Min Post-fertilization Research Articles

Reproduction, Early Development, and Larval Rearing of the Climbing Perch Anabas testudineus (Teleostei: Anabantidae) in Captivity

Induced spawning, early development, and larval rearing of the climbing perch Anabas testudineus were examined under captive conditions. Five female climbing perch [125.33 ± 3.27 mm SL; 70.67 ± 5.59 g BW] were paired with apparently mature males (108.50 ± 3.97 mm SL; 39.27 ± 4.70 g BW) and induced to spawn with 5,000 IU human chorionic gonadotropin (hCG) + 0.5 mL Ovaprim kg BW-1. All hCG + Ovaprim-injected fish spawned 9 – 11 h after hormone administration but saline-injected fish (control group) failed to spawn. Mean egg production per female, fertilization and hatching rates, and larval production per female were 7,667 ± 1,313, 98.11 ± 1.63, 61.01 ± 19.92, and 4,435 ± 1,041 %, respectively. Fertilized eggs (1.52 – 1.96 mm in dia) were almost spherical, non-adhesive, transparent, and buoyant due to a large, single oil globule (515 ± 33 μm in dia). First cleavage appeared 30 min post-fertilization (MPF) and egg development lasted for 20 – 24 h post-fertilization (HPF) (29°C). Newly hatched climbing perch larvae [1.73 ± 0.08 mm in total length (TL)] with yolk volume (YV) and oil globule volume (OGV) of 0.545 ± 0.104 and 0.072 ± 0.013 mm3, respectively, had no pigmented eyes or functional mouth parts and digestive tract, and immobile with the yolk sac pointed upward. Eyes became pigmented and both mouth and anus opened in 1 d old larvae (2.09 ± 0.04 mm TL) in preparation for exogenous feeding. Except for body depth (BD) and pre-anal length (PAL), a general increase in TL (1.73 – 3.72 mm), eye diameter (ED) (0.164 – 0.378 mm), head length (HL) (0.205 – 0.923 mm), and mouth gape (MG) size (0.308 – 0.552 mm) was observed, coinciding with yolk resorption in 7 d post-hatched larvae. Climbing perch larvae were reared at 4 stocking densities (25, 50, 75, 100 larvae L-1) and fed solely on live food (Brachionus rotundiformis from 2 – 12 d and Artemia nauplii from 13 – 40 d) or co-fed live food and microparticulate diet (MPD) (Artemia nauplii from 2 – 15 d and Artemia + MPD from 16 – 40 d). Mean survival rates at lower densities (25 – 50 larvae L-1) were significantly higher than those reared at 75 and 100 larvae L-1 (P < 0.05). Furthermore, climbing perch larvae co-fed Artemia nauplii and MPD showed better survival (28.67 – 81.00%) than those fed solely on live food (24.88 – 64.00%). However, at the end of the 40 d trial period, growth parameters did not vary significantly among densities (P > 0.05), except for the condition factor in larvae co-fed Artemia nauplii and MPD. Taken together, results demonstrate the effectiveness of hCG + Ovaprim for captive breeding and observations on the early development of climbing perch, and the feasibility of rearing the larvae at 25 – 50 L-1 on a combination of Artemia nauplii and MPD under laboratory conditions.

Alternative signal pathways underly fertilization and egg activation in a fish with contrasting modes of spawning

BackgroundThe processes of fertilization and egg activation are vital for early embryogenesis. However, while the mechanisms associated with key events during these processes differ among species and modes of spawning, the signal pathways underlying these processes are opaque for many fishes, including economically important species.ResultsWe investigated phenotypic traits, ultrastructure and protein expression levels in the eggs of the topmouth culter (Culter alburnus), a protected and economically important freshwater fish that exhibits two spawning modes, producing semi-buoyant eggs and adhesive eggs. Unfertilized eggs of C. alburnus were examined, as well as eggs at fertilization and 30 min post fertilization. Our results showed that in semi-buoyant eggs, energy metabolism was activated at fertilization, followed by elevated protein expression of cytoskeleton and extracellular matrix (ECM)-receptor interactions that resulted in rapid egg swelling; a recognized adaptation for lotic habitats. In contrast, in adhesive eggs fertilization initiated the process of sperm-egg fusion and blocking of polyspermy, followed by enhanced protein expression of lipid metabolism and the formation of egg envelope adhesion and hardening, which are adaptive in lentic habitats.ConclusionOur findings indicate that alternative signal pathways differ between modes of spawning and timing during the key processes of fertilization and egg activation, providing new insights into the molecular mechanisms involved in adaptive early embryonic development in teleost fishes.

Induction of gynogenesis with ultra-violet irradiated Koi carp (Cyprinus carpio haematopterus) sperm demonstrates the XX/XY sex determination system in Opsariichthys bidens

Opsariichthys bidens (Chinese hooksnout carp) is a small stream fish with high economic value. As the males of this species grow faster than females, it follows that mono-sex breeding has significant potential for commercial production. However, little is known about the sex determination system in O. bidens, this is the key process that is used to develop mono-sex breeding in fish. In the present study, we induced gynogenesis by fertilizing O.bidens eggs with ultra-violet (UV)- inactivated Cyprinus carpio haematopterus (Koi carp) sperm followed by cold shock treatment. Results showed that the optimal induction conditions was 25 min duration of cold shock treatment at 2 min post fertilization (mpf), the highest fertilization rate and hatching rate for gynogenic embryos were 68.24 ± 3.75% and 12.54 ± 1.59%, respectively. Embryos derived from gynogenesis developed normally, but the hybrid embryos failed (created with non-UV-inactivated Koi carp sperm and O.bidens eggs). Genetic analysis demonstrated only maternal inheritance in the gynogenetic offspring. Histological analysis further showed that the gonads of all gynogenetic fish were ovaries, thus suggesting that the sex-determining system of O. bidens is male heterogametic (XX-XY). The present study is the first to establish a procedure for inducing gynogenesis, we also identified the sex determination system (SD) of O. bidens, that will be possible to produce only male.

Early Life Cycle of the Pacific Thorny Oyster Spondylus crassisquama (Lamark, 1819)

Some genera of bivalve molluscs have an iconic ancestral history that predominates in the culture of a civilization through time. The Pacific thorny oyster Spondylus crassisquama is a living example of this. This study presents the description of the early life cycle until obtaining juveniles; and, for the latter, different types of substrates were evaluated. Larval culture was developed under laboratory conditions, chronologically recording the changes in larval morphology and behavior. The thorny oyster presents the first cell cleavage at 50 min postfertilization (PF). The transition between trochophore phase and the first D-veliger larvae was observed around 20–24 h PF. Above 75% of the larvae with presence of a serrate double ring showed ocular eyespot on the 16th day PF. Between the 16th and 18th days PF, the formation of the foot of the larvae was observed. The first postlarvae were observed on the 20th day with a total length of 553.0 ± 150.10 µm, distinguished by the appearance of the dissoconch. After 30 days PF, the first settled juveniles were observed with sizes of 1.6 ± 0.41 mm. Finally, the animals showed a preference for settlement on stones and concrete, and to a lesser extent on broken shells of S. crassisquama. This study shows that juvenile production is feasible under laboratory conditions; however, this requires more research to optimize the nursery process.

Study on artificial induction and early development of gynogenetic fat greenling Hexagrammos otakii

Fat greenling Hexagrammos otakii, belonging to Scorpaeniformes, is an important commercial marine fish species distributed in coast waters of China, Japan, and Korea. Artificial gynogenesis induction is one of the important techniques of chromosome manipulation and genetic breeding. The eggs are fertilized with genetic inactivated sperm, and genome information of the offspring is totally contributed by maternal genome. Until now, no gynogenesis induction was reported in H. otakii and other Scorpaeniformes species. The present study developed an effective gynogenesis induction method in H. otakii by applying hydrostatic pressure to the eggs activated with UV-irradiated homologous sperm. The optimal inducing conditions were 36,000 erg mm−2 for UV-irradiated and pressure of 65 MPa for 6 min at 20 min post fertilization. The relative hatching rates could reach 92%. The early development and growth before 6 month post hatching showed no difference between the gynogenetic and control diploid. The all-maternal inheritance was confirmed with 4 microsatellite loci. The genetic diversity of the control and gynogenetic diploid revealed with 20 microsatellite loci showed that the homozygosity of the gynogenetic diploid stocks was 0.84 in average, which was nearly double the value of the control diploid stocks (0.45 in average). Under scanning electron microscope, the egg envelop had a gelatinous-honeycomb before 0.5 h post fertilization (hpf). Then all or part of the gelatinous-honeycomb layer disappeared before 24 hpf. The uniform pores distributed to hexagonal pattern on the egg envelop were found in the control diploid and haploid groups. However, no pore on the egg envelop was found in the gynogenetic diploid from fertilization to hatching. These results would provide a valuable reference for gynogenesis induction in fish with viscous ovarian fluid coated eggs, although special incubation process to increase the survival rate should be further developed based on the characteristics of gynogenetic H. otakii.

Heat-induced triploids in Brycon amazonicus: a strategic fish species for aquaculture and conservation.

Triploidization plays an important role in aquaculture and surrogate technologies. In this study, we induced triploidy in the matrinxã fish (Brycon amazonicus) using a heat-shock technique. Embryos at 2 min post fertilization (mpf) were heat shocked at 38°C, 40°C, or 42°C for 2 min. Untreated, intact embryos were used as a control. Survival rates during early development were monitored and ploidy status was confirmed using flow cytometry and nuclear diameter analysis of erythrocytes. The hatching rate reduced with heat-shock treatment, and heat-shock treatments at 42°C resulted in no hatching events. Optimal results were obtained at 40°C with 95% of larvae exhibiting triploidy. Therefore, we report that heat-shock treatments of embryos (2 mpf) at 40°C for 2 min is an effective way to induce triploid individuals in B. amazonicus.

Meiotic Spindle Formation Following Inhibition of First Polar Body Formation in the Zhikong Scallop (Chlamys farreri Jones et Preston)

Fertilized Zhikong scallop (Chlamys farreri) eggs were treated with cytochalasin B (CB 0.5 mg/L) at 14–15 min postfertilization to inhibit first polar body formation. The eggs were then stained with fluorescein isothiocyanate (FITC) -anti-α-tubulin and propidium iodide (PI) to examine their microtubule patterns and chromosome, respectively. Fluorescent microscope observations of treated eggs sampled every 2–3 min during meiotic maturation revealed meiotic apparatus assembly and correlated chromosome segregation. In CB-treated groups, meiosis I proceeded normally and produced two groups of dyads, with 19 in each group. Both dyad groups were retained in the eggs as they entered meiosis II. Two, three, or four asters (centrosome with microtubules around it) in meiosis II rearranged the spindle in several patterns: bipolar [24.0 ± 4.1 μm (long axis) × 18.3 ± 4.1 μm (diameter: metaphase plate)], tripolar (18.6 ± 3.9 μm × 9.9 ± 1.3 μm), separated bipolar (18.3 ± 2.8 μm × 11.2 ± 1.8 μm), and other unclassified spindle patterns. Corresponding chromosome segregation, including bipolar (18.9%), tripolar (38.9%), double bipolar (16.5%), and unclassified (25.6%), was observed during meiosis II in CB-treated eggs. The data indicated that chromosome segregation patterns determined by spindle patterns were critically influenced by the number of centrosomes in meiosis II eggs following inhibition of polar body 1 (PB1) formation with CB.

Data-Theoretical Synthesis of the Early Developmental Process.

Biological development is often described as a dynamic, emergent process. This is evident across a variety of phenomena, from the temporal organization of cell types in the embryo to compounding trends that affect large-scale differentiation. To better understand this, we propose combining quantitative investigations of biological development with theory-building techniques. This provides an alternative to the gene-centric view of development: namely, the view that developmental genes and their expression determine the complexity of the developmental phenotype. Using the model system Caenorhabditis elegans, we examine time-dependent properties of the embryonic phenotype and utilize the unique life-history properties to demonstrate how these emergent properties can be linked together by data analysis and theory-building. We also focus on embryogenetic differentiation processes, and how terminally-differentiated cells contribute to structure and function of the adult phenotype. Examining embryogenetic dynamics from 200 to 400min post-fertilization provides basic quantitative information on developmental tempo and process. To summarize, theory construction techniques are summarized and proposed as a way to rigorously interpret our data. Our proposed approach to a formal data representation that can provide critical links across life-history, anatomy and function.

Detection of the Polar Body After Fertilization.

The polar body, with haploid DNA, is a small cell produced during the meiosis of an oocyte. Here, we describe the detailed procedures for the detection of the second polar body in zebrafish (Danio rerio) embryos after 10min post fertilization. A polar body can be easily distinguished as a small dot with a DAPI-stained nucleus surrounded by Phalloidin-labeled F-actin in each fertilized zebrafish embryo.

Early embryonic development of the vulnerable Shalyni barb,Pethia shalynius(Yazdani & Talukdar, 1975)

The present study provides the first detailed early embryonic development of the Shalyni barb, Pethia shalynius (Yazdani & Talukdar, 1975), a vulnerable cyprinid fish occurring in streams and lentic waters of Meghalaya, northeast India. Induced spawning by synthetic hormone injection in May 2019 was conducted to a pair of mature female and male P. shalynius under controlled conditions in a well-aerated aquarium. Fertilized eggs were spherical, 0.75–0.80 mm (approx.) in diameter, transparent, unpigmented and non-adhesive. A total of 22 developmental stages could be categorized under seven broad periods, viz. the zygote, cleavage, blastula, gastrula, segmentation, pharyngula and hatchling. The first cleavage occurred at 15 min post fertilization (mpf), followed by blastulation at 01:23 hr post-fertilization (hpf), gastrulation at 04:20 hpf, initial somite formation at 07:00 hpf, and pharyngula period at 19:20 hpf, respectively. Embryos hatched between 26–27 hpf and the newly-hatched larvae ranged 2.2–2.5 mm in total length. For naturally-declining populations of this vulnerable fish species, inferences drawn from the present study will help provide a baseline data for its conservation and management, and aid the research fields of developmental biology, biotechnology, molecular biology as well as taxonomy of this species.

Return temperature after heat shock affects the production of tetraploids in the yellowtail tetra Astyanax altiparanae

The aim of this study was to evaluate different post-shock temperatures for tetraploid induction in the yellowtail tetra Astyanax altiparanae. Newly fertilized eggs were divided into four groups, three were submitted to heat shock (40°C for 2 min) at 24 min post-fertilization (mpf) and another group remained without shock (control). Groups submitted to temperature shock were further separated at the following temperatures: 22°C, 26°C and 28°C. Survival among embryonic development was counted and at hatching the ploidy was analyzed by flow cytometry. The results showed that the post-shock temperature affects the parameters analyzed and, therefore, must be considered for optimization of the production of tetraploid in A. altiparanae. Those data are innovative and could be used in future studies of basic biology in this species.

Study on embryonic and larval developmental stages of Sucker head Garra gotyla (Gray 1830; Teleostei; Cyprinidae).

Garra gotyla is an indigenous coldwater fish of the cyprinid family and has wide geographical distribution in India as well as in other countries of Asia and Africa. Induced breeding in G. gotyla was carried out successfully for the first time and an attempt has been made to document developmental stages chronologically from the first minute of fertilization, through all stages of embryonic development until the fifth day post hatching. This experiment was carried out at 22-24°C water temperature at the Directorate of Coldwater Fisheries Research, Bhimtal, India. During the breeding trial, the fertilization rate was observed as 70-75% and hatching rate was 85-90%. The mature fertilized ova were measured as 0.8-1.0 mm in diameter and the perivitelline membrane became thick soon after fertilization and formation of the germ pole. The periods taken for complete developmental stages were recorded; cleavage stage 111 min (min post fertilization (pf)), blastulation stage 580 min (pf), neurulation and segmentation 1250 min (pf) and hatching was completed after 1420 min. The sac fry was measured as 3 mm in length and took almost 3 days for complete absorption of the yolk content. The major structural and differential changes observed are in head, tail, fins, alimentary canal, rudiments of each organ and appearance of melanophore pigmentation in the whole body. The 5-day-old larvae were measured as 6 mm in length with almost every organ fully differentiated. The present study will be utilized for large-scale production of fingerlings for stock enhancement in rivers, lakes and possibilities of genetic improvement and manipulation at the embryonic stage.

Attempt to produce a Nile tilapia tetraploid line by heat shock induction

Nile tilapia can achieve sexual maturity before reaching market size, which can be a great problem for production farms. To circumvent this problem the standard solution is to use androgen treatment to produce only male fish. However, the use of hormones has undesirable environmental issues due to residues in the water. One alternative would be by producing sterile triploid fish, which is conceptually possible by mating tetraploid x diploid. Therefore, our objective was to develop a tetraploid Nile tilapia broodstock. We performed three experiments, where females were hormonally induced spawning, oocytes were collected, fertilized with a semen pool, and eggs were incubated at 27 °C. In experiment I (E-I), we determined the first cleavage interval (FCI) to establish a suitable moment for heat shock. Using the obtained FCI of 90 min, we designed the Experiment II (E-II) with nine treatments: control (no shock); one shock (40 °C) at 60 min post-fertilization of 1, 2, 3 or 4 min duration; and two shocks (40 °C) at 50 and 70 min post-fertilization of 1, 2, 3 or 4 min. The ploidy status of juveniles was evaluated by flow cytometry, however, no tetraploid was found, thus, using the results from E-II, a third experiment was devised. In Experiment III (E-III), we applied nine treatments: control (no shock); one shock (40 °C) at 75, 80, 85 or 90 min post-fertilization; or two shocks (40 °C) at 75 and 85 min, 80 and 90 min, 85 and 95 min or 90 and 100 min post-fertilization, all shocks were of 4 min. Triploid, mosaic, and aneuploid tilapias were identified in the treated groups. Even though we analyzed a large number of animals (>2800) that were submitted to different heat shocks applied at suitable windows of tetraploid induction, no 100% 4 N juvenile was found, indicating that the generation of tetraploid Nile tilapia broodstock using the protocols described is impractical.

High percentages of larval tetraploids in the yellowtail tetra Astyanax altiparanae induced by heat-shock: The first case in Neotropical characins

Tetraploid fish are an important source of diploid gametes for polyploid production, and they can be induced by inhibition of the first mitotic cell division. Dispite its potential, very few protocols regarding tetraploidization and subsequent application to mass production of triploid fish for aquaculture exist. In the yellowtail tetra Astyanax altiparanae, triploids present sterility and increased carcass yield, suggesting the generation of tetraploid for large-scale production of such triploids. In this study, the efficacy of heat shock treatments (40° for 2 min) at 16, 18, 20, 22, 24, and 26 min post-fertilization (mpf) at an incubation temperature of 22 °C were compared on tetraploid induction of A. altiparanae. Ploidy status was confirmed by flow cytometry, karyotyping, and nuclear diameter of erythrocytes. Afterwards, sperm parameters and fertility capacity of tetraploid males were evaluated. The results showed that heat shock decreased the hatching rate, especially at 22 (19.1 ± 9.6%), 24 (16.0 ± 9.1%), and 26 mpf (10.3 ± 6.2%) in comparison with the control group (62.2 ± 7.7%). The flow cytometric analysis showed that the control group and the treatments with heat shock at 16 and 18 mpf presented only diploid individuals. However, from 20 mpf onwards, tetraploid individuals were observed, and the highest percentage arose at 26 mpf (94.55%). When mature, tetraploid males are capable of producing diploid spermatozoa with fertilization capacity, generating a 100% triploid offspring. The results indicate that the heat shock treatment at 26 mpf (40 °C for 2 min) is optimum for tetraploid induction in the A. altiparanae. Additionally, tetraploid mature males generated diploid spermatozoa and can be used to mass production of triploid fish. The current data are the first report of tetraploids in family Characidae, presenting an application in basic and applied sciences.

Androgenetic haploid Japanese pufferfish (Takifugu rubripes) induced by cold shock

Here, we explored cold-shock-induced haploid androgenesis in Japanese pufferfish (Takifugu rubripes) and analysed chromosomes by fluorescence in situ hybridization (FISH), single nucleotide polymorphism (SNP) in sex determination locus and microsatellite genotypes to verify all-male inheritance. We also observed early embryonic development to have an insight into the cytological mechanism of cold-shock-induced gynogenesis. The chromosome number of control group was diploid (2n = 44), while in cold-shock group haploid (1n = 22) and triploid (3n = 66) embryos were detected. Cellular DNA content flow cytometry showed that the rate of haploid induction was 90%. One signal of nucleolar organizing region (NOR) was detected by silver nitrate staining and FISH using rDNA probe in androgenetic haploids from cold-shock group. SNP analysis revealed that about half of haploid embryos examined had the G genotype indicating male-specific Y chromosome. Microsatellite genotyping showed that 26 out of 29 haploid embryos from cold-shock group exclusively inherited paternally derived genotypes. Cytological observation revealed that both second polar body and egg nucleus were located on the blastodisc surface at 60 min post fertilization in cold-shock group, suggesting simultaneous extrusion of both nuclei.

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.

Early embryonic development of brackish water Killifish Aphanius hormuzensis (Teleostei, Aphaniidae) inhabiting coastal environment in Southern Iran

The embryonic and early developmental stages of Killifish Aphanius hormuzensis reared in a laboratory condition were investigated in detail using light microscopy. The adult male and female of A. hormuzensis were captured from the wild. They were kept in laboratory condition for 2 months and spontaneously spawned at 28 ± 1°C. The fertilized macrolecithal eggs were characterized with spherical shape, transparent and average 1.60 ± 0.20 mm in diameter. The embryonic development was started with fertilized egg activation and the first cleavage was observed in 2.5 hr ± 20 min post fertilization (hpf), followed by blastula formation and gastrulation stages in 7 and 11 hpf respectively. First somite was observed in 20 hpf and the heart beating was detected in 25 hpf. The early ontogeny of A. hormuzensis was followed by complements of organs formation and the newly hatched larva with 4.9 ± 0.10 mm in length was detected in 166 hpf. This study provides a basis for further research on reproductive biology, conservation and breeding of this species.

The effect of temperature on the initial development of Brycon amazonicus Spix & Agassiz, 1829 as tool for micromanipulation of embryos.

Primordial germ cell (PGC) transplant is a promising tool in aquaculture; however, successful use of this technique requires in depth knowledge of the early stages of embryo and larval development. The aim of this study was to analyse the effect of different temperatures (22, 26, and 30°C) on the early development of B. amazonicus. The newly fertilized eggs were distributed into tanks with controlled temperature and oxygenation. Samples were collected at pre-established times and analysed under light and fluorescence microscopy. Temperature influenced the speed and duration of each stage of early development, including hatching time. The highest pronuclei fusion rate was observed 8 min post-fertilization (mpf) at 22 and 26°C, and 6 mpf at 30°C. The duration of the 512-1000 blastomeres phase during in the blastocyst stage was 1 h 30 min at 22°C, and 25 min at 26 and 30°C. Hatching occurred at 24 h 30 mpf at 22°C, 16 h post-fertilization (hpf) at 26°C, and 11 h 30 mpf at 30°C. The rate of morphologically normal larvae was 88.34% at 22°C, 90.49% at 26°C, and 73% at 30°C. Malformations of the head, yolk sac, heart, and tail were observed in all temperatures. Nevertheless, B. amazonicus embryos were able to develop satisfactory in all three temperatures tested. These results enable embryo manipulation at different temperatures to optimize the micromanipulation time of embryos and larvae for biotechnological studies.

Triploid hard clamsMercenaria mercenariaproduced by inhibiting polar body I or polar bodyII

Two types of triploid hard clams Mercenaria mercenaria were produced by inhibiting polar body I (PB1) or polar body II (PB2) with cytochalasin B. Treatments were applied at 22–23°C, with PB1 inhibition starting at 4–7 min postfertilization and ending when PB2 was first observed in control groups, and with PB2 inhibition starting at 17–23 min postfertilization and ending when 80% of control eggs released PB2. Triploid induction success was evaluated by chromosome counting in 2–4 cell embryos and by flow cytometry at larval and juvenile stages. PB2 inhibition produced more triploids (82%–100%) than PB1 inhibition (71%–83%), although the difference was not significant (p ≥ .088). Triploid percentages in PB1- or PB2-inhibited groups showed a small but insignificant decline during the first 6 months. At month 3, PB1 and PB2 triploids were not different from their within-group diploids, but significantly larger than control diploids; PB1 triploids were significantly larger than PB2 triploids (p ≤ .003). At month 6, PB1 triploids were not different from either within-group or control-group diploids, while PB2 triploids were significant larger than both within-group and control diploid; PB1 triploids were smaller than PB2 triploids. At month 16, PB1 and PB2 triploids in one remaining replicate were not different from their within-group diploids. Overall, this study shows that triploids can be efficiently produced by PB1 or PB2 inhibition, and their growth performance relative to diploids is variable depending on age and replicates or parental genotype.

Investigating appearance and regulation of the MXR phenotype in early embryo stages of the Mediterranean mussel (Mytilus galloprovincialis)

Multixenobiotic resistance (MXR) efflux transporters constitute a broad-spectrum physiological defense system allowing marine bivalves to cope with environmental challenges. There is, however, scarce information on the type and role that different MXR transporters may have in embryos, which represent the most sensitive stages of bivalves to environmental stress. In this study regulation of MXR-related transporters was investigated in early developmental stages of the Mediterranean mussel (Mytilus galloprovincialis). In vitro fertilization experiments using gametes from naturally-spawning broodstocks were performed to follow embryo development from fertilized eggs (30min post fertilization, pf) to fully developed D-shape veligers (48hpf). Quantitative PCR analyses indicated that ABCB and ABCC transcripts encoding the MXR-related transporters P-glycoproteins (P-gp) and Multidrug resistance proteins (Mrp), respectively, were expressed soon after 30minpf, with ABCC being more expressed than ABCB. Copy numbers of both transcripts were increased in trochophorae and D-veligers. MXR efflux activity assessed using the fluorescent substrate rhodamine 123 and selective P-gp or Mrp inhibitors showed that the P-gp mediated efflux was detected only in D-veligers, while a significant Mrp mediated efflux was detected soon after 30minpf and remained almost unchanged in trochophorae and D-veligers. MXR modulation by propranolol and carbamazepine showed that the pharmaceuticals may act as transcriptional regulators and substrates. Results reported lead to hypothesize that while P-gp aids in xenobiotic efflux performing a prominent protective role, Mrp could be a dual-functioning transporter performing both protective and physiological functions in mussel development.