Breeding and larval rearing of the camouflage grouper Epinephelus polyphekadion (Bleeker) in the hypersaline waters of the Red Sea coast of Saudi Arabia

This is the first report on the successful year-round natural spawning and larval rearing of Epinephelus polyphekadion (Bleeker) in captivity and under hypersaline water conditions of 42-43%₀ salinity in the Red Sea coast of Saudi Arabia. Although the fish spawned naturally once or twice a year during 1992-94 culture period, incorporation of cod-liver oil in the broodstock diet during the 1995 culture period enabled the fish to spawn continuously for 2-3 days in each month during March, April, May and August. The egg fertilization and hatching rates also increased during the 1995 spawning period. The egg fertilization rate varied from 90 to 100% with a mean of 96.5 ± 3.38%. The egg hatching rate varied from 70 to 95% with a mean of 83.1 ± 10.12%. The fertilized egg diameter averaged 757.3 ± 37.36 μm. There was a linear relation between the fertilized egg size and the egg hatching rate. The increase in the hatching rate relevant to the egg size was statistically significant (P < 0.01). The egg development time until hatching lasted for 19 h at 29°C. The newly hatched larval size ranged from 1.55-1.71 mm with a mean of 1.65 ± 0.052 mm in total length. The larval growth was slow in the early stages and the growth curve until metamorphosis showed a curvilinear pattern. Wide variations in larval size, range 22-47 mm with a mean of 33.40 ± 7.01 mm, were observed during the metamorphosis stage at day 50. No significant difference (P > 0.05) in growth and survival was observed between the larvae reared using white and grey coloured tanks. The larval survival up to metamorphosis was 1.6-4.7% with a mean of 2.98 ± 1.56% in the grey coloured tanks and 1.6-1.9% with a mean of 1.73 ± 0.16% in the white tanks. The results demonstrated the possibility of breeding E. polyphekadion under captive culture conditions. However, methods to improve the larval survival have to be pursued further for commercial farming of this species.     

Passive monitoring of phenological acoustic patterns reveals the sound of the camouflage grouper,Epinephelus polyphekadion

1. Passive acoustic monitoring (PAM) is a non‐invasive technique that uses hydrophones to monitor populations and ecosystem dynamics. Although many applications of PAM have been developed in recent years, it has never been used to identify a calling marine species.
2. The south pass of Fakarava Atoll, French Polynesia, hosts spawning events of many reef fish species, including the camouflage grouper Epinephelus polyphekadion, with a spawning aggregation abundance exceeding 17 000 individuals during the full moons of June and July.
3. The current study aimed to use PAM to distinguish camouflage grouper sounds among the vocal activities of all fish recorded during the aggregation periods. Audio recordings analysis resulted in the identification of 29 sound types, some of which showed diel and lunar patterns.
4. Temporal analysis of these sounds in relation to spawning activities allowed the identification of camouflage grouper calls. These calls can be described as a single pulse or a series of ‘boom(s)’ with a pulse duration of ~44 ms and a low dominant frequency of 103 ± 31 Hz. Video recordings show that the camouflage grouper produces the ‘booms’ to initialize spawner ascent and to promote synchronous gamete release into the water column.
5. The study highlights for the first time that PAM can be used to identify the previously unknown sound of a fish species. Moreover, we can use it to understand the phenology of some biological activities for improving the resolution of fish biodiversity assessments.

     

Improved fertilization rates by using a large volume tank in red spotted grouper (Epinephelus akaara)

Two types of spawning experiments were carried out using a small or a large tank in order to determine the reason for the low fertilization rate in artificially reared red spotted grouper. The mean fertilization rate in the large tank (45%) was significantly higher than the one in the small tank (22%). The fertilization rates were high when a pair spawned underwater (84%) compared with the `jump-type' spawning behavior (31%). Spawning in the large deep tank was effective for improving the fertilization rate in this species.     

Establishment of cell lines from a tropical grouper,Epinephelus awoara (Temminck & Schlegel), and their susceptibility to grouper irido- and nodaviruses

Four tropical marine fish cell lines have been established from the eye, fin, heart and swim bladder of grouper, Epinephelus awoara (Temminck & Schlegel). Optimum media and temperature conditions for maximum growth were standardized. The eye and swim bladder cells were mostly epithelial, but the fin and heart cells were mostly fibroblastic. The viability of cells was 95% after 1 year of storage in liquid nitrogen (-196 degrees C). Besides these four cell lines, previously established grouper brain, kidney and liver cell lines were also used for a viral susceptibility study which showed that all the cell lines were sensitive to grouper iridovirus, whereas only brain, fin and liver cell lines were susceptible to the yellow grouper nervous necrosis virus (a nodavirus). Electron microscopy studies of the grouper irido- and nodaviruses in ultrathin sections of infected cells showed an abundance of viral particles in the cytoplasm of the virus-infected cells indicating the effective replication of these two viruses. It is suggested that these cell lines can be used for the isolation of putative fish specific viruses and provide a valuable tool to study the mechanisms of host-pathogen interactions. Furthermore, these cell lines upon transfection, using pEGFP-C1 and pEGFP-aMT2.5 (ayu metallothionein promoter), produced significant fluorescent signals indicating their utility for exogenous studies.     

Lunar-related spawning in honeycomb grouper, Epinephelus merra

Lunar-related spawning in honeycomb grouper was investigated by histological and behavioral analyses. Ovarian development and spawning of the fish were related to lunar periodicity. And also the fish migrated to out side of the reef area and spawned for a few days after the full moon.     

Experimental evaluation of inorganic fertilization in larval giant grouper (Epinephelus lanceolatus Bloch) production

A major constraint in successful larviculture of groupers has been the small gape of the larvae and hence the requirement for small prey at first feeding. In this study, we examined how maintaining a phosphate concentration of 100 μg P L^?1 and an inorganic nitrogen (N) level of 700 μg N L^?1 via weekly fertilization with inorganic fertilizers affected phytoplankton, zooplankton and giant grouper larval survival in relation to a control group that was provided with rotifers immediately after larvae hatched. Unicellular algae, zooplankton within the size ranges of 10–50 μm and 50–100 μm and survival of giant grouper larvae were all significantly higher in the fertilized treatment compared with the control. Stomach analysis revealed that ciliates and flagellates were actively consumed by larval fish in the fertilized group, whereas few rotifers were consumed in the control. We conclude that the inorganic fertilization method provides high densities of suitable‐sized prey for larval groupers at the onset of exogenous feeding before they are able to consume larger, commercially available rotifers and copepods.     

The effects of the tank colour on growth performance and physiological responses in fingerling grouper,Epinephelus coioides

In this study, the effects of different background colours on growth performance and physiological parameters of Epinephelus coioides were investigated. One hundred eighty E. coioides (average weight 18.16 ± 0.07 g) were distributed in black, white and blue 300‐L tanks and fed to satiation for 60 days. The tank colour had no significant effect on the weight gain, condition factor, hepatosomatic index and specific growth rate (p > .05). The lowest amount of viscerosomatic index and the highest carcass protein were recorded in the white tank (p < .05). Daily food intake, feed conversion ratio and plasma glucose were significantly higher in the blue tank (p < .05). The highest levels of haematocrit and plasma triglyceride were observed in the white tank, while the total protein and albumin were higher in the black tank compared to the other tanks. The tank colour had no significant effects on the plasma cortisol level (p > .05). Our findings showed that colours used in this study did not evoke stress and thus had no effect on growth, but some metabolic adaptation occurred in each colour to achieve optimum growth.     

Establishment and characterization of a new cell line derived from kidney of grouper, Epinephelus akaara (Temminck & Schlegel), susceptible to Singapore grouper iridovirus (SGIV)

A marine fish cell line derived from the kidney of red-spotted grouper, Epinephelus akaara, designated as EAGK was established and characterized. The EAGK cells multiplied well in Leibovitz's L-15 medium containing 10% foetal bovine serum at 25 °C and have been subcultured for more than 90 passages. Karyotyping, chromosomal typing and ribosomal RNA (rRNA) genotyping analysis revealed that EAGK had a modal diploid chromosome number of 82 and was a fibroblast cell line originated from grouper. A severe cytopathic effect was observed in EAGK cells incubated with Singapore grouper iridovirus (SGIV), but not with soft-shelled turtle iridovirus, viral nervous necrosis virus or spring viraemia of carp virus. SGIV replication was further confirmed by immunofluorescence, electron microscopy and virus titre determination. Bright fluorescence was observed after transfection with fluorescent protein reporter plasmids, indicating that EAGK cells can be used to identify gene functions in vitro. In addition, the cell organelles including mitochondria and endoplasm reticulum changed and aggregated around virus factories after SGIV infection, suggested that the EAGK cell line could be an important tool for investigation of iridovirus-host interactions.     

Propagation of yellow grouper nervous necrosis virus (YGNNV) in a new nodavirus-susceptible cell line from yellow grouper, Epinephelus awoara (Temminck & Schlegel), brain tissue

A nodavirus was isolated from diseased yellow grouper, Epinephelus awoara , larvae cultured in southern Taiwan. The histopathology and RT–PCR results confirmed that it was a fish nodavirus; its coat protein gene sequence was similar to that of red spotted grouper nervous necrosis virus (RGNNV) and it is named yellow grouper nervous necrosis virus (YGNNV). A new nodavirus-susceptible cell line, grouper brain (GB) was established and characterized from the brain tissue of yellow grouper. The GB cells multiplied well in Leibovitz's L-15 medium supplemented with 10% foetal bovine serum at temperatures between 24 and 32 °C, and have been subcultured more than 80 times, becoming a continuous cell line. The GB cell line consists of fibroblast-like cells and some epithelioid cells. The cell line yielded titres of YGNNV up to 10^8.5 TCID₅₀ mL^–1. The GB cells effectively replicated the virus at 28 °C, which could be purified to homogeneity by caesium chloride gradient centrifugation. Electron microscopy studies showed that purified virus particles were 25–30 nm in diameter. The cytoplasm of infected cells was filled with aggregates of virus particles. These results indicate that the GB cell line is a significant tool for the study of fish nodaviruses.     

石斑鱼新型网箱健康养殖技术

从新型网箱健康养殖与传统网箱养殖的比较分析入手,进而从养殖场的选择、网箱的结构、苗种的放养、网箱养殖日常管理及疾病预防这几个方面分析石斑鱼新型网箱健康养殖技术,以供参考。     

七带石斑鱼染色体核型研究

采用PHA体内直接注射法制备了七带石斑鱼(Epinephelus septemfasciatus)头肾组织染色体标本并分析其核型。结果表明,七带石斑鱼二倍体染色体数为48,24对染色体均为端部着丝粒染色体,其染色体臂数(NF)为48,核型公式为:2n=48t,NF=48。未发现与性别相关的异型染色体,第24对染色体长度明显小于其他染色体。通过与其他22种石斑鱼染色体核型进行比较,发现七带石斑鱼具有石斑鱼属鱼类的原始核型特征,属于石斑鱼属鱼类的原始类群。     

石斑鱼虹彩病毒病发生风险评估模型的建立和验证

为评估石斑鱼养殖过程虹彩病毒病发生的风险,实验结合Delphi法和层次分析法(analytic hierarchy process,AHP)构建新加坡石斑鱼虹彩病毒(SGIV)发生的风险评估模型.风险因素评估指标体系包括1个目标层(石斑鱼虹彩病毒病发生风险),5个准则层(水质、石斑鱼健康状况、饲养管理、养殖模式和养殖环...     

Improvement in lipid metabolism and stress tolerance of juvenile giant grouper,Epinephelus lanceolatus (Bloch), fed supplemental choline

Six fish meal basal diets supplied with 0 (control), 0.25, 0.5, 1, 2.5 and 5 g kg^?1 of choline chloride, resulting in choline levels of 2.57, 2.67, 2.94, 3.84, 4.99 and 7.71 g kg^?1, respectively, were fed to giant grouper, Epinephelus lanceolatus, for 56 and 30 days to evaluate the growth and lipid metabolism, and stress tolerance respectively. In the first trial, fish fed different levels of choline‐containing diets for 56 days had no significant difference in weight gain, survival and feeding efficiency. Fish fed increased levels of dietary choline, however, tended to have decreases in the hepatic somatic index; lipids, triglycerides, and cholesterol in liver; and triglycerides and cholesterol in serum. A decrease of lipid content in dorsal muscle was recorded in fish fed the diets containing choline >2.94 g kg^?1. Additionally, dietary choline improved the reactions of fish to ammonia stress, including survival and behavioural responses, in fish fed diets containing choline levels >2.94 g kg^?1. These findings indicate that choline plays important roles in lipid metabolism and stress tolerance in giant grouper.     

Induced sex reversal of dusky grouper, Epinephelus marginatus (Lowe)

The present paper describes sex reversal in dusky grouper, Epinephelus marginatus (Lowe). Over a 4-month feeding period, 5 mg kg^?1 of the male hormone 17-α methyltestosterone was combined with frozen fish as feed. After 2.5 months of treatment, the gonads of all six fish treated in the experiment presented tissues with oocytes and spermatogonia, and after 3 months, there were only a few remnants of oogonial tissue left in the gonads. All fish in the experiment gave sperm of varying quality after 4 months of the trial. The quantity of the hormone administered to the fish over these 4 months was 89-113 mg kg^?1. Spermatozoid motility varied from 95% to 40%, and lasted for about one hour in sea water. Using the combined sperm of three males, fertilization ranged from 50% to 95%. Fertilized eggs and hatched larvae continued to develop normally.     

Two iridovirus-susceptible cell lines established from kidney and liver of grouper, Epinephelus awoara (Temminck & Schlegel), and partial characterization of grouper iridovirus

Two iridovirus-susceptible cell lines were established and characterized from grouper Epinephelus awoara kidney and liver tissues. These cell lines have been designated GK and GL, respectively. The cells multiplied well in Leibovitz's L-15 medium, supplemented with 10% foetal bovine serum, at temperatures between 20 and 32 °C, and have been subcultured more than 120 times, becoming continuous cell lines. The cell lines consist of a heterogeneous mixture of fibroblastic and epithelial cells. The viability of cells, stored frozen in liquid nitrogen (−196 °C), was 95% after 1 year. Chromosome morphologies of GK and GL cells were homogeneous. Both cell lines were susceptible to grouper iridovirus, and yielded high titres of up to 108 TCID50 mL⁻¹. In addition, both cell lines effectively replicated the virus, which could be purified to homogeneity by cesium chloride gradient centrifugation. Electron microscopy studies showed that purified virus particles were 170±10 nm in diameter, and were hexagonal in shape. Virus-infected cells showed an abundance of virus particles inside the cytoplasm. These results show that the GK and GL cell lines effectively replicate grouper iridovirus, and can be used as a tool for studying fish iridoviruses.     

Conditioning,maturation and year‐round natural spawning of orange‐spotted grouper,Epinephelus coioides (Hamilton, 1822), in recirculating aquaculture system

The present experiment was aimed at studying the conditioning, maturation and natural spawning of orange‐spotted grouper, Epinephelus coioides, in a recirculatory aquaculture system (RAS). Thirty fish (n = 30; 3.35 ± 0.05 kg) were stocked in a circular tank of 125 m³ capacity fitted with an RAS for conditioning and broodstock development. After 15 days, 15 fish were implanted with 17 α methyl testosterone and letrozole at the rate of 5 mg and 0.2 mg/kg body weight, respectively, for conversion from female to male. The gonadal development started after 1 month, and by 90th day, 63.53 ± 3.78% and 2.07 ± 0.84% of the oocytes attained a size of 400–500 μm and 500–600 μm respectively. Natural spawning commenced in the RAS from 4th month onward after stocking and spawning continued round the year. The spawning pair showed courtship behaviour with a typical vertical burst of swimming just before release of gametes. The total number of eggs spawned during 1 year was 47.23 million with spawning frequency varying form 5 to 13 times per month. The association of spawning events with new moon day (lunar cycle) weakened as time progressed. The mean monthly fertilization and hatching rates varied from 77.80 ± 3.34% to 83.70 ± 1.76% and 82.80 ± 4.21% to 88.33 ± 1.39% respectively. The study proved that RAS is an efficient system that provides a stable, controllable and conducive environment for year‐round natural breeding of orange‐spotted grouper.     

Infection and pathology in Queensland grouper,Epinephelus lanceolatus, (Bloch), caused by exposure to Streptococcus agalactiae via different routes

Since 2007, 96 wild Queensland groupers, Epinephelus lanceolatus, (Bloch), have been found dead in NE Australia. In some cases, Streptococcus agalactiae (Group B Streptococcus, GBS) was isolated. At present, a GBS isolate from a wild grouper case was employed in experimental challenge trials in hatchery‐reared Queensland grouper by different routes of exposure. Injection resulted in rapid development of clinical signs including bilateral exophthalmia, hyperaemic skin or fins and abnormal swimming. Death occurred in, and GBS was re‐isolated from, 98% fish injected and was detected by PCR in brain, head kidney and spleen from all fish, regardless of challenge dose. Challenge by immersion resulted in lower morbidity with a clear dose response. Whilst infection was established via oral challenge by admixture with feed, no mortality occurred. Histology showed pathology consistent with GBS infection in organs examined from all injected fish, from fish challenged with medium and high doses by immersion, and from high‐dose oral challenge. These experimental challenges demonstrated that GBS isolated from wild Queensland grouper reproduced disease in experimentally challenged fish and resulted in pathology that was consistent with that seen in wild Queensland grouper infected with S. agalactiae.