Tetraploid induction in tropical oysters,Crassostrea belcheri (Sowerby) and Crassostrea iredalei (Faustino)

Tetraploid induction has been conducted on temperate oysters but not on tropical oysters. In this study, different heat shocks (32, 35 and 38°C) and cold shocks (1, 4 and 7°C) were used to induce tetraploidy in two tropical oyster species, Crassostrea belcheri and Crassostrea iredalei, through meiosis I inhibition. Temperature shocks were applied on the newly fertilized eggs at 8–10 min post fertilization and terminated when second polar bodies began to form in the control eggs. The ploidy of the larvae and spat was determined via direct chromosome count. The percentage of larval survival until Day 20 was low (between 0.4% and 42.9%) for both temperature shocks and oyster species. No surviving larva was recorded for induction at 1, 4 and 38°C. Tetraploid spat was only recorded in C. iredalei but the percentage is low through heat shock induction of 32 and 35°C. This study shows that the tetraploid induction success rate was slightly higher in C. iredalei compared to C. belcheri. No surviving tetraploid spat were recorded for both oyster species through the cold shock method. This study shows that heat shock can be used to inhibit meiosis for the production of tetraploids but more experiments need to be conducted to determine the optimum temperature when dealing with tropical oysters.     

Triploid hard clams Mercenaria mercenaria produced by inhibiting polar body I or polar body II

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.     

Volitional Spawning of Black Sea Bass Centropristis striata Induced with Pelleted Luteinizing Hormone Releasing Hormone-Analogue

The black sea bass is a high‐value marine serranid and is a prime candidate for intensive cultivation. Reliable methods for controlled spawning are needed to accelerate the development of hatchery technologies that result in mass production of healthy juveniles. During 1998–2001, spawning studies were conducted at The University of North Carolina at Wilmington (UNCW) and at the South Carolina Department of Natural Resources (SCDNR), Charleston, using pelleted luteinizing hormone releasing hormone analogue (LHRH‐a). From April through July 2001, 28 vitellogenic‐stage females, with mean oocyte diameters (MOD) ranging from 277–448 μm, were implanted with a 95% cholesterol‐5% cellulose pellet containing LHRH‐a (‐50 μg/kg body wt) at UNCW. In 10 individual spawning trials, females with MOD of 305–448 μm and maximum oocyte diameter × 475 μm spawned volitionally beginning 2–3 d post‐implantation (PI) and continued spawning over an average of 1.9 d (range = 1–4 d). Individual females released a mean total of 149,000 eggs (117,000 eggs/kg) with a mean buoyancy rate of 40.5% (floaters). Fertilization and hatching rates were 98% and 27.2% of floaters, respectively, yielding 14,600 yolksac larvae/female (12,600 yolksac larvae/kg body wt), and overall egg viability averaged 8.9%. In eight group spawning trials (2–3 females/group), average performance of females, including fecundity (103,800 eggs/female; 105,500 eggs/kg body wt), buoyancy rate (42.5%), fertilization and hatching rates (97.7% and 24.3% of floaters), numbers of yolksac larvae produced (10,900 yolksac larvae/female; 10,100 yolksac larvae/kg body wt), and overall egg viability (10.6%) was comparable to what was seen in individual spawning trials. From 1998–2000, a total of 58 vitellogenic stage (70% of oocytes 500 pm) females were implanted with pelleted LHRH‐a (‐50 μg/kg body wt) in nine group spawning trials (2–19 females/group) at SCDNR. Volitional spawning typically began 18–42 h PI and recurred every 1–3 d for an average duration of 9 d. Female groups released a mean of 560,000 eggs (84,000/female; 132,000/kg body wt) over the spawning period, with mean buoyancy rate of 25.7% floaters. Fertilization and hatching rates were 17.7% and 11.6 % of floaters, respectively, yielding 4,300 yolksac larvae/female (4,600 yolksac larvae/kg body wt). Overall egg viability was 2.9%. Captive wild‐caught black sea bass were induced to undergo repetitive volitional spawning by implantation of pelleted‐LHRH‐a, consistent with a multiple clutch group synchronous pattern of ovarian development. Group spawning appears to be a practical way to compensate for variable fecundity and egg viability of individual females. Research is needed to identify optimum hormone treatments and eligibility requirements.     

Reproductive ability of triploid ornamental (koi) carp (Cyprinus carpio L.) × goldfish (Carassius auratus L.) hybrids and characteristics of their offspring

The purpose of this study was to investigate reproductive ability of backcross triploid koi (Cyprinus carpio L.) × goldfish (Carassius auratus L.) hybrids. These triploids have been obtained by crossing of F₁ hybrid females producing diploid eggs with males of parental species. Triploid hybrid females, when crossed with goldfish or koi males, produced mostly aneuploid fish with ploidy range from approximately 2.2n–3.2n with a mean value 2.5n; some fish in crosses of triploid females with koi males were tetraploid (4.0n). Since analysed fish had in their genomes one haploid set from parental males, the data indicate that triploid hybrid females mostly produced aneuploid eggs with ploidy range from approximately 1.2n–2.2n and a mean ploidy around 1.5n while some eggs were triploid. Triploid hybrid males were completely sterile and have not released any sperm after hormonal injection. Despite their low viability, some aneuploid fish obtained from triploid hybrid females were raised in indoor recirculating systems until the age of 2 years and their reproductive ability has been evaluated. One aneuploid female with ploidy 2.1n produced larvae with ploidy range from 2.9n to 3.4n with a mean ploidy of 3.1n when crossed with a koi male; about 60% of obtained larvae had ploidy from 3.0n to 3.2n. These data indicate that this female produced mostly eggs with unreduced ploidy level.     

Induction of triploidy in the South African abalone using cytochalasin B

An investigation into triploidy induction in the South African abalone, Haliotis midae, was conducted. It was found that 0.5 mg l–1 of Cytochalasin B (CB) in seawater induced triploidy when administered to coincide with the normal timing of the release of either polar body one (PB1) or two (PB2). This concentration of CB produced 70.9% triploid induction in the PB2 treatment and 48.4% induction at PB1. Significant numbers of tetraploid larvae were found in the PB1 treatment. These resulted from the presence of excess sperm (polyspermy) but only when CB was present. Although larval survival after triploid induction was lower than the control animals, it was considered high enough for use in commercial hatcheries. © Rapid Science Ltd. 1998     

Evaluation of cytochalasin B (CB) treatments for triploidy induction in the blacklip abalone, Haliotis rubra (Leach, 1814)

The effectiveness of cytochalasin B (CB) treatments for inducing triploidy was evaluated in the blacklip abalone Haliotis rubra (Leach, 1814) in two orthogonal design experiments. The first experiment employed three dosages (DSs) of 0.25, 0.5 and 1.0 mg CB L^?1, three starting times (STs) of 5, 15 and25 min post fertilization and three treatment durations (TDs) of 10, 20 and 40 min, for a total of 27 treatments. The second experiment comprised of two DSs of 0.25 and 0.5 mg CB L^?1, five STs of 5, 15, 20, 25and 30 min post fertilization, and three TDs of 10, 20 and 40 min, for a total of 30 treatments. Water temperature was held at 17.5–18.5°C. Day 3 larvae were sampled for triploidy using flow cytometry (FCM) and survival. Optimal inductions were treatments starting at 15 or 20 min post fertilization and continuing for 40 min, and those initiated 25 or 30 min post fertilization for 20 or 40 min, using 0.5 mg CB L^?1. These treatments were all targeted at inhibition of the second polar body (PB2) formation and yielded triploidy rates of 84.8–89.5% coupled with (relative) survival rates of 20.1–52.1% in the first experiment, and corresponding rates of 86.5–96.5% and 33.0–74.1%, respectively, in the second experiment. A common and essential feature of these optimal conditions is that treatment must fully span the period of time for most of the eggs to extrude PB2. Treatments that resulted in suppression of the first polar body (PB1) formation induced triploidy levels below 71.5% and 57.6% in experiments 1 and 2 respectively. Treatments that had overlapping effects on both PB1 and PB2 extrusion led to triploidy rates above 80% but very low survival rates of 1.8% and 5.4% in experiments 1 and 2 respectively.     

Comparative studies on survival and growth performance among diploid,triploid and tetraploid dojo loach <Emphasis Type="Italic">Misgurnus anguillicaudatus</Emphasis>

To determine the cytotype with better traits for the aquaculture practices of the dojo loach Misgurnus anguillicaudatus from the viewpoint of fish farming improvement, factorial crosses (2n♀ × 2n♂, 2n♀ × 4n♂, 4n♀ × 2n♂, 4n♀ × 4n♂) were conducted between natural diploids (D) and tetraploids (T), producing DD, DT, TD, and TT groups (female listed first). The potential benefits of the different cytotypes in culture were evaluated by comparing growth performance and survival rate for a 15-month rearing trial under the same production conditions. The average fertilization rate in DT and TT was significantly lower than in the DD and TD groups, possibly indicating the poor fertilizing capacity of the tetraploid sires. Survival rate in DT and TD was slightly lower than in DD but significantly higher than in the TT groups. Tetraploid females produced obviously larger eggs than diploids and, subsequently, significantly longer initial body length of TT and TD than DD and DT fry. However, from the second month of the growth trial, TT suffered higher mortality than other cytotypes, which significantly influenced morphometric growth parameters. The TD group exhibited superior growth performance throughout the experiment. The mean body length of DT was comparable with that of DD fish during the first 7 months but began to outgrow DD from the 9th month. This study suggests that the relatively better growth of tetraploid and higher survival rate of diploid can be integrated via inter-ploidy hybridization to get TD triploids with better culture traits.     

Ploidy level,morphological characteristics and performance of interploid hybrids by Megalobrama amblycephala♀× allo‐tetraploids ♂

Interploid hybrids were produced by mating allo‐tetraploid males, an interspecific tetraploidy induced by blunt snout bream (Megalobrama amblycephala) ♀× black bream (Megalobrama terminalis) ♂, with blunt snout bream females. The fertilization rate was high in the interploid hybrids. Although significant (P<0.05) mortalities were observed at hatching, swim‐up and 30‐day stages, respectively, the survival percentage of the interploid hybrids became stabilized after the 60‐day stage. In different interploid hybridization batches, 64–97% of juveniles were identified as 3n and 1–36% as 2n. Less than 2% of tetraploids were obtained in these interploid hybridization batches. The daily growth rate of the interploid 3n increased 7.5%, compared with that of diploid blunt snout breams during the 180‐day culture. Moreover, a method using seven morphological parameter ratios was set up with a relatively high classification accuracy of 93.8% to harmlessly discriminate interploid 3n from allo‐tetraploids, black bream and blunt snout bream.     

Chromosome set manipulations in the black carp

The mollusc-eating black carp (Mylopharyngodon piceus) has economic and health-care potential for biological control of nuisance aquatic molluscs. The present study investigates the production of gynogenetic-monosex and triploid-sterile populations of black carp. The goal was to provide a method which would eliminate unwanted biological and environmental impacts of introducing this exotic species into areas with nuisance mollusc infestation. Meiotic gynogenesis was induced by inseminating black carp eggs with UV-irradiated (800 Jm⁻²) sperm of common carp (Cyprinus carpio) or Japanese ornamental (koi) carp. Diploidy was restored through retention of the second polar body (2PB), by shocking activated eggs at 1–8 min post-fertilization (embryological age of 0.07–0.57τ₀, a parameter defined by the cell cycle duration) at 1 min intervals, with heat-shocks (41.0±1.0 °C for 1 min) or pressure-shocks (7500–7600 psi for 1.5 min). Highest survival was found when embryos were heat-shocked 1.5–4.5 min post-fertilization (0.10–0.25τ₀). The highest survival of free-swimming larvae from pressure-shocked eggs, was achieved at 7500 psi at 1–2 min post-fertilization (0.08–0.16τ₀). Triploidy was induced by retention of 2PB following normal fertilization. Batches of 30 000 eggs were fertilized with intact sperm and pressure-shocked (6000–8500 psi for 1.5 min) 2 min post-fertilization (0.15–0.16τ₀). The highest survival of triploid swim-up larvae was 5.1% in eggs shocked with 7500 psi. In random samples of individual larvae taken from each treatment, triploidy was analysed by cytofluorometry of the cellular DNA content. In DNA analysis performed in fingerlings (N≥15), 50% of the fish were triploids.     

Induction of triploidy and tetraploidy in stinging catfish, Heteropneustes fossilis (Bloch), using heat shock

Induction of triploidy and tetraploidy was attempted in Heteropneustes fossilis using heat shock. The optimal age of zygote, temperature level and duration of thermal shock required for effective induction of triploidy and tetraploidy was investigated in a series of experiments. A maximum of 82±7% triploids (3n=87) were obtained when fertilized eggs (2.5‐min old) were heat shocked at 40°C for 4‐min duration. A maximum of 40±8% tetraploids (4n=116) was obtained when the fertilized eggs (30‐min old) were heat shocked at 40°C for 4‐min duration. The triploid and tetraploid red blood cells (RBCs) nucleus volumes were 1.4 and 2.1 times greater, respectively, than that of the diploid RBC nucleus.     

Blocking polar body with cytochalasin B in the fertilized eggs of the small abalone, Haliotis diversicolor supertexta (Lischke), and the development and ploidy of the resultant embryos

The effects of blocking polar body I (PB1) or polar body II (PB2) with four different dosages of cytochalasin B (CB) on the development and ploidy of resultant embryos were studied in the small abalone, Halitis diversicolor supertexta (Lischke). To block the release of PBI, the fertilized eggs were treated with 0.25, 0.5, 1.0 or 2.0 mgL^?1 of CB for 10min beginning at 3 min post-fertilization at 24°C. To block the release of PB2, the fertilized eggs were treated under the same conditions as PB1, except that the treatment was begun 10min post-fertilization. In the control group, only 41.8% of the cells had a diploid number of 32 chromosomes, although spontaneous haploids (9.0%). tripolids (7.5%) and aneuploids (41.7%) were also observed. In CB treatment of PB1 and PB2 groups. 5.0-28.6% of the cells remained as diploid. triploids (10.0-18.9%) and aneuploids (41-1-61.0%). With regard to the development of the resultant embryos, the proportion of normal embryos in the control group was 87%, while in the treatment groups, the proportions of normal embryos in the FBI and PB2 groups were 57-58% and 53-56% in the 0.25 mg L^?1 and 0.5mg L^?1 CB treatments, respectively. From this data on induced triploids and the resultant development of normal embryos, the proportions suggest that 0.25-0.5 mg L^?1 of CB for 10min was sufficient for blocking the release of FB1 or PB2 to produce triploids in the small abalone.     

Studies on triploid oysters in Australia: Evaluation of cytochalasin B and 6-dimethylaminopurine for triploidy induction in Sydney rock oysters Saccostrea commercialis (Iredale and Roughley)

Naturally spawned Sydney rock oysters Saccostrea commercialis (Iredale and Roughley),were used to determine the appropriate stage of development for inducing triploidy and to compare the effectiveness of cytochalasin B (CB) and 6-dimethylaminopurine (6-DMAP) in dose-optimization trials. Induction should commence at 50% first polar body (PB1) extrusion in eggs (approximately 17-19 min post-fertilization at 25^oC). By day 5 the highest triploidy percentage and yield (number of triploid larvae per 100 fertilized eggs) were achieved in the ranges of 0.75-1.5 mg CB 1^-1 (1.6-3.1 μm CB)or 200-400 μm 6-DMAP (32.6-65.3 mg 6-DMAP l^-1). However, CB treatment resulted in greater survival and triploidy percentage than 6-DMAP in Sydney rock oysters.     

Evaluation of First-Feeding Regimens for Larval Nassau Grouper Epinephelus straitus and Preliminary, Pilot-Scale Culture through Metamorphosis

Two 10-day hatchery experiments were conducted to evaluate s-type (Hawaiian strain) and ss-type (Thailand strain) rotifers Brachionus plicatilis and cryogenically preserved oyster Crassostrea gigas trochophores as first feeds for larval Nassau grouper Epinephelus striatus. Newly hatched grouper larvae were reared at densities of 11.2–20.8/L in 500-L tanks at 36–38 ppt salinity, 25–26 C, and under a 11-h light: 13-h dark photoperiod. Beginning on day 2 posthatching (d2ph), prey were maintained at a density of 20 individuals/mL, while phytoplankton (Nanochloropsis oculata) was maintained at 500 × 10³ cells/mL. In experiment 1, survival and growth were higher (P < 0.05) for fish fed small s-type rotifers (mean lorica length = 117 μm; fish survival = 7.96%) selected by sieving than for fish fed non-selected rotifers (mean lorica length = 161 μm; fish survival = 2.13%). These results demonstrated the advantage of small prey size and suggested that super-small (ss-type) rotifer strains would be beneficial. In experiment 2, three feeding regimens were compared: 1) ss-type rotifers (mean lorica length = 147 μm); 2) oyster trochophores (mean diameter = 50 μm) gradually replaced by ss-type rotifers from d5ph; and 3) a mixed-prey teatment of 50% oyster trochophores and 50% ss-type rotifers. Survival was higher (P < 0.05) for larvae fed mixed prey (15.6%) than for those fed rotifers (9.73%) or trochophores and rotifers in sequence (2.55%), which also showed the slowest growth. Oyster trochophores, although inadequate when used exclusively, enhanced survival when used in combination with rotifers, possibly by improving size selectivity and dietary quality. In a pilot-scale trial, larvae were cultured through metamorphosis in two 33.8-m³ outdoor tanks. Fertilized eggs were stocked at a density of 10 eggs/L and larvae were fed ss-type rotifers from d2ph-d20ph, newly hatched Artemia from d15ph-d18ph, 1-d-old Artemia nauplii from d18ph-d62ph. Survival on d62ph was 1.17%, with a total of 5,651 post-metamorphic juveniles produced.     

Augmentation of free amino acids in eggs of red snapper,Lutjanus campechanus as part of the induced spawning protocol

An attempt was made to increase free amino acids (FAA) concentrations in eggs of red snapper Lutjanus campechanus as part of an induced spawning protocol. Mature female red snapper were given intramuscular injections of human chorionic gonadotropin (HCG) at a 1100 IU kg^?1 of female body weight. Immediately after the HCG injection, one set of females received an intramuscular injection of FAA in a buffered saline solution at a dose of 20 mg FAA cocktail kg^?1 of body weight. The FAA cocktail was 25% isoleucine, 25% leucine, 25% lysine and 25% valine on a molecular weight basis. A second set of fish did not receive the FAA supplement. Both sets of fish were equally successful in spawning with ovulation occurring 21–32.5 h post injection. Injection of FAA into brooders as eggs began final maturation and hydration resulted in an increase in leucine (nmoles mg egg^?1) and a slight increase in isoleucine (nmoles egg^?1) in recently ovulated eggs. FAA supplementation via brood injection altered FAA utilization rates by embryos and during larval development, resulted in a greater yolk sac and oil globule reserve in larvae as they approached the time of first feeding.     

Reproduction, gamete supply and larval rearing of New Zealand turbot Colistium nudipinnis (Waite 1910) and brill Colistium guntheri (Hutton 1873): a potential new aquaculture species

New Zealand turbot Colistium nudipinnis (Waite 1910) and brill Colistium guntheri (Hutton 1873) were studied to assess their potential for aquaculture development. The reproductive cycle of wild fish showed a long spawning season from winter to summer, during which it is possible to obtain gametes. Both species have a diurnal ovulatory cycle, and gamete collection, by stripping and fertilizing at sea, was most successful within 2–3 h before and after sunset. Male reproductive anatomy suggests that these flatfish spawn in close proximity and that pair formation is highly likely. The eggs of both species have multiple oil droplets, turbot eggs being slightly larger (0.99 mm diameter) with more droplets (18–55) than brill eggs (0.97 mm, 13–26 droplets). Hatching occurred approximately 84 h after fertilization at 14 °C. Newly hatched turbot averaged 2.2 mm in length, and brill averaged 2.1 mm. First feeding began 4 days post-hatch (DPH). During larval rearing, rotifers were replaced by Artemia nauplii at 10 DPH. Metamorphosis commenced at 12–15 DPH and was completed and the larvae settled by 45 DPH. Weaning to inert foods began at 20–22 DPH (50 mg weight) and was completed by 57 DPH. Survival of turbot was 22.8% from fertilized egg to hatching, 7.3% through incubation to 22 DPH and 2.1% through incubation to fully weaned juveniles. Weaning success for turbot from metamorphosis to 57 DPH was 31.5%.     

合浦珠母贝三倍体的卵诱导四倍体

将合浦珠母贝三倍体的卵与二倍体的精子授精,用０．５μｇ／ｍＬ细胞松弛素Ｂ抑制精卵第一极体的释放诱导四倍体。研究了处理起始时间及持续时间对胚胎孵化率和四倍体诱导率的影响及幼虫的生长及存活。实验结果表明：持续时间与胚胎孵化率呈负相关,而与四倍体诱导率呈正相关,持续时间一般为１５￣１８ｍｉｎ,处理起始时间一般在第一极体出现前３￣５ｍｉｎ。在胚胎期,四倍体诱导率平均为２０％。在幼虫培养阶段,幼虫死亡严重,     

GnRHa‐induced Multiple Spawns and Volition Spawning of Captive Spotted Rose Snapper,Lutjanus guttatus,at Mazatlan,Mexico

Sexual maturation and induced spawning treatments were carried out with captive spotted rose snapper, Lutjanus guttatus. A total of 3013 × 10⁶ eggs (64.7% were floating) were produced from eight treated females in 42 spawns induced with GnRHa implants during the course of the present study. GnRHa ethylene‐vinyl acetate copolymer effective doses were 204 ± 11 µg/kg in June 2005, and 224 ± 13 µg/kg in July 2005. General fertilization was 50.9 ± 34.5% and 12–14 h after spawning, viability of floating eggs was 90.4 ± 12.4%. Mean incubation period at 29–31 C was 18–20 h, and mean hatching was 94.4 ± 8.2% (73–100%). Newly hatched larvae were 2.18 ± 0.15 mm in total length (TL). One month after the last hormone experiment, previously GnRHa‐treated and untreated fish began spawning voluntarily. Hormone‐treated breeders had higher fecundity than untreated fish, producing 72.5 million eggs versus 13.9 million eggs for the untreated fish, over the following 11 mo. Combined data of volitional spawning for total egg fertilization, viability, hatching, and larval TL were 77.7 ± 1.8%, 90.3 ± 1.3%, 87.9 ± 2%, and 2.50 ± 0.12 mm, respectively. These results can ensure the sustainability of a commercial hatchery.     

Heat-shock-induced tetraploid and diploid/tetraploid mosaic in pond loach, Misgurnus anguillicaudatus

Tetraploid fish, which are considered as key resources of diploid gametes for further breeding and ploidy manipulation, can be artificially induced by inhibition of the mitotic cell division with hydrostatic pressure or temperature treatments. Although many attempts have been made to induce artificial tetraploid strains, successful establishment of viable and fertile tetraploid strains are rare. In pond loach, Misgurnus anguillicaudatus, natural tetraploid individuals are distributed in wild populations and diploid gametes from the tetraploid fish have been used for the induction of polyploid individuals, but artificially induced tetraploid strains have not been established yet. In the present study, we optimised starting timing of the heat-shock treatment (41 °C for 2 min) to inhibit a mitotic cell division in fertilised eggs of the normal diploid pond loach between 21 and 51 min after insemination at 20 °C. After the treatment, we observed external appearance of hatching larvae and flow cytometrically determined ploidy status of the resultant larvae. Although tetraploid and diploid/tetraploid mosaic larvae were obtained, the optimum timings for induction of tetraploidy varied amongst crosses. Various kinds of ploidy such as haploidy, diploidy, triploidy, pentaploidy, hexaploidy, aneuploidy and mosaic were detected in non-optimum heat-shock timings for tetraploidisation. Survivors, a tetraploid and a diploid/tetraploid mosaic male, matured at the age of 1-year-old, but they produced functional haploid spermatozoa.     

ARTIFICIAL REPRODUCTION OF Penaeus japonicus BATE AS A BASIS FOR THE MASS PRODUCTION OF EGGS AND LARVAE1

Penaeus japonicus Bate is absent on the Italian coasts, but it is considered a very promising species for national aquaculture. To develop the shrimp-culture on a commercial basis, a convenient method for producing eggs and larvae of Penaeus japonicus was studied. A stock of 156 females, age class 1+ and F₃, underwent unilateral eyestalk ablation and they were stocked with males in three plastic tanks (bottom area 14 m²) at a density of 25 individuals/m² and a sex ratio of 1:1. Conditions of environment and management consisted of: temperature 21±3°C; natural light and photoperiod; total exchange of sea water every day; feeding on mussels. A month after eyestalk ablation the females were inspected every 7–10 days; mature females were transferred to the spawning tanks. Females with well developed gonads averaged 25.0% of the population of females at every inspection; 51.3% of spawners were impregnated with spermatophores; others were inseminated artificially by the method of introducing spermatophores into the thelycum. In six inspections (carried out during a period of 89 days), 3,028,525 eggs were released; a female released 17,500 eggs on the average (maximum was 123,000 eggs, minimum 4,340). The mean rate of fertilized eggs was 52.7%—67.7% from naturally inseminated females and 7.5% from the artificially inseminated ones. The mean hatching rate was 30.9%—40.1% from natural insemination and 3.3% from artificial insemination. Total amount of viable nauplii was 820,550.     

Lipid Nutrition and Feeding of Cobia Rachycentron canadum Larvae

This study examined the fatty acid composition of cobia Rachycentron canadum eggs and yolksac larvae, as well as the ovaries of wild caught females as an initial guide to lipid nutritional requirements. A 2-wk feeding study also was conducted to investigate the effectiveness of four dietary treatments on the growth and survival of cobia larvae. Cobia eggs in the tailbud stage contained 31.4 ± 1.3 μg lipid/egg. After hatching, the amount of lipid decreased significantly (P < 0.05) from 28.3 ± 0.3 to 23.2 ± 0.1 μg lipid/larvae during the yolksac larval stage (days 1 to 3 after hatching). Ovaries from wild caught adults and captive spawned eggs and yolksac larvae contained high levels of PUFAs with docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and arachidonic acid (ARA) accounting for approximately 80% of the total suggesting that cobia larvae may have a high dietary requirement for these fatty acids. For the feeding study, larvae were fed: 1) Artemia only; 2) enriched rotifers for 1 d only + microparticulate diet (day 313); 3) enriched rotifers for 3 d (day 3–5) + Artemia (day 3–13); and 4) enriched rotifers for 6 d (day 3–8) + Artemia (day 3–13). Cobia larvae began feeding on rotifers 3 d after hatching and on newly hatched Artemia nauplii by the fifth day following the onset of exogenous feeding (day 7). On day 7, no differences in larval growth were found among larvae fed rotifers for 3 versus 6 d, whereas larvae fed only Artemia or rotifers for I d followed by microparticulate diet were significantly smaller (P < 0.05) and did not survive beyond day 9 and 13, respectively. The results of the feeding study indicate that cobia larvae require rotifers for a minimum of 4 d following the onset of exogenous feeding.