta, Jaureguizar, Baigun, Fontoura, Agostinho, Almeida-Val, Val, Torres, Jimenes-Segura and Giarrizzo2010; Agostinho et al., Reference Agostinho, Gomes, Santos, Ortega and Pelicice2016; Vitule et al., Reference Vitule, Da Costa, Frehse, Bezerra, Occhi, Daga and Padial2017) are negatively affecting Neotropical ichthyofauna.
These deleterious phenomena specifically affect the genus Brycon, with several member species being listed as endangered and under threat of extinction (e.g. B. orbignyanus, B. insignis, B. vermelha, B. opalinus and B. nattereri) (ICMBio, 2016). Given these circumstances, biotechnical tools, such as germ cell transplantation and fish sterilization, could be important to guarantee genetic conservation (de Siqueira-Silva et al., Reference de Siqueira-Silva, Saito, dos Santos-Silva, da Silva Costa, Psenicka and Yasui2018). Germ cell transplantation (or surrogate technology) involves the generation of a germline chimera through the transference of germ cells from a target species to an appropriate host with the goal of enabling the production of viable gametes from the donor fish (Saito et al., Reference Saito, Goto-Kazeto, Arai and Yamaha2008; Lacerda et al., Reference Lacerda, Batlouni, Costa, Segatelli, Quirino, Queiroz, Kalapothakis and França2010; Nóbrega et al., Reference Nóbrega, Greebe, Van De Kant, Bogerd, de França and Schulz2010; Yasui et al., Reference Yasui, Fujimoto and Arai2010). This technique requires a sterile host so that the transplanted germ cells can develop without endogenous competition (Golpour et al., Reference Golpour, Siddique, Siqueira-Silva and Pšenička2016). Triploidization is one of the main techniques currently utilized to produce sterile hosts for germ cell transplantation (de Siqueira-Silva et al., Reference de Siqueira-Silva, Saito, dos Santos-Silva, da Silva Costa, Psenicka and Yasui2018). The presence of three sets of chromosomes disrupts meiosis, generally making triploid fish sterile (Cuñado et al., Reference Cuñado, Terrones, Sánchez, Martínez and Santos2002). This makes triploid fish suitable hosts for the application of surrogate technology (Okutsu et al., Reference Okutsu, Shikina, Kanno, Takeuchi and Yoshizaki2007) focused on the preservation of endangered species (Golpour et al., Reference Golpour, Siddique, Siqueira-Silva and Pšenička2016; Goto and Saito, Reference Goto and Saito2019).
Furthermore, the use of triploids also reduces environmental concerns over exotic, transgenic, or hybrid species (Benfey, Reference Benfey2016; do Nascimento, Reference do Nascimento, de Siqueira-Silva, Pereira-Santos, Fujimoto, Senhorini, Nakaghi and Yasui2017a; Piferrer et al., Reference Piferrer, Beaumont, Falguiere, Flajšhans, Haffray and Colombo2009). For aquaculture purposes, triploid fish can improve growth rates, carcass yields, and meat quality (do Nascimento et al., Reference do Nascimento, de Siqueira-Silva, Pereira-Santos, Fujimoto, Senhorini, Nakaghi and Yasui2017a, Reference do Nascimento, Pereira-Santos, Piva, Manzini, Fujimoto, Senhorini, Yasui and Nakaghi2017b). Triploidy is commonly induced using chemical, pressure, or temperature shocks (Arai, Reference Arai2001), which seek to inhibit the process of second polar body extrusion (Dunham, Reference Dunham2004). Another strategy is to use diploid gametes from tetraploid individuals (do Nascimento et al., Reference do Nascimento, Pereira-Santos, Levy-Pereira, Monzani, Niedzielski, Fujimoto, Senhorini, Nakaghi and Yasui2020). However, fertile tetraploids are scarce, difficult to work with (Yoshikawa et al., Reference Yoshikawa, Morishima, Fujimoto, Arias-Rodriguez, Yamaha and Arai2008), and are limited to a few species (Piferrer et al., Reference Piferrer, Beaumont, Falguiere, Flajšhans, Haffray and Colombo2009).
In Neotropical fish, the induction of triploidy can be applied to several species including Brycon amazonicus, also known as matrinxã. B. amazonicus is a tropical fish with excellent flesh quality, a rapid growth rate, and high market value. As a result, it is of great aquacultural importance (Gomes, Reference Gomes2010). This species demonstrates migratory behaviour, annual maturation, and total spawning (Zaniboni-Filho et al., Reference Zaniboni-Filho, Reynalte-Tataje and Weingartner2006). Additionally, B. amazonicus is omnivorous and can be easily adapted to captivity (Honczaryk and Inoue, Reference Honczaryk and Inoue2009). Therefore, triploid matrinxã may be used to improve aquacultural yields, or to serve as hosts for germ cells from endangered species of the genus Brycon. Therefore, the aim of this study was to induce triploidy in B. amazonicus using temperature shocks.
Materials and methods
Broodstock induction
Specimens of B. amazonicus were maintained in earthen ponds (1000 m2) at the Centro Nacional de Pesquisa e Conservação da Biodiversidade Aquática Continental/Instituto Chico Mendes de Conservação da Biodiversidade (CEPTA/ ICMBio), Pirassununga City, São Paulo State, Brazil (21°55′58′′S, 47°22′31′′W). The fish were fed twice a day using a commercial diet (4200 kcal kg–1 and 45% crude protein).
The experiment was performed during the species’ natural spawning season from November to December. Specimens for breeding were captured and selected based on external characteristics. The presence of a swollen abdomen and reddish urogenital papilla was used as a selection criterion for females, while the release of sperm upon the application of gentle abdominal pressure was used as a selection criterion for males. The fish were then carefully transported to spawning tanks with constant water flow. Females (n = 6) and males (n = 6) were induced to spawn by injection of carp pituitary gland extract (CPE) at 5.5 mg kg–1 body weight. The hormone was administered in two different applications for females. The first injection was comprised of 10% of the overall CPE dose. The second injection was conducted 10 h after the first injection and was comprised of the other 90% of the CPE dose. Males received a single dose of CPE (3.0 mg kg–1) at the same time as the second dose for females. When the females began to spawn, the gametes (eggs and sperm) were hand stripped using abdominal massage in separated containers. Fertilization was achieve by immediately mixing the gametes with water from the incubators.
Triploidization
The fertilized eggs were divided into four plastic containers whose bottoms contained a 100-µm nylon mesh. The fertilized eggs were maintained in water at 26°C. At 2 min post fertilization (mpf), three groups of eggs were subjected to heat-shock treatments lasting 2 min at either 38°C, 40°C or 42°C. The fertilized eggs of the fourth container were not subjected to heat-shock treatment and were used as a control group. Immediately following heat shock, all containers were moved to incubators with constant water flow and aeration. These procedures were performed separately for each couple, resulting in six replicates.
Developmental analysis
To conduct an analysis of fertilized eggs during early development, a sample from each treatment group (c. 100 eggs) was placed into plastic containers (bottom with 100 µm nylon mesh) at the top of each incubator. Survival rates were then noted at each of the following stages: cleavage, blastula, gastrula, somite, and hatching. Rates of normal and abnormal larvae were also determined (see Figure 1). These procedures were performed with a stereomicroscope (Nikon SMZ 1500,Tokyo, Japan) and accompanying software Nis-Ar Elements (Nikon, Tokyo, Japan). After hatching, samples of 100 larvae from each treatment group were selected for ploidy analysis using flow cytometry.
Morphology of normal (A) and abnormal (B) larvae of Brycon amazonicus.
Fish rearing
Larvae from the treatment determined to be optimal were reared until approximately 2 years old. For the first 5 months, the larvae were reared in 5000 litre concrete tanks with constant water flow. During this period, the larvae received commercial powdered food (4200 kcal kg–1 and 45% crude protein) five times a day. Afterwards, the fish were transferred to 1000 m2 earthen ponds, and were fed with commercial pellets (3 mm, 91.62% dry matter, 45% crude protein, 8% crude fat, 2.8% crude fibre, and 12.10% mineral matter) twice a day until apparent satiation.
Flow cytometry
Triploid rates (%) during the larval stage and in adults were detected using flow cytometry according to the protocol developed by Xavier et al. (Reference Xavier, Senhorini, Pereira-Santos, Fujimoto, Shimoda, Silva, dos Santos and Yasui2017). The samples were first lysed in a detergent solution (9.53 mM MgSO4.7H2O, 47.67 mM KCl, 15 mM Tris, 74 mM sucrose, and 0.8% of Triton X-100). Afterwards, nuclei were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). Samples were then analyzed using a flow cytometry CyFlow Ploidy Analyzer (Partec, GMBh, Germany). By comparing histogram peaks, triploid percentages could be determined for each treatment.
Blood smear
Fish (five diploids and five triploid specimens, each c. 2 years old) were collected from the maintenance tank and analyzed using flow cytometry as described earlier (see section previously). The animals were anaesthetised in a eugenol solution (1 g l–1) and blood was collected from a caudal puncture using a syringe containing one drop of EDTA (5%). One drop of the collected blood was smeared using a cover slip by dragging across a slide. The slides were then stained with the rapid panoptic kit (Laborclin, Pinhais, Brazil) and observed with an optical microscope (Nikon NI, Tokyo, Japan) and accompanying software Nis-Ar Elements (Nikon, Tokyo, Japan). The captured images were analyzed using ImageJ software (National Institutes of Health, USA, http://rsb.info.nih.gov/ij/) for measurement (n = 100) of area (µm2), perimeter (µm), major axis (µm), and minor axis (µm).
Statistics
Results are shown as the mean ± standard error. The data were checked for normality using a Lilliefors test. Afterwards, one-way analysis of variance (ANOVA) and a Tukey post hoc test were performed on early developmental data. A t-test was applied for erythrocyte measurements. The software STATISTICA v.10.0 (Statsoft, Tulsa, USA) was used with a significance level of α = 0.05.
Results
Early development
Data on the development of heat-shocked embryos are shown in Table 1. No difference among treatments were observed for cleavage (P = 0.9656), blastula (P = 0.5520), or normal stages (P = 0.1059). During the gastrula stage (P = 0.0065) and somite stage (P = 0.0018), the lowest survival rates were observed for fertilized eggs heat shocked at 42°C. Fertilized eggs from this group did not result in any hatching events. Morphology of normal and abnormal larvae are described in Figure 1.
Survival (%) of Brycon amazonicus in percentage (± SE) after heat-shock treatment for induction of triploid fish
Heat shock were applied at 38, 40 and 42°C at 2 mpf for 2 min.
a,bDifferent superscript letters within a column designates statistical differences using the Tukey test (ANOVA; P < 0.05).
Mosaic: fish with 2n and 3n cells; SE: standard error.
Flow cytometry
Flow cytometry data demonstrated that the control group and fertilized eggs heat shocked at 38°C resulted in mainly diploid individuals (Table 1). However, the group heat shocked at 40°C, resulted in high percentages of triploid individuals (95.00%). A few mosaics were also observed for 38°C (Table 1).
Blood smear
Table 2 summarizes the results obtained from erythrocyte nuclei measurements. Triploid fish showed significantly increased dimensions for area (P = 0.0001), perimeter (P = 0.0001), major axis (P = 0.0001), and minor axis (P = 0.0001). Representative examples of blood smears from diploid and triploid individuals are shown in Figure 2.
Nuclear measurements of erythrocytes (± SE) from diploid and triploid of Brycon amazonicus
SE: standard error.
a,bDistinct letters indicate significant difference (t-test; P < 0.05).
Blood smears of diploid (left) and triploid (right) individuals of Brycon amazonicus. Erythrocytes nuclei from diploids (A) presented small size in comparison with triploids (B). Scale: 10 µm.
Discussion
In this study, high percentages of triploids were obtained for Brycon amazonicus using heat-shocked fertilized eggs at 40°C for 2 min. This protocol prevents the extrusion of the second polar body, as observed in previous studies (Adamov et al., Reference Adamov, Nascimento, Maciel, Pereira-Santos, Senhorini, Calado, Evangelista, Nakaghi, Guerrero and Fujimoto2017; Bertolini et al., Reference Bertolini, Lopez, do Nascimento, Arashiro, de Siqueira-Silva, dos Santos, Senhorini and Yasui2020; Yasui et al., Reference Yasui, Nakaghi, Monzani, Nascimento, Pereira dos Santos, Goes, Porto-Foresti and Senhorini2020). In nature, triploid fish can be spontaneously arise, as described in several studies concerning Neotropical fish (Molina et al., Reference Molina, Margarido and Galetti2007; Pansonato-Alves et al., Reference Pansonato-Alves, Oliveira and Foresti2011; Utsunomia et al., Reference Utsunomia, Pansonato-Alves, Paiva, Costa Silva, Oliveira, Bertollo and Foresti2014). However, for aquaculture purposes, triploidy must be induced artificially (Piferrer et al., Reference Piferrer, Beaumont, Falguiere, Flajšhans, Haffray and Colombo2009).
In our study, heat-shock treatments did not guarantee 100% triploidy rate. This is in agreement with previous reports (Adamov et al., Reference Adamov, Nascimento, Maciel, Pereira-Santos, Senhorini, Calado, Evangelista, Nakaghi, Guerrero and Fujimoto2017; Yasui et al., Reference Yasui, Nakaghi, Monzani, Nascimento, Pereira dos Santos, Goes, Porto-Foresti and Senhorini2020). This signifies the need to optimize the current protocol or make use of tetraploid individuals (do Nascimento et al., Reference do Nascimento, Pereira-Santos, Levy-Pereira, Monzani, Niedzielski, Fujimoto, Senhorini, Nakaghi and Yasui2020) to enable the mass production (100%) of triploid fish.
Additionally, the larval stage survival rate obtained was lower compared with similar studies (Adamov et al., Reference Adamov, Nascimento, Maciel, Pereira-Santos, Senhorini, Calado, Evangelista, Nakaghi, Guerrero and Fujimoto2017; Bertolini et al., Reference Bertolini, Lopez, do Nascimento, Arashiro, de Siqueira-Silva, dos Santos, Senhorini and Yasui2020). The induction of triploid fish may lead to mosaics, as observed here and with Pacific salmon (Teplitz et al., Reference Teplitz, Joyce, Doroshov and Min1994), therefore affecting survival rates. Additional research into the formation of mosaics using karyotyping could be interesting in this case.
Erythrocyte measurements have proven to be a simple and inexpensive method for the discrimination of diploid and triploid individuals (Flajšhans et al., Reference Flajšhans, Gela, Kocour, Buchtová, Rodina, Pšenička, Kašpar, Piačková, Sudová and Linhart2010; Fukushima et al., Reference Fukushima, Bailone, Weiss, Martins and Zaniboni-Filho2012; Goo et al., Reference Goo, Im, Gil, Lim and Park2015). Using flow cytometry as a validation technique, blood smear analysis was capable of identifying adult triploid individuals of B. amazonicus. Therefore, the combination of both tools (erythrocyte nuclei analysis and flow cytometry) offers an accurate procedure for the identification of triploids.
As stated previously, triploid fish have promising aquaculture applications, as they may offer increased carcass yields (%) and improved meat quality (do Nascimento et al., Reference do Nascimento, Pereira-Santos, Piva, Manzini, Fujimoto, Senhorini, Yasui and Nakaghi2017b). However, such fish are also suitable for the conservation of endangered species as they reduce the effect of escaped fish (Dunham, Reference Dunham2004). Another interesting application is the use of sterile triploid fish as hosts for germ cell transplantation studies (Okutsu et al., Reference Okutsu, Shikina, Kanno, Takeuchi and Yoshizaki2007). Triploids of B. amazonicus, for example, could be used to host germ cells from other Brycon species threatened with extinction, such as B. orbignyanus, B. insignis, B. opalinus, B. vermelha and B. nattereri (ICMBio, 2016).
In conclusion, triploidy was efficiently induced in B. amazonicus using heat-shock treatments (40°C, 2 min) of fertilized eggs 2 min post fertilization; with 95% of resulting larvae exhibiting triploidy.
Acknowledgements
We acknowledge CEPTA/ICMBio for kindly providing the facilities and experimental fish.
Financial support
The authors are grateful to CTG (China Three Gorges Corporation) for the financial support of this research; São Paulo Research Foundation (FAPESP) (Young Investigators Award Grant #2010/17429–1; Young Researcher Scholarship #2011/11664-1) and CNPq #471140/2012-0.
Ethical standards
All procedures were approved by the Ethics Committee by the Guide for the Care and Use of Laboratory Animals of the University of São Paulo (CEUA 4299290819).
Conflict of interest
None.
