Effects of different temperatures on the embryonic development of the Lebranche mullet Mugil liza.

We herein investigated the influence of temperature on the embryonic development (from fertilisation to hatching) of Mugil liza larvae. For this purpose, oocytes (>600 µm) and sperm were obtained from breeding stock at the laboratory of marine fish culture (LAPMAR). After fertilisation, 1200 eggs were distributed in 12 cylindrical experimental units of 400 mL under four different temperatures 18, 22, 26 and 30 ºC, all in triplicate. Every 15 min until hatching, about 10 eggs were randomly sampled in each treatment. The eggs were visualized and photographed, and the classification of embryonic stages was performed. Temperature influenced the main events of the embryonic development of M. liza. More accelerated development was observed according to the increase in temperature until the gastrula phase. At temperatures of 22 and 26 °C, embryonic development occurred from fertilisation to hatching of the larvae. In the 18 °C treatment, it was verified that most of the embryos ceased development during the final phase of cleavage and the beginning of blastula formation, while in the 30 °C treatment patterns of embryo malformation were also verified, with erratic divisions of the blastomeres, resulting in irregular cells. Unlike what was observed at a temperature of 18 °C, none of the embryos incubated at 30 °C reached the blastopore closure phase, stopping in the gastrula. The larvae hatched in the treatments at 22 and 26 °C were viable and exhibited intense swimming, with a large amount of reserve material (yolk) and an evident drop of oil.

Surface contraction waves or cell proliferation waves in the presumptive neurectoderm during amphibian gastrulation: Mexican axolotl versus African clawed frog.

This essay represents a critical analysis of the literary data on various types of waves occurring in the amphibian embryos during gastrulation. A surface contraction wave travels through the presumptive neurectoderm during Mexican axolotl gastrulation. This wave coincides temporally and spatially with involution of the inducing chordomesoderm and with the prospective neural plate. By contrast, there is no similar surface contraction wave during African clawed frog gastrulation. However, the clawed frog displays the waves of DNA synthesis and mitosis in the presumptive neurectoderm during gastrulation, whereas no such waves were discovered in axolotl gastrulae. These sets of experimental data are in accordance with the contemporary concept of considerable ontogenetic diversity of the class Amphibia.

Embryonic and early larval development of two marine angelfish, Centropyge bicolor and Centropyge bispinosa.

Marine angelfish (family: Pomacanthidae) are among the most sought-after fish species in the saltwater aquarium trade. However, there is a lack of information in the literature on their early ontogeny. The objective of this study was to describe the embryonic and early larval development of two dwarf angelfish, the bicolour angelfish, Centropyge bicolor and the coral beauty angelfish, Centropyge bispinosa. The eggs of these two species were obtained from spontaneous spawning of the broodstock fish in captivity and incubated at 26.0 ± 0.2°C throughout the study. Fertilized eggs (n = 15) of both species are transparent, pelagic and spherical; the mean diameters of the eggs were measured at 703.6 ± 7.8 µm for C. bicolor and 627.6 ± 7.8 µm for C. bispinosa. The eggs of both species possessed a narrow perivitelline space, smooth and thin chorion, a homogenous and non-segmented yolk as well as a single oil globule. Overall, the observed embryonic development pattern of C. bicolor and C. bispinosa was very similar, and the main difference was the embryonic pigmentation pattern, which only became evident close to hatching. Larvae of both species started hatching at 13 h 30 min after fertilization, and the larval characteristics of both species also showed high levels of similarities. However, the mouth opening time for C. bicolor was 72 h after hatching (AH) and 96 AH for C. bispinosa. In general, the observed early ontogeny of C. bicolor and C. bispinosa also resembled that of other Centropyge species documented in the literature.

Embryonic development in Zungaro jahu.

The aim of this study was to characterize the embryonic development of Zungaro jahu, a fresh water teleostei commonly known as 'jaú'. Samples were collected at pre-determined times from oocyte release to larval hatching and analysed under light microscopy, transmission electron microscopy and scanning electron microscopy. At the first collection times, the oocytes and eggs were spherical and yellowish, with an evident micropyle. Embryo development took place at 29.4 ± 1.5°C and was divided into seven stages: zygote, cleavage, morula, blastula, gastrula, organogenesis, and hatching. The differentiation of the animal and vegetative poles occured during the zygote stage, at 10 min post-fertilization (mpf), leading to the development of the egg cell at 15 mpf. From 20 to 75 mpf, successive cleavages resulted in the formation of 2, 4, 8, 16, 32 and 64 blastomeres. The morula stage was observed between 90 and 105 mpf, and the blastula and gastrula stage at 120 and 180 mpf; respectively. The end of the gastrula stage was characterized by the presence of the yolk plug at 360 mpf. Organogenesis followed, with differentiation of the cephalic and caudal regions, elongation of the embryo by the cephalo-caudal axis, and somitogenesis. Hatching occurred at 780 mpf, with mean larval total length of 3.79 ± 0.11 mm.

Analysis of Cell Size in the Gastrula of Ten Frog Species Reveals a Correlation of Egg with Cell Sizes, and a Conserved Pattern of Small Cells in the Marginal Zone.

We investigated the relationship between egg and cell sizes in the early gastrula of ten species of frogs with eggs of 1,100-3,500 µm diameters. We asked whether differences in cell size of the vegetal region, blastocoel roof, and marginal zone of the early gastrula were associated with egg size. Alternatively, we proposed that cell size differences may associate with gastrulation characteristics. The analyzed species were as follows: Xenopus laevis, Engystomops randi, Engystomops coloradorum, Espadarana callistomma, Epipedobates machalilla, Epipedobates anthonyi, Epipedobates tricolor, Dendrobates auratus, Gastrotheca riobambae, and Eleutherodactylus coqui. A positive correlation between egg and cell size was detected in the three regions of the gastrula. The correlation was strong in the vegetal region and blastocoel roof, and weak in the marginal zone. Large eggs allowed the evolution of frog terrestrial reproductive modes by storing nourishment for the developing embryos. Large cells, laden with yolk, occur in the vegetal region. However, small cell size characterized the marginal zone and blastocoel roof. We proposed that small cells of the marginal zone are required for involution and blastopore formation. The evolution pressure toward small cells in the marginal zone contributed to maintain the blastopore as a universal feature of frog gastrulation in eggs of different sizes and gastrulation modes. Our comparative analysis revealed two fundamental and conserved properties of the frog early gastrula, the correlation of egg with cell sizes, and the general small size of cells in the marginal zone.

Morphology of gametes, post-fertilization events and the effect of temperature on the embryonic development of Astyanax altiparanae (Teleostei, Characidae).

The aim of this study was to describe the morphology of gametes, post-fertilization events and subsequent temperature effects on the early developmental stages of the neotropical species Astyanax altiparanae. The sperm of this species presents a typical morphology of teleost sperm with a spherical head (diameter = 1.88 µm), midpiece (diameter = 0.75 µm) and a single flagellum (length = 18.67 µm). The extrusion of the second polar body and fusion of male and female pronucleus were reported for the first time in this species. Additionally, we observed the formation of the fertilization cone, which prevents polyspermic fertilization. Developmental stages at 22°C, 26°C and 30°C gave rise to fertilization rates at 91.12, 91.42 and 93.04% respectively. Hatching occurred at 25 hpf at 22°C, 16 hpf at 26°C and 11 hpf at 30°C and the hatching rates were 61.78%, 62.90% and 59.45%, respectively. At 22°C, the second polar body was extruded at ≈6 mpf and the male and female pronucleus fused at ≈10 mpf. This fundamental information is important for the field and opens up new possibilities in fish biotechnology, including micromanipulation and chromosome-set manipulation.

Initial development of Prochilodus hartii (Pisces: Prochilodontidae) submitted to induced reproduction.

The Prochilodontidae prepare for reproduction in captivity, but neither ovulation nor spawning occurs, thus requiring induced reproduction for breeding and restocking. This study analyzed for the first time the embryogenesis and larval ontogeny of P. hartii submitted to induced reproduction by hypophysation with crude common carp pituitary extract. The extrusion of oocytes and sperm was performed manually and fertilization was done using the dry method. After fertilization, the eggs were kept in incubators at 23°C. A stereomicroscope was used to measure egg diameter and to monitor embryo development. Samples of larvae were collected daily for 7 days for histological and biometric analyses. The recently extruded oocytes, non-hydrated, are spherical, grey, and non-adhesive with a diameter of 1480 ± 39 µm and after hydration, have a diameter of 2860 ± 120 µm. The positive response to hypophysation was 100% for females and 80% for males. Spawning occurred 7 h after the third hormonal dosage. The fertilization rate was 77% at 23°C. Blastopore closure occurred at 6 h 45 min and embryonic development was completed 36 h 10 min after fertilization. After 204 h post fertilization (hpf) the larvae reached a standard length of 6.56 ± 0.14 mm with the yolk sac completely resorbed. In P. hartii, the oral cavity opening occurred 132 hpf. The results of this study provide knowledge to better understand induced reproduction, breeding, and management of P. hartii, a species with a high potential for pisciculture, and which is commercially important in the Jequitinhonha River basin.

Structural analysis of embryogenesis of Leiarius marmoratus (Siluriformes: Pimelodidae).

Embryological studies in fish species are useful to the understanding of their biology and systematics. The available biological data in Leiarius marmoratus are scarce and additional information about its reproductive biology is needed, mainly because this species has been commercially exploited and used in production of hybrid lineages. In order to evaluate the temporal-morphological embryonic modifications in L. marmoratus, samples of nearly 200 embryos were collected at random at different stages of development, starting from fecundation (time zero). Embryos were fixed in modified Karnovsk's solution and 2.5% glutaraldehyde, processed and analysed under optic and electron microscopy. The incubation period of L. marmoratus was equal to 14.42 h at a mean temperature of 28.3 ± 0.07°C. The following stages of embryonic development were established: zygote, cleavage, gastrula, organogenesis and hatching. These stages were divided into phases, as follows: cleavage - phases of 2, 4, 8, 16, 32 and 64 cells and morula; gastrula - phases of 25, 50, 75 and 90% of epiboly and blastopore closure; and organogenesis - neurula, segmentation and pre-larval phases. The embryogenesis of L. marmoratus was typical of neotropical teleosteans, with peculiarities in species development.

Early development of Betta splendens under stereomicroscopy and scanning electron microscopy.

Betta splendens is a very important ornamental species. The current paper describes the embryonic and larval development of B. splendens under stereomicroscopy and scanning electron microscopy. Eggs and larvae from natural spawning were collected at different developmental stages at previously established intervals and analysed. The eggs of B. splendens are yellowish, clear, spherical, demersal, translucent and telolecithal with a large amount of yolk. Between 0-2 h post-initial collection (hpIC), the eggs were at the egg cell, first cleavage and morula stages. The blastula stage was identified at 2-3 hpIC and the early gastrula phase was observed at 3-4 hpIC with 20% epiboly, which was finalized after 13-18 hpIC. When the pre-larvae were ready to hatch, the appearance of somites and the free tail were observed, at 23-25 hpIC. At 29 hpIC, the majority of larvae had already hatched at an average temperature of 28.4 ± 0.2°C. The newly hatched larvae measured 2.47 ± 0.044 mm total length. The mouth opened at 23 h post-hatching (hPH) and the yolk sac was totally absorbed at 73 hPH. After 156 hPH, the heart was pumping blood throughout the entire larval body. The caudal fin, operculum and eyes were well developed at 264 hPH. When metamorphosis was complete at 768 hPH, the larvae became juveniles. The current study presents the first results about early development of B. splendens and provides relevant information for its reproduction, rearing and biology.

The gastrocoel roof plate in embryos of different frogs.

The morphology of the gastrocoel roof plate and the presence of cilia in this structure were examined in embryos of four species of frogs. Embryos of Ceratophrys stolzmanni (Ceratophryidae) and Engystomops randi (Leiuperidae) develop rapidly, provide comparison for the analysis of gastrocoel roof plate development in the slow-developing embryos of Epipedobates machalilla (Dendrobatidae) and Gastrotheca riobambae (Hemiphractidae). Embryos of the analyzed frogs develop from eggs of different sizes, and display different reproductive and developmental strategies. In particular, dorsal convergence and extension and archenteron elongation begin during gastrulation in embryos of rapidly developing frogs, as in Xenopus laevis. In contrast, cells that involute during gastrulation are stored in the large circumblastoporal collar that develops around the closed blastopore in embryos of slow-developing frogs. Dorsal convergence and extension only start after blastopore closure in slow-developing frog embryos. However, in the neurulae, a gastrocoel roof plate develops, despite the accumulation of superficial mesodermal cells in the circumblastoporal collar. Embryos of all four species develop a ciliated gastrocoel roof plate at the beginning of neurulation. Accordingly, fluid-flow across the gastrocoel roof plate is likely the mechanism of left-right asymmetry patterning in these frogs, as in X. laevis and other vertebrates. A ciliated gastrocoel roof plate, with a likely origin as superficial mesoderm, is conserved in frogs belonging to four different families and with different modes of gastrulation.

Development beyond the gastrula stage and digestive organogenesis in the apple-snail Pomacea canaliculata (Architaenioglossa, Ampullariidae).

Development of Pomacea canaliculata from the gastrula stage until the first day after hatching is described. Trochophore embryos are developed after gastrulation, showing the prototroch as a crown of ciliated orange-brownish cells. However, no true veliger embryos are formed, since the prototroch does not fully develop into a velum. Afterward, the connection between the fore- and midgut is permeated and the midgut becomes full of the pink-reddish albumen, which is stored into a central archenteron's lake, from where it is accumulated into the large cells forming the midgut wall ("giant cells"). Electron microscopy of giant cells in late embryos showed that albumen is engulfed by large endocytic vesicles formed between the irregular microvilli at the top of these cells. By the end of intracapsular development, giant cells become gradually replaced by two new epithelial cell types which are similar to those found in the adult midgut gland: the pre-columnar and the pre-pyramidal cells. Pre-columnar cells have inconspicuous basal nuclei and are crowned by stereocilia, between which small endocytic vesicles are formed. Pre-pyramidal cells have large nuclei with 2-3 nucleoli and show a striking development of the rough endoplasmic reticulum. The genesis of the three cell lineages (giant, pre-columnar and pre-pyramidal cells) is hypothetically attributed to epithelial streaks that occur at both sides of the midgut since early stages of development.

Development beyond the gastrula stage and digestive organogenesis in the apple-snail Pomacea canaliculata (Architaenioglossa, Ampullariidae)

Development of Pomacea canaliculata from the gastrula stage until the first day after hatching is described. Trochophore embryos are developed after gastrulation, showing the prototroch as a crown of ciliated orange-brownish cells. However, no true veliger embryos are formed, since the prototroch does not fully develop into a velum. Afterward, the connection between the fore- and midgut is permeated and the midgut becomes full of the pink-reddish albumen, which is stored into a central archenteron's lake, from where it is accumulated into the large cells forming the midgut wall ("giant cells"). Electron microscopy of giant cells in late embryos showed that albumen is engulfed by large endocytic vesicles formed between the irregular microvilli at the top of these cells. By the end of intracapsular development, giant cells become gradually replaced by two new epithelial cell types which are similar to those found in the adult midgut gland: the pre-columnar and the pre-pyramidal cells. Pre-columnar cells have inconspicuous basal nuclei and are crowned by stereocilia, between which small endocytic vesicles are formed. Pre-pyramidal cells have large nuclei with 2-3 nucleoli and show a striking development of the rough endoplasmic reticulum. The genesis of the three cell lineages (giant, pre-columnar and pre-pyramidal cells) is hypothetically attributed to epithelial streaks that occur at both sides of the midgut since early stages of development.

Development beyond the gastrula stage and digestive organogenesis in the apple-snail Pomacea canaliculata (Architaenioglossa, Ampullariidae)

Development of Pomacea canaliculata from the gastrula stage until the first day after hatching is described. Trochophore embryos are developed after gastrulation, showing the prototroch as a crown of ciliated orange-brownish cells. However, no true veliger embryos are formed, since the prototroch does not fully develop into a velum. Afterward, the connection between the fore- and midgut is permeated and the midgut becomes full of the pink-reddish albumen, which is stored into a central archenterons lake, from where it is accumulated into the large cells forming the midgut wall ("giant cells"). Electron microscopy of giant cells in late embryos showed that albumen is engulfed by large endocytic vesicles formed between the irregular microvilli at the top of these cells. By the end of intracapsular development, giant cells become gradually replaced by two new epithelial cell types which are similar to those found in the adult midgut gland: the pre-columnar and the pre-pyramidal cells. Pre-columnar cells have inconspicuous basal nuclei and are crowned by stereocilia, between which small endocytic vesicles are formed. Pre-pyramidal cells have large nuclei with 2-3 nucleoli and show a striking development of the rough endoplasmic reticulum. The genesis of the three cell lineages (giant, pre-columnar and pre-pyramidal cells) is hypothetically attributed to epithelial streaks that occur at both sides of the midgut since early stages of development.(AU)

Observation of the embryonic development in Pseudoplatystoma coruscans (Siluriformes: Pimelodidae) under light and scanning electron microscopy.

Pseudoplatystoma coruscans is a very popular species for tropical fish culture as it has boneless meat of delicate taste and firm texture. Few studies on fish reproductive biology refer to the morphological features of eggs. The goal, therefore, of this present work was to perform a structural and ultrastructural analysis of fertilization and embryonic development in P. coruscans. The incubation period, from fertilization to hatching, lasts 13 h at 28/29 degrees C and 18 h at 27 degrees C. The oocytes had a mean diameter of 0.95 mm and hatched larvae were 2.55 mm in diameter. Analysing their development, we observed round, yellow oocytes that bore a double chorion membrane and a single micropyle. At 10 s after fertilization, several spermatozoa were detected attached to the oocyte surface. After 1 min of development, a fertilization cone that obstructed the micropyle could be observed. Segmentation started between 20 and 30 min after fertilization, when the egg cell was then formed. The first cleavage occurred between 30 and 45 min after fertilization, prior to reaching the morula stage (75 and 90 min after fertilization). The epiboly movement started at 120 and 180 min after fertilization and ended at 360 and 480 min after fertilization. Differentiation between cephalic and caudal region was detected after 420 and 600 min after fertilization and larvae hatched between 780 and 1080 min after fertilization. Seven main embryonic development stages were identified: egg cell, cleavage, morula, blastula, gastrula, segmentation with differentiation between cephalic and caudal regions, and hatching.

A comparative analysis of frog early development.

The current understanding of Xenopus laevis development provides a comparative background for the analysis of frog developmental modes. Our analysis of development in various frogs reveals that the mode of gastrulation is associated with developmental rate and is unrelated to egg size. In the gastrula of the rapidly developing embryos of the foam-nesting frogs Engystomops coloradorum and Engystomops randi, archenteron and notochord elongation overlapped with involution at the blastopore lip, as in X. laevis embryos. In embryos of dendrobatid frogs and in the frog without tadpoles Eleutherodactylus coqui, which develop somewhat more slowly than X. laevis, involution and archenteron elongation concomitantly occurred during gastrulation; whereas elongation of the notochord and, therefore, dorsal convergence and extension, occurred in the postgastrula. In contrast, in the slow developing embryos of the marsupial frog Gastrotheca riobambae, only involution occurred during gastrulation. The processes of archenteron and notochord elongation and convergence and extension were postgastrulation events. We produced an Ab against the homeodomain protein Lim1 from X. laevis as a tool for the comparative analysis of development. By the expression of Lim1, we were able to identify the dorsal side of the G. riobambae early gastrula, which otherwise was difficult to detect. Moreover, the Lim1 expression in the dorsal lip of the blastopore and notochord differed among the studied frogs, indicating variation in the timing of developmental events. The variation encountered gives evidence of the modular character of frog gastrulation.

Gastrulation of Gastrotheca riobambae in comparison with other frogs.

Blastopore formation, the embryonic disk, archenteron and notochord elongation, and Brachyury expression in the marsupial frog Gastrotheca riobambae was compared with embryos of Xenopus laevis and of the dendrobatids Colostethus machalilla and Epipedobates anthonyi. In contrast with X. laevis embryos, the blastopore closes before elongation of the archenteron and notochord in the embryos of G. riobambae and of the dendrobatid frogs. Moreover, the circumblastoporal collar (CBC) thickens due to the accumulation of involuted cells. An embryonic disk, however, is formed only in the G. riobambae gastrula. We differentiate three gastrulation patterns according to the speed of development: In X. laevis, elongation of the archenteron and notochord begin in the early to mid gastrula, whereas in the dendrobatids C. machalilla and E. anthonyi the archenteron elongates at mid gastrula and the notochord elongates after gastrulation. In G. riobambae, only involution takes place during gastrulation. Archenteron and notochord elongation occur in the post gastrula. In the non-aquatic reproducing frogs, the margin of the archenteron expands anisotropically, resulting in an apparent displacement of the CBC from a medial to a posterior location, resembling the displacement of Hensen's node in the chick and mouse. The differences detected indicate that amphibian gastrulation is modular.

Regulatory network for cell shape changes during Drosophila ventral furrow formation.

Rapid and sequential cell shape changes take place during the formation of the ventral furrow (VF) at the beginning of Drosophila gastrulation. At the cellular level, this morphogenetic event demands close coordination of the proteins involved in actin cytoskeletal reorganization. In order to construct a regulatory network that describes these cell shape changes, we have used published genetic and molecular data for 18 genes encoding transcriptional regulators and signaling pathway components. Based on the dynamic behavior of this network we explored the hypothesis that the combination of three recognizable phenotypes describing wild type or mutant cell types, during VF invagination, correspond to different activation states of a specific set of these gene products, which are point attractors of the regulatory network. From our results, we recognize missing components in the regulatory network and suggest alternative pathways in the regulation of cell shape changes during VF formation.

Morphogenesis of extraembryonic membranes and placentation in Mabuya mabouya (Squamata, Scincidae).

Topological and histological analyses of Mabuya mabouya embryos at different developmental stages showed an extraembryonic membrane sequence as follows: a bilaminar omphalopleure and progressive mesodermal expansion around the whole yolk sac at gastrula stages; mesodermal split and formation of an exocoelom in the entire embryonic chamber at neurula stages; beginning of the expansion of the allantois into the exocoelom to form a chorioallantoic membrane at pharyngula stages; complete extension of the allantois into the exocoelom between limb-bud to preparturition stages. Thus, a placental sequence could be enumerated: bilaminar yolk sac placenta; chorioplacenta; allantoplacenta. All placentas are highly specialized for nutrient absorption from early developmental stages. The bistratified extraembryonic ectoderm possesses an external layer with cuboidal cells and a microvillar surface around the whole yolk sac, which absorbs uterine secretions during development of the bilaminar yolk sac placenta and chorioplacenta. During gastrulation, with mesodermal expansion a dorsal absorptive plaque forms above the embryo and several smaller absorptive plaques develop antimesometrially. Both structures are similar histologically and are active in histotrophic transfer from gastrula stages until the end of development. The dorsal absorptive plaque will constitute the placentome and paraplacentome during allantoplacental development. At late gastrula-early neurula stages some absorptive plaques form chorionic concavities or chorionic bags that are penetrated by a long uterine fold and seem to have a specialized histotrophic and/or metabolic role. The extraembryonic mesoderm does not ingress into the yolk sac and neither an isolated yolk mass nor a yolk cleft are formed. This derived pattern of development may be related to the drastic reduction of the egg size and obligatory placentotrophy from early developmental stages. Our results show new specialized placentotrophic structures and a novel arrangement of extraembryonic membrane morphogenesis for Squamata.

The expression of Brachyury (T) during gastrulation in the marsupial frog Gastrotheca riobambae.

Gastrulation in the marsupial frog Gastrotheca riobambae has been analyzed by the distribution of the Brachyury (T) protein. Comparison with other amphibians provides mechanistic insights, since G. riobambae develops slowly and has the most divergent mode of amphibian gastrulation, producing an embryonic disk. The T pattern indicates that the prospective mesoderm is superficial, as in many amphibians. The dorsal blastopore lip could not be identified by the expression of T, or by morphological criteria, thus it is unknown whether Gastrotheca embryos have a dorsal organizer before or after blastopore closure. The circumblastoporal and notochordal expression of T, which are temporally contiguous in Xenopus, are separated in Gastrotheca, implying that distinct regulatory mechanisms may control the expression of T in its two domains. The separation of the T pattern also indicates that involution at the blastopore is separate from notochord formation. In addition, extension of the archenteron and notochord occurs after blastopore closure, suggesting that dorsal convergence and extension have been delayed until after blastopore closure. Therefore, dorsal convergence and extension need not be the cause of blastopore closure in Gastrotheca. The separation of gastrulation events in embryos that have not been experimentally manipulated, such as those of Gastrotheca, helps in understanding the distinct nature of gastrulation processes.