Optimization of triploidy induction in the blacklip abalone, Haliotis rubra (Leach, 1814), using 6-dimethylaminopurine

Optimal conditions of 6‐dimethylaminopurine (6‐DMAP) for triploidy induction in the blacklip abalone Haliotis rubra (Leach, 1814) were investigated, targeting inhibition of second polar body (PB2) formation. Two experiments were conducted at a water temperature of 17.5–18.5°C where factorial combination of (1) four dosages (DSs) of 100, 150, 200 and 250 μM 6‐DMAP, four starting times (STs) of 15, 20, 25 and 30 min post fertilization, and two treatment durations (TDs) of 20 and 30 min and (2) three DSs of 50, 100 and 150 μM 6‐DMAP, three STs of 15, 20 and 25 min post fertilization, and three TDs of 10, 20 and 30 min, were applied respectively. Day 3 larvae were sampled for triploidy and survival. Percent triploidy was verified using flow cytometry (FCM). Results show that optimal inductions that combine both high rates of triploidy and reasonable survival were those treatments commenced 15 or 20 min post fertilization and continued for 20 or 30 min, using 100 μM 6‐DMAP. These conditions induced rates of triploidy and relative survival of 80.5–93.3% and 36.5–40.2%, respectively, in the first experiment, and corresponding rates were 79.1–93.6% and 20.7–43.0% in the second experiment. High percent triploidy were also obtained in a number of treatments using 150 μM 6‐DMAP, but with overall survival rates generally lower than those using 100 μM 6‐DMAP.     

Triploidy induction in Australian greenlip abalone Haliotis laevigata (Donovan) with cytochalasin B

Triploid induction in Australian greenlip abalone, Haliotis laevigata (Donovan), was conducted by blocking the formation of the second polar body using cytochalasin B (CB). Twenty minutes after fertilization, the zygotes of greenlip abalone were treated with four CB concentrations (0, 0.25, 0.5 and 0.75 mg L⁻¹) for 10, 15 and 20 min. The ploidy of resultant larvae was determined using flow cytometry at 72-h post fertilization. Our study showed that fertilization, hatching, survival and induced triploidy of abalone larvae were significantly affected by the CB concentration and treatment duration. The effective range of CB concentration for triploid induction on greenlip abalone was 0.5–0.75 mg L⁻¹ with an induction duration of 10–15 min. The results indicate that the most effective treatment combination for triploid induction in greenlip abalone is 0.5 mg CB L⁻¹ for 15 min starting at 20-min post fertilization.     

Tetraploid induction by meiosis inhibition in the dwarf surfclam Mulinia lateralis (Say 1822): effects of cytochalasin B duration

Different durations of a cytochalasin B (CB) treatment were tested for tetraploid induction by meiosis inhibition in the dwarf surfclam Mulinia lateralis Say. Cytochalasin B, 0.67 mg L^?1, was applied to newly fertilized eggs at 8–10‐min post fertilization and removed when in the untreated eggs: (1) Polar body 1 (PB1) was released in 90% of the eggs and polar body 2 (PB2) began to form (T1); (2) Polar body 2 was released in about 25–30% of the eggs (T2); (3) Polar body 2 was released in about 70–75% of the eggs (T3); or (4) eggs began to enter mitosis I or the polar lobe began to form (T4). Three replicates were produced using different sets of parents. The ploidy of resultant larvae and juveniles was determined by flow cytometry. Blocking PB1 alone in T1 groups produced mostly tetraploids, and longer CB treatments in T2 and T3 resulted in increasing numbers of pentaploids. In T4 groups where both PB1 and PB2 were inhibited, larvae were predominantly pentaploids. Pentaploid larvae were arrested at the trochophore stage. The majority of tetraploid larvae died as trochophores, although a small fraction reached D‐stage. Among 478 juvenile clams sampled from a T1 group, three (0.6%) were confirmed as tetraploids. This study shows that tetraploid embryos can be produced at high efficiencies (40–90%) by blocking meiosis I. Tetraploids produced by meiosis inhibition in normal eggs are viable in M. lateralis, but their survival beyond metamorphosis is extremely low.     

Thermal shock induction of triploidy in Atlantic cod (Gadus morhua L.)

The effects of thermal treatments on induction of triploidy in Atlantic cod have been investigated. Cold shock [−1.7±0.1°C at 20 min post fertilization (PF) for 2 h] was based on a previously developed protocol, and heat shocks, below the lethal threshold of 24°C, were at 16, 18 or 20°C applied 20, 30 or 40 min PF for 20 min. Cold shock did not affect larval survival and was ineffective for producing triploids (range 0–4%). A heat shock of 20°C at 20 min PF generated the highest percentages (range 66–100%) of triploid larvae at hatching, with survival ranging from 10% to 20% relative to the controls. Lower heat shock temperatures or delayed shocks increased survival but decreased the number of triploids, providing no net gain in triploid yield (range 1–9%). Heat shocks applied later than 20 min PF produced 2–4% tetraploid larvae at hatching. A thermal shock of 20°C initiated at 20 min PF and lasting 20 min proved to be the most generally efficient treatment for induction of triploidy in Atlantic cod.     

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.     

Induced triploidy in the European clam, Ruditapes decussates (L.), and performance of triploid larvae

Abstract. For the first time, effective treatments using cytochalasin B were developed to induce triploidy in the European clam, Ruditapes decussatus (L.). The percentage of triploid embryos was assessed by karyological or image analysis. Two treatments (0·5 or 1 mg of cytochatasin B (CB) per ml of dimethyisulfoxide in 1 litre of sea water) were applied at different times after fertilization (10 to 25min), for two different periods (15 and 20min). Best results were obtained for a CB concentration of 1 mg/1. When treatment was applied 15min after fertilization for a 20min period, 94% and 95% of triploid embyros were obtained in two repeated experiments. At metamorphosis, the treated larvae appeared to be no smaller than the control larvae in all experiments. However, in general, significantly higher mortalities for CB-treated batches were found when compared with the untreated batch.     

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.     

Effect of recovery salinity on survival of acutely stressed halibut (Hippoglossus hippoglossus L) larvae

First‐feeding halibut larvae (245‐day degrees; 40 days post hatch), reared at 34 g L^?1 salinity and 7°C, were subjected to handling and allowed to recover in a range of salinities (0–34 g L^?1) and at 10°C. Survival of the unfed larvae was determined daily for 18 days. Mortality rates approached 0 after 4 days in all treatments and presumed starvation‐induced mortality started at about 11 days post handling. By 20 days post treatments, all larvae had died. Salinities in the range of 10–20 g L^?1 produced significantly (anova , P<0.01) higher initial survival (71–95%) than salinities above 20 g L^?1 (24–48%) or below 10 g L^?1 (0–19%) and this survival pattern changed little in unfed larvae for the first 10 days following the stressor. For example, 24 hour post handling, survival of halibut was improved from 28.7±16.5% (mean±standard error, n=3) at 34.0 g L^?1 to 95.2±4.8% at 13 g L^?1. A second‐order polynomial regression of 4‐day post‐handling survival data (y=?0.002x ²+0.0603x+0.0699, r²=0.3936) predicted a maximum survival at 15.1 g L^?1 salinity. These results have important implications for halibut aquaculture and research when handling of larvae is unavoidable. For practical applications, we recommend reducing salinity of receiving waters to 15–20 g L^?1 with a slow (3–4 days) reacclimation to ambient conditions.     

Induction of Triploidy and Tetraploidy in Nile Tilapia, Oreochromis niloticus (L.)

Abstract.— Induction of triploidy and tetraploidy in Nile tilapia, Oreochromis niloticus , was investigated by heat shock, cold shock, hydrostatic pressure, and/or chemicals (cytochalasin A, B, and D). Additionally, efficacy of combined protocols was determined. Heat shock 10 min after fertilization induced triploidy when incubation temperature was 24 C but not when incubation temperature was 31 C. Heat shock of 40–41 C at 4–6 min after fertilization was effective in inducing up to 100% triploidy with hatchability similar to controls. Cold shock at 13 C for 45 min five min after fertilization induced 85–100% triploids. Heat shock and multiple heat shocking were the most effective treatments for the induction of tetraploidy. Two heat treatments of 41 C applied at 65 and 80 min after fertilization for 5 min each produced approximately 80% tetraploidy in hatched fry. Immersion of fertilized eggs in cytochalasin A, B, or D at concentrations up to 10 μg/L applied at various times and durations was ineffective in inducing triploidy or tetraploidy.     

Triploidization in rohu × mrigal hybrid and comparison of growth performance of triploid hybrid

The present study was aimed at the identification of treatment optima to induce triploidy in ‘Labeo rohita (rohu) × Cirrhinus cirrhosus (mrigal)’ hybrid using heat shock treatment. The eggs were exposed at four different temperature regimes viz., 38, 39, 40 and 41°C for 1–3 min, applied 3–5 min after fertilization. After 4 min of fertilization, heat shock treatments for 1 and 1.5 min durations were found the best inducing triploidy up to 100% and 96% respectively. Survival rates upto yolk sac absorption were found to be 73% and 71% in rohu and mrigal, 68% and 67% in the reciprocal diploid hybrids and 61% and 60% in the reciprocal triploid hybrids (RTH). Triploidy was confirmed by chromosome counting that revealed the diploid chromosome number of rohu and mrigal at 2n = 50 and in their triploid hybrid chromosome number was found to be 3n = 75. Growth rate of the RTH showed a significant difference (P < 0.05) from the single species and the diploid hybrids. Triploids also showed higher survival rate over the diploids.     

Cryopreservation of Persian sturgeon (Acipenser persicus) sperm: effects of cryoprotectants,antioxidant, membrane stabilizer,equilibration time and dilution ratio on sperm motility and fertility

Three experiments were performed to develop protocols for cryopreservation of Persian sturgeon Acipenser persicus, sperm. In the first experiment, sperm from six males was individually split in three subsamples and cryopreserved using Modified Tsvetkova's extender (mT) supplemented with dimethyl sulfoxide (DMSO), methanol (MeOH), glycerol (Gly) and ethylene glycol (EG) at concentration of 5%, 10%, 15% and 20%. In the second set of experiments, the effects of six equilibration times (0, 5, 10, 20, 40 and 60 min) and dilution ratios (volume sperm: volume extender 1:0.5, 1:1, 1:2, 1:3, 1:5 and 1:10) and the additive advantage of bovine serum albumin (BSA; 0, 2.5, 5 and 10 mg mL^?1) and ascorbic acid (0, 2.5, 5 and 10 U mL^?1), on the post‐thaw survival of sperm (triplicate set of six fish) were evaluated. Then, sperm was diluted in 1:1 mT extender with 10 mg mL^?1 BSA with selected cryoprotectants (15% MeOH and 10% DMSO) for 5 min. After a month of storage in liquid nitrogen, post‐thawed sperm motility; fertilization and hatching rate and viability of derived larvae were measured (Exp.3). Evaluation of cryoprotectants efficiency showed that MeOH 15% and DMSO 10% were suitable for cryopreservation of Persian sturgeon sperm. Gly and EG resulted in very low post‐thaw motility rates even at lowest concentration. No significant difference was observed among the four different equilibration times (0, 5, 10, 20 min) (P > 0.05) although higher equilibration times than 20 min resulted low post‐thaw motility (P < 0.05). The motility of frozen–thawed sperm did not significantly change when dilution ratio was increased from 1:0.5 to 1:3 (P > 0.05). However, higher dilution ratios (1:5 and 1:10) reduced the percentage of motile sperm. Supplementation of the cryoprotectant solution with 10 mg mL^?1 BSA significantly improved post‐thaw motility (P < 0.05), but ascorbic acid did not improve post‐thaw motility (P > 0.05). The results of experiment 3 showed that the highest fertilization (30.2 ± 5.75) and hatching rates (28.2 ± 5.25) were observed when samples were frozen with 15% MeOH (P > 0.05). Our study indicates that the use of mT extender consisting of 10 mg mL^?1 BSA in 15% MeOH diluted with sperm at 1:1 ratio for 5 min can be recommended cryopreservation method for Persian sturgeon sperm.     

CB诱导熊本牡蛎三倍体及其存活率与倍化率的变化关系

为诱导熊本牡蛎三倍体,研究了细胞松弛素B (CB)浓度、诱导起始时间、诱导持续时间等因素对卵裂率、D幼率、三倍体率的影响,并分析了幼虫、稚贝及成贝的存活率和三倍体率的变化特征。结果显示,CB浓度为0.5~0.6 mg/L,诱导起始时间为40%受精卵释放第一极体,诱导持续时间为20 min时可获得87%的三倍体率。卵裂率、D幼率、三倍体率的最大影响因素分别为CB浓度、诱导持续时间、诱导起始时间与诱导持续时间。三倍体率与卵裂率无显著负相关性,而与D幼率呈显著正相关。因此,减小CB浓度或诱导持续时间,可同时获得较高的三倍体率与幼虫产量。3~15日龄三倍体组与对照组的存活率分别由71.27%与96.09%降低至34.14%与58.80%,成贝期450日龄(9月)三倍体组与对照组的存活率分别为53.62%与44.67%。3~9日龄三倍体率从87%降低至77%,而90~450日龄三倍体率平均值为59.21%±4.99%,表明幼贝与成贝期三倍体率变化较小,三倍体率的维持与存活率无显著相关性。     

Induction of triploidy in spotted sand bass (Paralabrax maculatofasciatus Steindachner, 1868) by cold shock

Conditions for the induction of triploidy with cold shock of fertilized eggs of the spotted sand bass Paralabrax maculatofasciatus (Steindachner) were investigated. Different temperatures (12, 8 and 4 °C), timing of cold shock application (5, 10 and 15 min after fertilization) and duration of the shock (5, 10, 15 and 20 min) were tested. Triploidy was determined using flow cytometry at 12 h after larvae hatched. Triploids were produced only when the cold shock treatment was applied 5 min after fertilization. No significant difference was observed in the percentage of triploidy between temperature and the shock duration. At 8 and 4 °C, 100% triploidy was obtained at different durations of cold shock. Survival was significantly lower at 12 or 4 °C than at 8 °C. No significant difference was observed for shock duration at the temperature of 8 or 12 °C; however, at 4 °C, survival was significantly lower at longer durations. We recommend induction of triploidy by applying cold shock at 8 °C for a duration of 15–20 min starting at 5 min after fertilization, in the spotted sand bass.     

Induced triploidy in grass carp, Ctenopharyngodon idella Val

Induction of triploidy in grass carp was accomplished by means of thermal shocks to eggs shortly after fertilization. Triploidy occurred most often with cold shocks at 5–7°C and at durations of 25–30 min starting 2.0–4.5 min after fertilization. Estimated percent triploid ranged from 50 to 100% on five occasions. With one exception, cold shocks of 5–7°C for less than 25 min did not induce triploidy, and cold shock durations of 30 min or longer generally resulted in 100% mortality. A heat shock of 40°C for 1 min, 4.75 min after activation, was the only heat treatment which produced triploidy (8%) with 81% surviving to the blastula stage. Fertilized eggs immersed in a solution of cytochalasin B (10 mg/l, 0.1% DMSO) for 10 min, 12 min after activation, resulted in 54% of the eggs surviving to the blastula stage with none found to be triploid.     

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     

Induction of triploidy in the turbot (Scophthalmus maximus): II. Effects of cold shock timing and induction of triploidy in a large volume of eggs

Triploidy was induced in the turbot (Scophthalmus maximus, L.) by applying cold shocks shortly after fertilization. The combined effects of the timing of cold shock commencement after fertilization, cold shock duration and cold shock temperature were investigated. Ploidy was assessed by counting the number of nucleoli per nucleus (NOR) in larvae and also by measuring erythrocyte size in juveniles. A clear peak in triploidy induction was obtained when shocks were started between 6 and 7 min after fertilization at a pre-shock temperature of 13–14°C. With this timing, shocks of 20-min duration at 0°C gave >90% triploidy, with survival about 80% of the untreated controls. In order to ensure both high triploidy rates and high survival, it was necessary to carefully maintain the water temperature just below 0°C. Experiments with small and large volumes of eggs were performed in order to determine how changes in the relative volumes of eggs and chilled water could affect survival and triploidy induction. The best combination to induce triploidy in the turbot was as follows: shock commencement 6.5 min after fertilization, shock duration 25 min, and shock temperature between 0 and −1°C. With this combination, 100% triploidy could consistently be induced with survival 60% of the untreated control. This was successfully applied to a large volume of eggs (300 ml; 1 ml 800 eggs) in order to mass-produce triploid turbot. Triploids had lower survival rate than diploids at hatching but similar thereafter, with the ability to complete the different stages of larval rearing, indicating the viability to produce triploid turbot under farming conditions.     

Induction of triploidy in the zebrafish, Brachydanio rerio (Hamilton)

Abstract. Triploidy was induced in the zebrafish, Brachydanio rerio (Hamilton), by varying all possible combinations of the time after fertilization (AF) (1-3min after insemination), temperature (36-42°C) and shock duration (1-7min). A thermal shock of 41°C for 4min, 2.5min AF ensured 100% triploidy and maximum (51%) survival. Induction of triploidy was confirmed by measurement of erythrocyte nuclear volume and chromosome counting. There was no significant difference in the growth rate of triploid and diploid fishes. All surviving triploids developed into males, and produced a few spermatozoa unable to fertilize normal eggs. A study on thermal and other characteristics required to ensure 100% triploidy rate in fish indicates that these characteristics are species specific.     

The larval rearing of the marine ornamental crab, Mithraculus forceps (A. Milne Edwards, 1875) (Decapoda: Brachyura: Majidae)

The goal of this study is to develop a larviculture protocol for Mithraculus forceps, a popular marine aquarium species. Different temperatures (25±0.5°C and 28±0.5°C), stocking densities (10, 20, 40 and 80 larvae L^?1), prey densities (newly hatched Artemia of 1, 4, 7 and 12 nauplii mL^?1) and metamorphosis to crab conditions (Systems A and B) were tested. The best survivorship and faster development were obtained when the larvae were reared at a density of 40 larvae L^?1 for 7 days post hatching (DPH) in System A, at 28°C and fed with 7 mL^?1 of newly hatched Artemia nauplii. After 7 DPH all the megalopa were moved to System B and the same temperature and prey density were maintained. At the end of the experiment, 12 DPH, survivorship of 74.1±4.8% was obtained.     

The use of clove oil, metomidate, tricaine methanesulphonate and 2-phenoxyethanol for inducing anaesthesia and their effect on the cortisol stress response in black sea bass (Centropristis striata L.)

Juvenile and adult black sea bass (Centropristis striata L.) were exposed to various concentrations of four anaesthetics to determine practical dosages for handling as well as for procedures such as bleeding, ovarian biopsy or tag implantation. In experiment 1, juveniles exposed to either 2.0 mg L^?1 metomidate, 15 mg L^?1 clove oil, 70 mg L^?1 tricaine methanesulphonate (TMS) or 200 mg L^?1 2‐phenoxyethanol (2‐PE) reached stage II of anaesthesia in 3–5 min and could be handled for weighing and measuring. All fish had completed recovery to stage III within 6 min. In experiment 2, the established concentrations of each anaesthetic were tested on juveniles to determine their ability to prevent a reflex to a subcutaneous needle puncture. All of the fish exposed to clove oil (20 mg L^?1) and 40% of the TMS‐treated (70 mg L^?1) fish reacted while none of the fish anaesthetized in metomidate (2.0 mg L^?1) or 2‐PE (200 mg L^?1) responded to the needle puncture. In experiment 3, metomidate (5.0 mg L^?1), clove oil (30 mg L^?1) TMS (125 mg L^?1) or 2‐PE (300 mg L^?1) were all effective for performing an ovarian biopsy or tag implantation on adults. In experiment 4, TMS (125 mg L^?1) exacerbated the cortisol response to a short handling stressor during a 30 min exposure. Fish anaesthetized in 2‐PE (300 mg L^?1), metomidate (5.0 mg L^?1) or clove oil (40 mg L^?1) had increased cortisol levels associated with the handling stressor but there were no further increases during the remainder of the experimental period. The results demonstrate that these anaesthetics are effective for sedation and anaesthesia of black sea bass and that the best choice is dependant upon the procedures to be performed.     

Combined effects of salinity and potassium concentration on juvenile mulloway (Argyrosomus japonicus, Temminck and Schlegel) in inland saline groundwater

This paper reports on experiments conducted to examine the combined effects of salinity and potassium concentration on survival and growth of juvenile mulloway (Argyrosomus japonicus, Temminck and Schlegel) in inland saline groundwater. Three separate experiments were conducted in 20 (±1)°C water. In the first experiment, mulloway were held in 60 L aquaria (triplicate) with salinities of 5, 15, 25 or 35 g L^?1 and potassium concentrations of 20%, 40%, 60% or 80% of the concentration present in oceanic water of the equivalent salinity in a 4 × 4 factorial combination for 7 days. Response surface contour diagrams were generated from survival data to estimate optimal conditions. The results showed that maximum survival of juvenile mulloway occurred at salinities of >14 g L^?1 and potassium concentrations of >38%. Survival was lowest at salinities of <7 and >33 g L^?1 and potassium concentrations of <25%. The second experiment was conducted with mulloway held in 60 L aquaria at salinities of 15, 25 or 35 g L^?1 and potassium concentrations of 40%, 60%, 80% or 100% in a 3 × 4 factorial combination for 44 days. Optimal conditions for maximum survival and growth of mulloway were within a salinity range of 15–35 g L^?1 and potassium concentration above 40%. The third experiment was conducted in three 500 L tanks to record the survival and growth of mulloway fingerlings held at 20 (±1)°C, 23 g L^?1 salinity and potassium concentrations of 50% for 8 months. Survival and growth of mulloway fingerling in inland saline groundwater were similar to those reported from a semi‐intensive floating tank system in inland saline water and sea cage trials in oceanic water.