Blastula Stage Research Articles

Toxic effects of a mancozeb-containing commercial formulation at environmental relevant concentrations on zebrafish embryonic development.

The toxicological knowledge of mancozeb (MZ)-containing commercial formulations on non-target species is scarce and limited. Therefore, the objective of this work was to represent a realistic application scenario by evaluating the toxicity of environmental relevant and higher concentrations of a commercial formulation of MZ using zebrafish embryos. Following determination of the 96-h LC50 value, the embryos at the blastula stage (~ 2h post-fertilisation, hpf) were exposed to 0.5, 5, and 50μgL-1 of the active ingredient (~ 40× lower than the 96-h LC50). During the exposure period (96h), lethal, sublethal, and teratogenic parameters, as well as behaviour analysis, at 120hpf, were assayed. Biochemical parameters such as oxidative stress-linked enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR)), reactive oxygen species (ROS) levels, and glutathione levels (GSH and GSSG), as well as the activity of degradation (glutathione S-transferase (GST) and carboxylesterase (CarE)), neurotransmission (acetylcholinesterase (AChE)), and anaerobic respiration (lactate dehydrogenase (LDH))-related enzymes, were analysed at the end of the exposure period. Exposed embryos showed a marked decrease in the hatching rate and many malformations (cardiac and yolk sac oedema and spinal torsions), with a higher prevalence at the highest concentration. A dose-dependent decreased locomotor activity and a response to an aversive stimulus, as well as a light-dark transition decline, were observed at environmental relevant concentrations. Furthermore, the activities of SOD and GR increased while the activity of GST, AChE, and MDA contents decreased. Taken together, the involvement of mancozeb metabolites and the generation of ROS are suggested as responsible for the developmental phenotypes. While further studies are needed to fully support the hypothesis presented, the potential cumulative effects of mancozeb-containing formulations and its metabolites could represent an environmental risk which should not be disregarded.

The embryological and larval development of the sea urchin Diadema savignyi (Audouin, 1809) (Diadematoida: Diadematidae) from the South China Sea

ABSTRACTThis paper provides the first descriptions of embryonic and larval development of the sea urchin Diadema savignyi (Audouin, 1809) (Diadematoida: Diadematidae). Diadema savignyi developed from relatively small eggs, 79.24 ± 6.37 µm. At 26–27°C, the embryonic development of D. savignyi took about 7 h until hatching from the fertilization envelope at the blastula stage and beginning of the larval life. Then the larvae went through the following stages of development: spherical blastula (7–12 h), gastrula and prism (23–31 h), pluteus with one pair of larval arms (48 h), pluteus with two pairs of larval arms (50–72 h), and late pluteus (14–18 d). In the late stages of larval development, the proportions of the larval body change greatly. The duration of development of D. savignyi from fertilization to the juvenile stage was 45–47 days. The larval development of D. savignyi and other species having the Echinopluteus transversus type of larvae, whose development was studied earlier, is reviewed and described on the basis of own data and those published previously in literature in order to compare the development of plutei within the family Diadematidae.

Chromatin accessibility and histone acetylation in the regulation of competence in early development

As development proceeds, inductive cues are interpreted by competent tissues in a spatially and temporally restricted manner. While key inductive signaling pathways within competent cells are well-described at a molecular level, the mechanisms by which tissues lose responsiveness to inductive signals are not well understood. Localized activation of Wnt signaling before zygotic gene activation in Xenopus laevis leads to dorsal development, but competence to induce dorsal genes in response to Wnts is lost by the late blastula stage. We hypothesize that loss of competence is mediated by changes in histone modifications leading to a loss of chromatin accessibility at the promoters of Wnt target genes. We use ATAC-seq to evaluate genome-wide changes in chromatin accessibility across several developmental stages. Based on overlap with p300 binding, we identify thousands of putative cis-regulatory elements at the gastrula stage, including sites that lose accessibility by the end of gastrulation and are enriched for pluripotency factor binding motifs. Dorsal Wnt target gene promoters are not accessible after the loss of competence in the early gastrula while genes involved in mesoderm and neural crest development maintain accessibility at their promoters. Inhibition of histone deacetylases increases acetylation at the promoters of dorsal Wnt target genes and extends competence for dorsal gene induction by Wnt signaling. Histone deacetylase inhibition, however, is not sufficient to extend competence for mesoderm or neural crest induction. These data suggest that chromatin state regulates the loss of competence to inductive signals in a context-dependent manner.

A cell-based boundary model of gastrulation by unipolar ingression in the hydrozoan cnidarian Clytia hemisphaerica

In Cnidaria, modes of gastrulation to produce the two body layers vary greatly between species. In the hydrozoan species Clytia hemisphaerica gastrulation involves unipolar ingression of presumptive endoderm cells from an oral domain of the blastula, followed by migration of these cells to fill the blastocoel with concomitant narrowing of the gastrula and elongation along the oral-aboral axis. We developed a 2D computational boundary model capable of simulating the morphogenetic changes during embryonic development from early blastula stage to the end of gastrulation. Cells are modeled as polygons with elastic membranes and cytoplasm, colliding and adhering to other cells, and capable of forming filopodia. With this model we could simulate compaction of the embryo preceding gastrulation, bottle cell formation, ingression, and intercalation between cells of the ingressing presumptive endoderm. We show that embryo elongation is dependent on the number of endodermal cells, low endodermal cell-cell adhesion, and planar cell polarity (PCP). When the strength of PCP is reduced in our model, resultant embryo morphologies closely resemble those reported previously following morpholino-mediated knockdown of the core PCP proteins Strabismus and Frizzled. Based on our results, we postulate that cellular processes of apical constriction, compaction, ingression, and then reduced cell-cell adhesion and mediolateral intercalation in the presumptive endoderm, are required and when combined, sufficient for Clytia gastrulation.

Morphological Embryonic Development Stages of Barbonymus gonionotus (Bleeker, 1850)

Morphological study on embryonic development of Barbonymus gonionotus (Bleeker, 1850) was described under laboratory condition. Fertilization occurred at 16 hours after injection. Ten minutes after fertilization, the one cell stage of embryos and unfertilized eggs were observed. The developmental stages were observed and divided into early cleavage stage, morula stage, blastula stage, gastrula stage, somite formation stage, hatching stage and larval stage. The one-cell embryo of fertilized egg develops subsequently through the first, second, third, fourth and fifth cleavage. After thirty- two cell stage, morula stage was observed after 25 minutes postfertilization followed by blastula stage. Gastrula stage appeared at 2 hours 50 minute after fertilization. After gastrula stage, the embryo transformed into bud stage at 5 hours. Further development of a pair of mesodermal somite formation, appearance of notochord and heart, were observed after 9 hours after fertilization. Thirty-three somite formation stage was observed at about 12 hours and 39 minutes. Anal structure was observed and located between 22 and 23 myomeres. After 15 hours, the development of all cells was completed. Hatching occurred about 16 hours after fertilization, the membrane

The maternal coordinate system: Molecular-genetics of embryonic axis formation and patterning in the zebrafish.

Axis specification of the zebrafish embryo begins during oogenesis and relies on proper formation of well-defined cytoplasmic domains within the oocyte. Upon fertilization, maternally-regulated cytoplasmic flow and repositioning of dorsal determinants establish the coordinate system that will build the structure and developmental body plan of the embryo. Failure of specific genes that regulate the embryonic coordinate system leads to catastrophic loss of body structures. Here, we review the genetic principles of axis formation and discuss how maternal factors orchestrate axis patterning during zebrafish early embryogenesis. We focus on the molecular identity and functional contribution of genes controlling critical aspects of oogenesis, egg activation, blastula, and gastrula stages. We examine how polarized cytoplasmic domains form in the oocyte, which set off downstream events such as animal-vegetal polarity and germ line development. After gametes interact and form the zygote, cytoplasmic segregation drives the animal-directed reorganization of maternal determinants through calcium- and cell cycle-dependent signals. We also summarize how maternal genes control dorsoventral, anterior-posterior, mesendodermal, and left-right cell fate specification and how signaling pathways pattern these axes and tissues during early development to instruct the three-dimensional body plan. Advances in reverse genetics and phenotyping approaches in the zebrafish model are revealing positional patterning signatures at the single-cell level, thus enhancing our understanding of genotype-phenotype interactions in axis formation. Our emphasis is on the genetic interrogation of novel and specific maternal regulatory mechanisms of axis specification in the zebrafish.

Zebrafish gastrulation: Putting fate in motion.

Gastrulation entails specification and formation of three embryonic germ layers-ectoderm, mesoderm and endoderm-thereby establishing the basis for the future body plan. In zebrafish embryos, germ layer specification occurs during blastula and early gastrula stages (Ho & Kimmel, 1993), a period when the main morphogenetic movements underlying gastrulation are initiated. Hence, the signals driving progenitor cell fate specification, such as Nodal ligands from the TGF-β family, also play key roles in regulating germ layer progenitor cell segregation (Carmany-Rampey & Schier, 2001; David & Rosa, 2001; Feldman et al., 2000; Gritsman et al., 1999; Keller et al., 2008). In this review, we summarize and discuss the main signaling pathways involved in germ layer progenitor cell fate specification and segregation, specifically focusing on recent advances in understanding the interplay between mesoderm and endoderm specification and the internalization movements at the onset of zebrafish gastrulation.

The dynamics of DNA methylation during epigenetic reprogramming of primordial germ cells in medaka (Oryzias latipes)

ABSTRACTPrimordial germ cells (PGCs) are precursors of eggs and sperm. How the PGCs epigenetically reprogram during early embryonic development in fish is currently unknown. Here we generated a series of PGC methylomes using whole genome bisulfite sequencing across key stages from 8 days post fertilization (dpf) to 25 dpf coinciding with germ cell sex determination and gonadal sex differentiation in medaka (Oryzias latipes) to elucidate the dynamics of DNA methylation during epigenetic reprogramming in germ cells. Our high-resolution DNA methylome maps show a global demethylation taking place in medaka PGCs in a two-step strategy. The first step occurs between the blastula and 8-dpf stages, and the second step occurs between the 10-dpf and 12-dpf stages. Both demethylation processes are global, except for CGI promoters which remain hypomethylated throughout the stage of PGC specification. De novo methylation proceeded at 25-dpf stage with the process in male germ cells superseding female germ cells. Gene expression analysis showed that tet2 maintains high levels of expression during the demethylation stage, while dnmt3ba expression increases during the de novo methylation stage during sexual fate determination in germ cells. The present results suggest that medaka PGCs undergo a bi-phasic epigenetic reprogramming process. Global erasure of DNA methylation marks peaks at 15-dpf and de novo methylation in male germ cells takes precedence over female germ cells at 25 dpf. Results also provide important insights into the developmental window of susceptibility to environmental stressors for multi- and trans-generational health outcomes in fish.

Combinatorial action of NF–Y and TALE at embryonic enhancers defines distinct gene expression programs during zygotic genome activation in zebrafish

Animal embryogenesis is initiated by maternal factors, but zygotic genome activation (ZGA) shifts regulatory control to the embryo during blastula stages. ZGA is thought to be mediated by maternally provided transcription factors (TFs), but few such TFs have been identified in vertebrates. Here we report that NF–Y and TALE TFs bind zebrafish genomic elements associated with developmental control genes already at ZGA. In particular, co-regulation by NF–Y and TALE is associated with broadly acting genes involved in transcriptional control, while regulation by either NF–Y or TALE defines genes in specific developmental processes, such that NF–Y controls a cilia gene expression program while TALE controls expression of hox genes. We also demonstrate that NF–Y and TALE-occupied genomic elements function as enhancers during embryogenesis. We conclude that combinatorial use of NF–Y and TALE at developmental enhancers permits the establishment of distinct gene expression programs at zebrafish ZGA.

Evaluation of BMP-mediated patterning in a 3D mathematical model of the zebrafish blastula embryo.

Bone Morphogenetic Proteins (BMPs) play an important role in dorsal-ventral (DV) patterning of the early zebrafish embryo. BMP signaling is regulated by a network of extracellular and intracellular factors that impact the range and signaling of BMP ligands. Recent advances in understanding the mechanism of pattern formation support a source-sink mechanism, however it is not clear how the source-sink mechanism shapes patterns in 3D, nor how sensitive the pattern is to biophysical rates and boundary conditions along both the anteroposterior (AP) and DV axes of the embryo. We propose a new three-dimensional growing Partial Differential Equation (PDE)-based model to simulate the BMP patterning process during the blastula stage. This model provides a starting point to elucidate how different mechanisms and components work together in 3D to create and maintain the BMP gradient in the embryo. We also show how the 3D model fits the BMP signaling gradient data at multiple time points along both axes. Furthermore, sensitivity analysis of the model suggests that the spatiotemporal patterns of Chordin and BMP ligand gene expression are dominant drivers of shape in 3D and more work is needed to quantify the spatiotemporal profiles of gene and protein expression to further refine the models.

Embryonic and larval development of serpae tetra Hyphessobrycon eques (Steindachner, 1882)

In this study, the embryonic and larval development stages of one of the most important ornamental fish serpae tetra (Hyphessobrycon eques) are described. The early life stage is documented from fertilization until the beginning of the juvenile period. The fertilized eggs (the average diameter = 938.55 ± 35.20 µm) were incubated at a water temperature of 26 ± 0.5°C. The cleavage finished in 1:10 hr (=h) and the early blastula stage occurred at 1:26 hr post fertilization (hpf). The gastrulation started at 3:05 hpf, and 50% epiboly was observed at 3:25 hpf. Segmentation stage was monitored at 7:26 hpf. Embryonic developmental stage was completed and hatching occurred 20–21 hpf. The total length (TL) of newly hatched larvae was 2.64 ± 0.21 mm. The larval development of serpae tetra was divided into four different periods: Yolk-sac larva (1–4 DAH, TL = 2.77 ± 0.09 mm - 3.85 ± 0.11 mm), preflexion larva (5–12 DAH), flexion larva (13–15 DAH, TL = 5.78 ± 0.46 mm on the 15th day) and post-flexion larva (16–30 DAH, TL = 10.7 ± 0.27 mm on the 28th–30th days). The mouth and anus are closed at 1 DAH. The mouth and anus opened at 4 DAH. Exogenous feeding started on the 4th day. The first gulping of the swim bladder was on days 3. The larva begins to swim freely, and the yolk sac was completely consumed at 4 DAH. Histological structures of the eye and brain of new hatched larva were clearly identified at 1 day after hatching (DAH). According to histological findings, the digestive system (stomach, intestine) started to develop and the liver could be seen on the ventral side of the swim bladder at 5 DAH. No histological difference was observed between the anterior intestine and the posterior intestine at 15–16 DAH. The larval metamorphosis was completed, and the larvae transformed into juveniles at 28–30 DAH.

Survival of Polyploid hybrid salamander embryos

BackgroundAnimals with polyploid, hybrid nuclei offer a challenge for models of gene expression and regulation during embryogenesis. To understand how such organisms proceed through development, we examined the timing and prevalence of mortality among embryos of unisexual salamanders in the genus Ambystoma.ResultsOur regional field surveys suggested that heightened rates of embryo mortality among unisexual salamanders begin in the earliest stages of embryogenesis. Although we expected elevated mortality after zygotic genome activation in the blastula stage, this is not what we found among embryos which we reared in the laboratory. Once embryos entered the first cleavage stage, we found no difference in mortality rates between unisexual salamanders and their bisexual hosts. Our results are consistent with previous studies showing high rates of unisexual mortality, but counter to reports that heightened embryo mortality continues throughout embryo development.ConclusionsPossible causes of embryonic mortality in early embryogenesis suggested by our results include abnormal maternal loading of RNA during meiosis and barriers to insemination. The surprising survival rates of embryos post-cleavage invites further study of how genes are regulated during development in such polyploid hybrid organisms.

Different of saline embriogenesis and eggs hatching rate of the giant gourami (Osphronemus gouramy)

Water salinity is one of the environmental factors that affect the hatching rate of the fish. The aims of this study were to find out the influence of salinity to embryogenesis and hatching rate of giant gourami at the different salinities. This study used four treatments and three replications. The treatments were A 0 ppt, B 1-2 ppt, C 3-4 ppt, and D 5-6 ppt. The salinity was made by adding salt water to the fresh water in aquarium. The number of eggs in each treatment was 32 grains respectively. The parameters observed were the embryogenesis, fertility, hatching rate, hatching time, and water quality. The results showed that salinity influenced the embryogenesis duration of eggs during blastula and gastrula stages, but did not influence to the hatching rate and hatching time of eggs.

Evolution of Metazoan Life Cycles and the Origin of Pelagic Larvae

The problem of the origin of the metazoan life cycle is analyzed on the base of various hypotheses on the origin of multicellular animals. According to the Gastraea theory and the Phagocytella theory, the ancestral metazoan life cycle was holopelagic. In the framework of the hypotheses of the primary sedentarity, the metazoan ancestor had a pelago-benthic life cycle with floating larvae, the synzoospores. In accordance with this hypothesis, Eumetazoa originated from the progenetic larvae of the sedentary ancestor. The primary life cycle of Eumetazoa (i.e., metazoans with a nervous system, musculature, mouth, and gut) was holopelagic. Particularly this life cycle is typical for recent Ctenophora, which is the earliest branch of the eumetazoans. Cnidaria and Bilateria are sister groups. Their last common ancestor acquired the pelago-benthic life cycle de novo. The pelagic part of the life cycle of cnidarians is comprised of the blastula and gastrula stages only. Some anthozoans still maintain the planktotrophic gastrula larvae in their life cycles. Planulae of Medusozoa are simplified lecithotrophic larvae that had lost the function of spread because of the appearance of the medusa stage in the life cycle. In triploblastic Bilateria, the prolongation of the pelagic stage of the life cycle occurred due to the appearance of the ciliated bilaterally symmetrical larvae, which are actually the juveniles raised into the water column. This phylogenetic modus can be designated by the special term “larvalization.” Thus, the ciliated pelagic larvae of all bilaterians have a common origin from the juvenile stages of the last common bilaterian ancestor. Their ciliated bands came from the modified ciliated tentacular apparatus of juvenile stages of the last common bilaterian ancestor. Homologous elements in the ciliated bands of trochozoan and deuterostomian larvae are traced.

참굴(Crassostrea gigas)의 배발생단계에 따른 인슐린유사성장인자 시스템의 발현

Insulin-like growth factors(IGFs) are important in vertebrate embryogenesis and fetal growth of mammals and fish. The Pacific oyster(Crassostrea gigas) is one of the most important aquaculture species in Asia. However, the production of nature seed fluctuates widely year to year. One recent cause of this decrease is increasing seawater pollution to deal with this problem, superior seed production is needed. In particular, early seed production, which is determined by embryogenesis, is vital for aquaculture success. Therefore, this study examined the role of growth factor IGF-I activity during C. gigas embryogenesis. One-year-old cultured C. gigas obtained from Gyeongsangnam-do, Jaranman, Korea(34°51′32.34″ N, 128°12′23.44″ E) were used to collect eggs and sperm. The eggs were maintained in three 20L tanks at 23℃. Each development stage was classified as being reached when more than 80% of the embryos reached that step. C. gigas larvae were sampled at the unfertilized egg, fertilized egg, blastula, gastrula, trochophore, and D-shaped larva stages. The activity of IGF-I was high in the blastula, and IGF-I receptors decreased as development progressed. Tyrosine-phosphorylation was also similar to the expression of IGF-I. Based on these results, we confirmed that the embryonic development of C. gigas is associated with IGF-I activity.

Blastula stage specification of avian neural crest

Cell fate specification defines the earliest steps towards a distinct cell lineage. Neural crest, a multipotent stem cell population, is thought to be specified from the ectoderm, but its varied contributions defy canons of segregation potential and challenges its embryonic origin. Aiming to resolve this conflict, we have assayed the earliest specification of neural crest using blastula stage chick embryos. Specification assays on isolated chick epiblast explants identify an intermediate region specified towards the neural crest cell fate. Furthermore, low density culture suggests that the specification of intermediate cells towards the neural crest lineage is independent of contact mediated induction and Wnt-ligand induced signaling, but is, however, dependent on transcriptional activity of β-catenin. Finally, we have validated the regional identity of the intermediate region towards the neural crest cell fate using fate map studies. Our results suggest a model of neural crest specification within a restricted epiblast region in blastula stage chick embryos.

PI3K inhibition highlights new molecular interactions involved in the skeletogenesis of Paracentrotus lividus embryos

The sea urchin embryo develops a well-defined biomineralized endoskeleton, synthesized exclusively by the skeletogenic cells, supported by ectodermal cues for the correct skeleton patterning. The biomineralization process is tightly regulated via a hierarchical order of gene expression, including transcription and growth factors, biomineralization proteins. Recently, the role of kinases and intracellular signaling pathways in sea urchin skeletogenesis has been addressed, although the downstream components still remain unknown. In this study, we investigated the role of phosphatidylinositide 3-kinase (PI3K)-mediated signaling pathway in Paracentrotus lividus, to identify its genes/proteins targets. The effects of LY294002 (LY), a PI3K-specific inhibitor, were evaluated at morphological and molecular levels. Treatment with 40 μM LY from the blastula stage completely blocked skeleton deposition, which was reversed by wash out experiments. Besides, LY caused a slight delay in the tripartite gut development. Despite the skeleton absence, a few skeleton-specific proteins/mRNAs were regularly expressed and localized in LY-treated embryos, as shown for MSP130 and SM50 by immunofluorescence and in situ hybridization experiments. QPCR analyses showed that LY differently affected the expression of genes coding for other biomineralization proteins, transcription and growth factors. SM30 and carbonic anhydrase expression was severely downregulated, while almost all the transcription factors analyzed were upregulated. Based on the present results and in silico analyses, we propose an “interactomic” model simulating PI3K connections in P. lividus embryos. Our findings define a novel regulatory step in the embryonic skeletogenesis, and provide valuable molecular data for further studies on the role of PI3K signaling in invertebrate biomineralization.

Embryonic development of Peruvian grunt <i>Anisotremus scapularis</i> (Perciformes: Haemulidae)

Peruvian grunt Anisotremus scapularis is distributed from Ecuador to Chile and it is considered an important aquaculture resource in Peru. Knowledge of embryonic development is crucial because it is part of the basic biology of a species. The aim of this study was to describe the embryonic stages of Peruvian grunt. The eggs were obtained by natural spawning and reared at 19 °C under laboratory conditions. Morphometric characteristics of the egg were evaluated: diameter (0.752 ± 0.025 mm) (mean ± sd) and oil globule diameter (0.165 ± 0.014 mm). The first division was observed approximately 45 min after fertilization. Blastula stage started after 4 h and the middle gastrula stage after 12:30 h. Early neurula was observed 17 h after fertilization. Cardiac beats and movements of the free embryonic tail were recorded after 30 h of incubation. Hatching occurred between 31 to 41 h and length of newly hatched larvae was 2.558 ± 0.051 mm. The embryonic development of this species is similar to previous studies regarding other marine fish. This study is the first report of embryonic development of A. scapularis, which is a valuable information that provide a baseline reference for the efforts for the culture of this species.

Morphological evidence of neurotoxic effects in chicken embryos after exposure to perfluorooctanoic acid (PFOA) and inorganic cadmium

Perfluorooctanoic acid (PFOA) and cadmium (Cd) are persistent environmental pollutants, bioaccumulative, and capable of crossing the placental barrier. There is evidence of developmental toxicity induced by PFOA and Cd, but their effects on early development are still poorly understood. In the present study, chicken embryos were exposed to PFOA, Cd, and a mixture of PFOA + Cd from blastula stage up to 72 h of incubation. Embryo mortality and morphology were assessed. PFOA and Cd exposure increased mortality and induced malformations, mainly in the cephalic region, the neural tube, and the ventral body wall. After mixed exposure the effect was observed mostly in the neural tube and the cephalic region. However, the mechanism remains unknown. Because of the resemblance between human and chick development, it was possible to correlate the observed alterations in chickens with known human congenital malformations. Therefore, PFOA and Cd are neurotoxic to chicken embryos inducing morphological abnormalities and are possibly neurotoxic to human embryos.

Strategies for aquaculture and conservation of Neotropical catfishes based on the production of triploid Pimelodus maculatus

Triploidization is a technique to produce sterile fish on a large scale. In the spotted catfish Pimelodus maculatus, sterile fish serve as a host of germ cell transplantation for subsequent reconstitution of endangered catfish species. This study aimed to establish an efficient protocol for triploidization in P. maculatus using temperature shock. In this study, we tested the efficacy of heat shock treatments (37 °C, 38 °C, and 39 °C) applied at 2 min post-fertilization (mpf) for the duration of 2 min. Intact embryos served as diploid control. Ploidy status was confirmed by flow cytometry, characterization of erythrocytes nuclei, and karyotyping. Developmental stages were verified at cleavage, blastula, gastrula, somite, and hatching stages, as well as the percentages of normal and abnormal larvae. No significant difference was observed between the temperature treatments for percentage of unfertilized oocytes (P = 0.1834), 2-cell stage (P = 0.2348), blastula (P = 0.2972), hatching (P = 0.0668), and abnormal larvae (P = 0.6177). Heat shock decreased the survival at the blastula stage (P = 0.0178), somite (P = 0.0469), and the percentage of abnormal larvae (P = 0.0261). All treatments presented high percentages of triploid individuals, and the highest values were observed for heat shock at 38 °C (96.7 %). Based on the results described above, an efficient protocol for triploidization in P. maculatus was established using heat shock (38 °C) at 2 mpf for 2 min.