Both of marine fish species Oryzias melastigma and Pagrus major all failing in early localization at embryo stage by vasa RNA.

Germ cells are essential for gonadal development. As precursors of germ cells, primordial germ cells (PGCs) are particularly important for germline formation. However, the research on distribution patterns of PGCs in marine fish is very limited, especially for economic species. The vasa gene has been widely used as marker to identify PGCs origination and migration because of vasa RNA is a component of germ plasm. In this study, we isolated full-length vasa cDNA (Omvas and Pmvas) from marine medaka (Oryzias melastigma) and red seabream (Pagrus major), detected vasa transcripts in different tissues by RT-PCR and described vasa expression patterns during embryogenesis and gametogenesis by in situ hybridization. At the same time, we also explored the relationship between early distribution of germ plasm components and species evolution. The results demonstrated that deduced amino acid sequence of Omvas and Pmvas shared several conserved motifs of Vasa homologues and high identity with other teleost, and vasa transcripts were exclusively detected in early germ cells of gonad. During embryogenesis, vasa RNA of both fishes, like medaka (Oryzias latipes), failed to localize at cleavage furrows and distributed uniformly throughout each blastomere. This study firstly discovered that the marine economic fish, red seabream, lost vasa RNA early specific localization at cleavage furrows and distinctive distribution in germ cells. In addition, compared with other teleost, we found that early distribution of germ plasm might not relate to species evolution. This will improve our understanding of vasa localization modes in teleost, and facilitate fish germ cell manipulation.

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.

The effect of spatial position and age within an egg-clutch on embryonic development and key metabolic enzymes in two clownfish species, Amphiprion ocellaris and Amphiprion frenatus.

Since the size of newly hatched larval fish is directly related to egg size, small differences in initial egg size can be critical to survival and further development of offspring. Underlying processes causing size variation in fish offspring are still not entirely understood. In this study we investigated whether the spatial position of an individual egg within a clutch affects size variation in two benthic spawning coral reef fishes, the clownfishes Amphiprion ocellaris and A. frenatus. To evaluate the effects of within-clutch position on embryonic development, egg growth metrics and protein content were analysed on day 2, 5 and 8 after deposition (adp). Additionally the activities of the key metabolic enzymes citrate synthase (CS) and lactate dehydrogenase (LDH) were investigated to evaluate the physiological status of the embryos. Central eggs of A. frenatus were significantly longer and heavier than peripheral eggs only on day 2 and 5 adp (2.07 mg, 2.59 mm vs. 1.84 mg, 2.49 mm). No significant differences were observed in A. ocellaris between eggs originating from a central or peripheral (5 mm from edge) position (1.33 mg, 2.26 mm vs. 1.15 mg, 2,18 mm). Diameter of the eyes did not differ between the two fish species nor between different positions, for any age group. The protein content of eggs (7.5% of wet weight) was independent of age, position and species. Enzymatic activity increased from 2 adp until peak activity was observed for both enzymes on day 8 adp, independent from position. The range of CS- and LDH-activity was 0.3-13.0 and 0.2-71.7 U g-1 wet weight, respectively. Significant differences in enzymatic activity were observed between age groups in both species, which in connection with significantly larger eggs of A. frenatus at day 2 and 5 adp could hint at a better O2 supply of central eggs. Potential implications for captive breeding are given.

Thermal influence on the embryonic development and hatching rate of the flameback pygmy angelfish Centropyge aurantonotus eggs.

The flameback pygmy angelfish Centropyge aurantonotus, highly appreciated and valued by the aquarium market, is heavily harvested and traded. Temperature is one of the abiotic factors that has the most influence on fish development, especially in the early stages of life. For captive production, it is essential to know the appropriate environmental parameters for each species. In this sense, this study aimed to evaluate the influence of temperature on the embryonic development and hatching rates of C. aurantonotus incubated at six different temperatures (20, 22, 24, 26, 28, 30°C). Embryonic development events were very similar in terms of morphological and chronological characteristics compared with other species of the genus Centropyge. Incubation time was inversely proportional to temperature. The treatment at 22°C required twice the time of that required by 30°C treatment for hatching to occur. The best incubation temperature range was 24-28°C. Values below 22°C and at 30°C showed lower hatching rates compared with other treatments. Based on these results, the recommended temperature at which to incubate C. aurantonotus eggs is between 24-28°C.

Boat noise affects the early life history of two damselfishes.

Anthropogenic noise can have a negative effect on the physiology and survival of marine fishes. Most research has focused on later life-stages, and few studies have investigated the effects of human-induced noise on embryogenesis. The current study investigated whether playback of motorboat noise affected the embryogenesis of the coral reef damselfishes, Amphiprion melanopus and Acanthochromis polyacanthus. Embryos reared under the playback of boat noise had faster heart rates compared to the ambient reef controls. The effects of noise on morphological development differed between species and the fundamental interrelationships between early life history characteristics changed dramatically under boat noise for Ac. polyacanthus. Noise treatments did not alter the survival rates of embryos under laboratory conditions. Although species specific, our findings suggest that anthropogenic noise causes physiological responses in fishes during embryogenesis and these changes have direct impacts on their development and these alterations may have carry-over effects to later life stages.

Evolutionarily conserved vasa identifies embryonic and gonadal germ cells in spinyhead croaker Collichthys lucidus.

In this study, a 2198 bp full-length cDNA of spinyhead croaker Collichthys lucidus vasa gene encoding 616 amino-acid residues was obtained. Multiple alignment revealed that C. lucidus vasa has eight conserved characteristic motifs of the DEAD box protein family and has the highest identity to large yellow croaker Larimichthys croceas. Reverse-transcription (RT)-PCR and Western blot analyses indicated that the vasa messenger (m)RNA and Vasa protein are specifically expressed in the gonads in both sexes. In situ hybridisation (ISH) demonstrated that vasa RNA is exclusively detected in the germ cells in C. lucidus gonads and its temporospatial expression reveals a dynamic pattern during oogenesis. Surprisingly, C. lucidus vasa 3'UTR can direct stable and specific GFP expression in the primordial germ cells (PGC) of medaka Oryzias latipes embryos. Taken together, these results suggest that because C. lucidus vasa expression delineates critical stages of oogenesis, it may be a useful molecular marker for the identification of gonadal germ cells, facilitating the isolation and utilization of germ cells in future study.

Ontogeny of urea and ammonia transporters in mahi-mahi (Coryphaena hippurus) early life stages.

The mechanism(s) of ammonia and urea excretion in freshwater fish have received considerable attention; however, parallel investigations of seawater fish, specifically in the early life stages are scarce. The first objective of this study was to evaluate the patterns of ammonia and urea excretion in mahi-mahi (Coryphaena hippurus) up to 102  hours post fertilization (hpf). Similar to other teleosts, mahi embryos are ureotelic before hatch and gradually switch to being ammoniotelic around the time of hatch. The second objective was to characterize mRNA levels of ammonia transporters (Rhag, Rhbg, Rhcg1 and Rhcg2), as well as urea transporter (UT) and sodium hydrogen exchangers (NHE3 and NHE2) during mahi development. As predicted, the mRNA levels of the Rhesus glycoprotein (Rh) genes, especially Rhag, Rhbg and the UT gene were highly consistent with the ontogeny of ammonia and urea excretion rates. Further, the localization of each transporter was examined in larvae collected at 60 and 102 hpf using in situ hybridization. Rhag was expressed in the gills, yolk sac, and operculum. Rhbg was expressed in the gills and upper mouth. Rhcg1 and NHE3 were co-localized in the sub-operculum, and Rhcg2 was expressed in the skin. Together, these results indicate that urea excretion is critical for ammonia detoxification during embryonic development and that Rh proteins are involved in ammonia excretion via gills and yolk sac, possibly facilitated by NHE3.

Spawning kinetics and egg/larval quality of greater amberjack (Seriola dumerili) in response to multiple GnRHa injections or implants.

The greater amberjack (Seriola dumerili) is one of the most promising finfish species for aquaculture, due to its cosmopolitan distribution and acceptability, high growth rates and large size. However, lack of reproduction control has been one of the main bottlenecks for its commercialization. The study examined two endocrine methods for the induction of oocyte maturation/ovulation and spawning, which are based on the induction of endogenous Luteinizing Hormone (LH) release from the pituitary, through the use of Gonadotropin Releasing Hormone agonist (GnRHa) either in the form of sustained-release delivery systems (implants) or injections (acute release). The stock (n = 28) consisted of wild fish captured in 2011 and each GnRHa administration method was conducted in two replicates, with 6 and 8 fish per tank, respectively, at a 1:1 sex ratio. Fish were given a GnRHa injection once a week (three administrations), or a GnRHa implant every 2 weeks (two administrations). Mean daily relative fecundity was significantly higher (t1.3 = -5.24, P = 0.012) in the implanted fish (15,170 ±â€¯2,738 eggs kg-1 female day-1) compared to the injected fish (6,119 ±â€¯2,790 eggs kg-1 female day-1). Total relative fecundity was also significantly higher (t1.3 = -9.93, P = 0.003) in the implanted fish (102,402 ±â€¯20,337 eggs kg-1 female) compared to the injected ones (26,517 ±â€¯9,938 eggs kg-1 female), but there were no differences in the quality of eggs in terms of fertilization, 24-h embryo survival, hatching and 5-d larval survival. The number of females with fully vitellogenic oocytes eligible for induction of spawning decreased from the initial to the final sampling, from 7 to 6 females for the GnRHa implant treatment and from 7 to 3 females for the GnRHa injection treatment. In addition to apparently promoting the proper endocrine changes leading to multiple cycles of oocyte maturation, ovulation and spawning, and thus producing larger numbers of eggs; the use of GnRHa implants may be more appropriate in greater amberjack than multiple injections because (a) it also stimulates vitellogenesis and (b) involves less handling of the fish.

Characterization of first blastomeres in Patagonian blenny (Eleginops maclovinus) (Perciformes: Eleginopidae).

SummaryThere is no information about the characteristics of early cleavage in the Patagonian blennie (Eleginops maclovinus), which can be used as a diagnostic tool for embryo quality. The purpose of this investigation, therefore, was to characterize the first blastomeres of E. maclovinus morphologically. Of a 'pool' of incubated eggs at 10.7 ± 0.5°C, 100 microphotographs of blastodiscs were extracted at different incubation periods from 0.25 to 5 h after fertilization and analyzed. Blastodiscs taken at 3.5, 4.0 and 5.0 h were characterized and classified into symmetric or asymmetric groups according to their morphology. The proportions of length (L) and width (W) of each blastomere were determined to establish its symmetry. Additionally, 20 microphotographs of blastodiscs of normal appearance were analyzed morphologically (control blastodisc: CB) and compared other blastodiscs (4.0 and 5.0 h). The results showed that before fertilization oocytes presented a somehow turgid aspect (maximum average diameter of 987 ± 41 µm) and after fertilization and hydration, their diameter increased to 1001.5 ± 11 µm (but not statistically significant) and presented a spherical shape. First cleavage ends after 3.5 h of development, forming two blastomeres 467 ± 45 µm length (L) and 328 ± 21 µm width (W) with a L/W ratio of 1.43 ± 0.19. The second cleavage ends after development at 4.5 h forming four blastomeres 238 ± 65 µm length and 227 ± 65 µm width with a ratio L/W of 1.06 ± 0.09. Five categories were identified during the blastomere characterization: 70% normal or symmetric; 8% with odd numbers of blastomeres; 6% unequal; 6% 'pie shaped' and 10% amorphous.

Effects of Deepwater Horizon crude oil on ocular development in two estuarine fish species, red drum (Sciaenops ocellatus) and sheepshead minnow (Cyprinodon variegatus).

Polycyclic aromatic hydrocarbons (PAHs) present in crude oil have been shown to cause the dysregulation of genes important in eye development and function, as well as morphological abnormalities of the eye. However, it is not currently understood how these changes in gene expression are manifested as deficits in visual function. Embryonic red drum (Sciaenops ocellatus) and sheepshead minnow (Cyprinodon variegatus) were exposed to water accommodated fractions (WAFs) of weathered crude oil and assessed for visual function using an optomotor response assay in early life-stage larvae, with subsequent samples taken for histological analysis of the eyes. Larvae of both species exposed to increasing concentrations of oil exhibited a reduced optomotor response. The mean diameters of retinal layers, which play an important role in visual function and image processing, were significantly reduced in oil-exposed sheepshead larvae, though not in red drum larvae. The present study provides evidence that weathered crude oil has a significant effect on visual function in early life-stage fishes.

Characterization and localization of primordial germ cells in Totoaba macdonaldi.

The totoaba, Totoaba macdonaldi, is an endangered fish of the Gulf of California with high economic and ecological potential. Therefore, our purpose was to characterize the Primordial Germ Cells (PGCs) of this Sciaenid with two objectives: (1) to provide the basis for PGCs cryopreservation to preserve the genetic resources and (2) to take the first step to know the gonadal genesis and sex differentiation of totoaba. Immunofluorescence analysis performed from 2-cell stage to 8-day after hatch (DAH) shows that Vasa protein is specific for PGCs. These cells were first observed in the peripheral and dorsal regions of the blastodisc at approximately the 50%-epiboly stage and migrated to both sides of embryo body during the development. Finally, at 7 DAH the PGCs of the hatching embryo reached the place where the gonad will be developed. Histology analysis of larvae showed a genital ridge with enclosed PGCs on the dorsal side of the peritoneum at 9 DAH, gonadal primordium growth was observed at 11 DAH as a result of the interaction between PGCs and somatic cells derived from the peritoneum. Results of qPCR showed that vasa expression was restricted to the embryonic and early larval development, highest values were observed in 2-cell and mid-blastula stage suggesting the maternal inheritance of vasa mRNA. These findings support the hypothesis of preformation in T. macdonaldi PGCs. The migration pattern of PGCs allow us to recommend the isolation and subsequent cryopreservation of these cells before 7 DAH when the embryonic and larval development is given at 21 °C.

RNA sequencing of early round goby embryos reveals that maternal experiences can shape the maternal RNA contribution in a wild vertebrate.

BACKGROUND: It has been proposed that non-genetic inheritance could promote species fitness. Non-genetic inheritance could allow offspring to benefit from the experience of their parents, and could advocate pre-adaptation to prevailing and potentially selective conditions. Indeed, adaptive parental effects have been modeled and observed, but the molecular mechanisms behind them are far from understood. RESULTS: In the present study, we investigated whether maternal RNA can carry information about environmental conditions experienced by the mother in a wild vertebrate. Maternal RNA directs the development of the early embryo in many non-mammalian vertebrates and invertebrates. However, it is not known whether vertebrate maternal RNA integrates information about the parental environment. We sequenced the maternal RNA contribution from a model that we expected to rely on parental effects: the invasive benthic fish species Neogobius melanostomus (Round Goby). We found that maternal RNA expression levels correlated with the water temperature experienced by the mother before oviposition, and identified temperature-responsive gene groups such as core nucleosome components or the microtubule cytoskeleton. CONCLUSIONS: Our findings suggest that the maternal RNA contribution may incorporate environmental information. Maternal RNA should therefore be considered a potentially relevant pathway for non-genetic inheritance. Also, the ability of a species to integrate environmental information in the maternal RNA contribution could potentially contribute to species fitness and may also play a role in extraordinary adaptive success stories of invasive species such as the round goby.

Effect of vitrification solutions on survival rate of cryopreserved Epinephelus moara embryos.

Embryo cryopreservation is important for long-term preservation of germplasm and assisted reproduction. However, it is still very difficult to obtain viable embryos from cryopreserved fish embryos. In this study, embryos of Epinephelus moara were used to investigate the effects of various cryopreservation methods. Embryos in stages 10 pairs somite (10S), 18 pairs somite (18S), 22 pairs somite (22S), tail-bud (TB), embryo twitching (ET) and pre-hatch (PH) were treated with five-step equilibrium penetration in 40% PMG3T vitrification solution, which contained 15.75% 1,2-propylene glycol, 10.50% Methanol, 8.75% Glycerol and 5.00% Trehalose. We found that 18S, 22S, TB and ET stage embryos had higher survival rates and were more tolerant to the vitrification solution. Five-step equilibrium treatments on the embryos at the tail-bud stage were performed using two vitrification solutions: 40% PMG3T and 40% PMG3S, which consisted of 15.75% 1,2-propylene glycol, 10.50% Methanol, 8.75% Glycerol and 5.00% Sucrose. The embryonic survival rate under PMG3S treatment (63.36%) was significantly higher than PMG3T treatment (43.93%) (P < 0.05). PMG3S and PMG3T with concentrations of 35%, 40% and 45% were tested on tail-bud stage embryos. Higher concentration of the vitrification solution led to significantly lower embryonic survival rate (P < 0.05). The survival rate was 36.79-72.05% in PMG3S, and 37.11-55.18% in PMG3T, and there were non-significant differences in embryonic development and malformation rates among the groups treated with different concentrations. The embryonic normal development rates in PMG3S and PMG3T were 21.27% and 11.04%, and the malformation rates were 36.13% and 31.04%, respectively. The optimum treatment condition was 40 min using 40% PMG3S on embryos at the tail-bud stage. Both PMG3S and PMG3T were used for cryopreserving embryos at 16 pairs somite, tail-bud and ET stage in liquid nitrogen, where we obtained 190 surviving embryos, and 44 fishes underwent normal development and hatched. The survival rate of cryopreserved embryos was 5.15%, the normal development rate was 1.31%, and the malformation rate was 3.66%. We found that PMG3S and PMG3T were effective for cryopreservation of Epinephelus moara embryos. The results provide a foundation for further explorations of fish embryo cryopreservation techniques.

Nanos3 not nanos1 and nanos2 is a germ cell marker gene in large yellow croaker during embryogenesis.

In this study, three nanos gene subtypes (Lcnanos1, Lcnanos2 and Lcnanos3) from Larimichthys crocea, were cloned and characterized. We determined the spatio-temporal expression patterns of each subtype in tissues as well as the cellular localization of mRNA in embryos. Results showed that deduced Nanos proteins have two main homology domains: N-terminal CCR4/NOT1 deadenylase interaction domain and highly conserved carboxy-terminal region bearing two conserved CCHC zinc-finger motifs. The expression levels of Lcnanos1 in testis were significantly higher than other tissues, followed by heart, brain, eye, and ovary. Nevertheless, both Lcnanos2 and Lcnanos3 were restrictedly expressed in testis and ovary, respectively. No signals of Lcnanos1 and Lcnanos2 expression were detected at any developmental stages during embryogenesis. On the contrary, the signals of Lcnanos3 were detected in all stages examined. Lcnanos3 transcripts were firstly localized to the distal end of cleavage furrow at the 2-cell stage. Subsequently, mounting positive signals started to appear in a small number of cells as the embryo developed to blastula stage and early-gastrula stage. As development proceeded, positive signals were found in the primitive gonadal ridge. These cells of Lcnanos3 positive signals implied the specification of the future PGCs at this stage. It also suggested that PGCs of croaker originate from four clusters of cells which inherit maternal germ plasm at blastula stage. Furthermore, we preliminarily analyzed the migration route of PGCs in embryos of L. crocea. In short, this study laid the foundation for studies on specification and development of germ cell from L. crocea during embryogenesis.

Refrigeração do sêmen da garoupa-verdadeira Epinephelus marginatus / Refrigerated storage of dusky grouper Epinephelus marginatus sperm

Foram estudados diferentes diluentes no processo de refrigeração do sêmen da garoupa-verdadeira. Inicialmente, a taxa e a duração da motilidade e a concentração espermática foram avaliadas, caracterizando a qualidade do sêmen fresco. Para os testes de refrigeração a 4ºC, diferentes diluentes foram testados em atmosfera normal e modificada (100% oxigênio). O sêmen fresco apresentou concentração espermática de 3,1±0,2 x 109 células mL-1, motilidade média de 90%, permanecendo móvel, em média, por 3.060 segundos. No experimento de refrigeração, a taxa e a duração média da motilidade foram mantidas adequadas durante 144 horas para o diluente A (70%; 3100 segundos) em atmosfera normal. Na atmosfera modificada, a qualidade do sêmen caiu drasticamente durante as primeiras 24 horas, independentemente do diluente empregado, não propiciando vantagem em sua utilização. A refrigeração do sêmen da garoupa-verdadeira permite manter por até 144 horas uma apropriada qualidade espermática.(AU)

This study aimed to evaluate different extenders in refrigerated storage of dusky grouper sperm. Initially, motility rate, motility time, and sperm density were analyzed to characterize the fresh sperm quality. Different diluents were used in the refrigerated storage sperm at 4ºC in normal atmosphere and modified atmosphere (100% oxygen). The fresh sperm has a spermatozoa density of 3.1±0.2 x 109 spermatozoa mL-1, motility rate 90% and motility time of 3,060 seconds. In the experiment of refrigerated storage, the motility rate and the motility time were maintained appropriate for 144 hours for the extender A (70%; 3,100 seconds) in normal atmosphere. In the modified atmosphere, the sperm quality decreased drastically during the first 24 hours, independently of the diluent used, not propitiating advantages. The refrigerated sperm of dusky grouper is a viable alternative for the short-term storage, being possible, to maintain for up to 144 hours an appropriate sperm viability.(AU)

Development, and effect of water temperature on development rate, of pikeperch Sander lucioperca embryos.

Knowledge of embryo development is essential to the application of reproductive biotechnology in aquaculture, including for pikeperch Sander lucioperca. We describe pikeperch embryo development and demonstrated effects of temperature on the duration of embryogenesis. Developmental stages in embryos incubated at 15 °C were identified as zygote, 0-1.5 h post-fertilization (hpf); cleavage, 2.5-7.5 hpf; blastula, 9-18.75 hpf; gastrula, 21-39, hpf; segmentation, 45-105 hpf; and hatching, 125-197 hpf. Additional groups of eggs were fertilized and incubated at 10, 15, 20, and 25 °C to document stages of development, development rate, and survival. The optimal fertilization and incubation temperature was shown to be 15 °C, with the highest fertilization, survival, and hatching rates. Embryo development was slower at 10 °C, with 45% of fertilized embryos surviving to hatching. Development was accelerated at 20 °C, and resulted in a 56% survival rate of fertilized embryos. At 25 °C, embryos did not develop to the blastula stage. Pikeperch could be a valuable percid model for research in which flexible incubation temperatures is required.

Developmental transcriptomic analyses for mechanistic insights into critical pathways involved in embryogenesis of pelagic mahi-mahi (Coryphaena hippurus).

Mahi-mahi (Coryphaena hippurus) is a commercially and ecologically important species of fish occurring in tropical and temperate waters worldwide. Understanding early life events is crucial for predicting effects of environmental stress, which is largely restricted by a lack of genetic resources regarding expression of early developmental genes and regulation of pathways. The need for anchoring developmental stages to transcriptional activities is highlighted by increasing evidence on the impacts of recurrent worldwide oil spills in this sensitive species during early development. By means of high throughput sequencing, we characterized the developmental transcriptome of mahi-mahi at three critical developmental stages, from pharyngula embryonic stage (24 hpf) to 48 hpf yolk-sac larva (transition 1), and to 96 hpf free-swimming larva (transition 2). With comparative analysis by multiple bioinformatic tools, a larger number of significantly altered genes and more diverse gene ontology terms were observed during transition 2 than transition 1. Cellular and tissue development terms were more significantly enriched in transition 1, while metabolism related terms were more enriched in transition 2, indicating a switch progressing from general embryonic development to metabolism during the two transitions. Special focus was given on the most significant common canonical pathways (e.g. calcium signaling, glutamate receptor signaling, cAMP response element-binding protein signaling, cardiac ß-adrenergic signaling, etc.) and expression of developmental genes (e.g. collagens, myosin, notch, glutamate metabotropic receptor etc.), which were associated with morphological changes of nervous, muscular, and cardiovascular system. These data will provide an important basis for understanding embryonic development and identifying molecular mechanisms of abnormal development in fish species.

A new type of homodiploid fish derived from the interspecific hybridization of female common carp × male blunt snout bream.

It is commonly believed that hybridization might lead to the formation of new polyploidy species, but it is unclear whether hybridization can produce a new homodiploid species. Here, we report the spontaneous occurrence of a new crucian carp-like homodiploid fish (2n = 100) that originated from the interspecific hybridization of female common carp (Cyprinus carpio, Cyprininae, 2n = 100) × male blunt snout bream (Megalobrama amblycephala, Cultrinae, 2n = 48). The phenotype and reproductive traits of this new crucian carp-like homodiploid fish were found to be very similar to those of the existing diploid species (diploid crucian carp; Carassius auratus). FISH and 5S rDNA analyses revealed that the genotype of the crucian carp-like homodiploid fish differs from those of its parents but is closely related to that of diploid crucian carp. The results provide evidence of the existence of a possible route through which the distant hybridization of this cross can generate crucian carp. The new type of homodiploid fish is of great value in fish genetic breeding and for studying the early evolutionary process.

Assessment of cathepsin mRNA expression and enzymatic activity during early embryonic development in the yellowtail kingfish Seriola lalandi.

In pelagic species such as Seriola lalandi, survival of both the eggs and embryos depends on yolk processing during oocyte maturation and embryo development. The main enzymes involved in these processes are the cathepsins, which are essential for the hydration process, acquiring buoyancy and nutrition of the embryo before hatching. This study aimed to investigate the mRNA expression profiles of cathepsins B, D and L (catb, catd and catl) and the activity of these enzymes during early development in S. lalandi. We included previtellogenic oocytes (PO). All three enzymes were highly expressed in PO, but the expression was reduced throughout development. Between PO and recently spawned eggs (E1) the transcript to catb and catd decreased, unlike catl. Cathepsin B activity, showed stable levels between PO until blastula stage (E4). High activities levels of cathepsins D and L were observed in E1 in comparison with later developmental stages. Cathepsin L activity remained constant until E1, consistent with observations in other pelagic spawners, where its participation in a second protolithic cleavage of the yolk proteins, has been proposed for this enzyme. Their profiles of both mRNA expression and enzymatic activity indicate the importance of these enzymes during early development and suggest different roles in egg yolk processing for the hydration process and nutrition in early embryos in this species.

Molecular characterization and expression analysis during embryo development of CD4-1 homologue in large yellow croaker Larimichthys crocea.

CD4+ helper T (Th) cells are a master component of the adaptive immune response. CD4 is one of the most effective surface markers for identifying Th cells. In the present study, we cloned and characterized a CD4-1 homologue, LycCD4-1, from large yellow croaker Larimichthys crocea. The full-length cDNA of LycCD4-1 is 1695 bp long, encoding a protein of 462 amino acids. The deduced LycCD4-1 protein has a typical domain architecture as found in mammalian CD4 molecules, including a signal peptide, four extracellular immunoglobulin-like (Ig-like) domains, a transmembrane region, and a CXC signaling motif in the cytoplasmic tail. Four N-glycosylation sites and 10 cysteine residues were also found in LycCD4-1, which may be essential for its tertiary structure and succeeding function. Homology comparison showed that LycCD4-1 has 27.9-58.4% identity to other teleost fish CD4-1 molecules, and 16.4-20% identity to those of higher vertebrates. Genomic analysis revealed that the LycCD4-1 gene consisted of nine exons and eight introns and exhibited a similar exon-intron organization to other species CD4 genes except for a different intron length. Phylogenetic analysis showed that LycCD4-1 form a cluster with CD4-1 molecules in other fish species. The LycCD4-1 was constitutively expressed in all tissues tested, with a higher expression in gills and spleen. LycCD4-1 mRNA expression in the spleen and head kidney tissue was increased by poly (I:C) at 48 h, whereas its expression levels were somewhat down-regulated at 6 h and 72 h after bacterial vaccine induction in spleen. Unexpectedly, LycCD4-1 mRNA could be detected in each stage of early embryo development since fertilized eggs, with a higher level before mid-gastrula and the highest level in high blastocysts. These results will be helpful for better understanding molecular characteristics of CD4-1 and tracing origin of CD4-1+ cell precursors in fish.