Requirements of vitamin C (l-ascorbyl-2-sulphate and l-ascorbyl-2-polyphosphate) and its effects on non-specific immune responses of grouper, Epinephelus malabaricus

An 8‐week feeding trial was conducted to determine two vitamin C derivatives, l ‐ascorbyl‐2‐sulphate (C2S) and l ‐ascorbyl‐2‐polyphosphate (C2PP), to satisfy vitamin C requirement and to test their effects on the non‐specific humoral immune responses of juvenile grouper, Epinephelus malabaricus. C2S and C2PP were each supplemented at 20, 50, 80, 150, 250 and 400 mg kg^?1 diet in the semi‐purified basal diet providing of 7, 16, 28, 55, 86, 142 mg ascorbic acid (AA) equivalent of C2S kg^?1 diet and 4, 9, 15, 31, 49, 75 mg AA equivalent of C2PP kg^?1 diet, respectively. Basal diet without AA supplemented was included as a control. Each diet was fed to triplicate groups of grouper (mean initial weight: 6.69 ± 0.07 g). Fish fed diets with ≥28 mg AA equivalent of C2S or ≥4 mg AA equivalent of C2PP kg^?1 had significantly (P < 0.05) greater weight gain (WG) than fish fed the unsupplemented control diet. Liver AA concentrations were higher in fish fed diets with ≥16 mg AA equivalent of C2S or ≥9 mg AA equivalent of C2PP kg^?1 than fish fed the control diet. Alternative pathway of complement activation (ACP) was higher in fish fed diets with ≥55 mg AA equivalent of C2S or ≥15 mg AA equivalent of C2PP kg^?1 than fish fed the control diet. Lysozyme activity was higher in fish fed ≥86 mg AA equivalent of C2S or ≥15 mg AA equivalent of C2PP kg^?1 than fish fed the control diet. Analysis by broken‐line regression of WG indicated that the adequate dietary vitamin C concentration from each vitamin C derivative in growing grouper is 46.2 mg AA equivalent of C2S kg^?1 diet and 17.8 mg AA equivalent of C2PP kg^?1 diet, and it also indicated that C2S is approximately 39% as effective as C2PP in meeting the vitamin C requirement for grouper. The data suggest that both C2S and C2PP supplementation support non‐specific immune responses of grouper.     

点带石斑鱼仔稚鱼消化系统黏液细胞的类型与分布

利用组织学切片及AB-PAS组化染色技术对点带石斑鱼0～28日龄仔稚鱼消化道(口咽腔、食道、胃、肠)不同类型黏液细胞的发生与发育进行了系统的研究.点带石斑鱼黏液细胞分成Ⅰ～Ⅳ共4种着色类型和囊状、梨状、杯状3种形态类型.结果表明,点带石斑鱼黏液细胞最早出现在3日龄仔鱼的口咽腔部位.12日龄之前的黏液细胞数量较少,且主要以Ⅱ型为主,形态上主要为囊状细胞.12～15日龄黏液细胞的数量出现急剧增长,其空间分布模式也随之基本形成.之后日龄的样本中,黏液细胞以Ⅲ、Ⅳ型为主,而Ⅰ、Ⅱ型黏液细胞所占比例较低.口咽腔、食道的黏液细胞以囊状、梨状为主,胃、肠黏液细胞早期为囊状,随后梨状细胞逐渐增多,最终以杯状细胞为主.食道前段、肠前段的黏液细胞存在显著的差异,但此两段均为黏液细胞的主要分布场所.     

Requirements of vitamin C (L-ascorbyl-2-monophosphate-Mg and L-ascorbyl-2-monophosphate-Na) and its effects on immune responses of grouper, Epinephelus malabaricus

An 8‐week feeding trial was conducted to evaluate two vitamin C derivatives, L‐ascorbyl‐2‐monophosphate‐Mg (C2MP‐Mg) and L‐ascorbyl‐2‐monophosphate‐Na (C2MP‐Na), to satisfy the vitamin C requirement and to test their effects on the immune responses of juvenile grouper, Epinephelus malabaricus. C2MP‐Mg and C2MP‐Na were each supplemented at 20, 50, 80, 150, 250, and 400 mg kg^?1 diet in the basal diet providing of 7, 18, 31, 51, 93, 145 mg ascorbic acid (AA) equivalent of C2MP‐Mg kg^?1 diet and 4, 10, 17, 31, 47, 77 mg ascorbic acid (AA) equivalent of C2MP‐Na kg^?1 diet, respectively. Basal diet without AA supplementation was included as control. Each diet was fed to triplicate groups of grouper (mean initial weight 3.20 ± 0.05 g). Fish fed diets supplemented with either C2MP‐Mg or C2MP‐Na had significantly (P < 0.05) greater weight gain (WG), feed efficiency and survival than those fed the unsupplemented control diet. Liver ascorbate concentrations in fish generally increased as dietary C2MP‐Mg or C2MP‐Na supplementation level increased. Haemolytic complement activity was higher in fish fed diets supplemented with 92 mg AA equivalent of C2MP‐Mg kg^?1 or 10–17 mg AA equivalent of C2MP‐Na kg^?1 than fish fed the unsupplemented control diet. Lysozyme activity was higher in fish fed ≥51 mg AA equivalent of C2MP‐Mg kg^?1 or ≥47 mg AA equivalent of C2MP‐Na kg^?1 than fish fed the unsupplemented control diet. Analysis by broken‐line regression of WG indicated that the adequate dietary vitamin C concentration from each vitamin C derivative in growing grouper is 17.9 mg AA equivalent of 2MP‐Mg kg^?1 and 8.3 mg AA equivalent of C2MP‐Na kg^?1, and it also indicated that C2MP‐Mg is about 46% as effective as C2MP‐Na in meeting the vitamin C requirement of grouper.     

饥饿对点带石斑鱼饵料转换期仔鱼生长和发育的影响

在盐度29、水温27.8~28.8℃的条件下,进行点带石斑鱼仔鱼饥饿实验,确定仔鱼初次摄食饥饿不可逆点(PNR);观察饥饿条件下点带石斑鱼饵料转换期(初孵仔鱼,5、8、10、15和20日龄)的存活与生长、油球利用、鳍棘生长和游泳行为的变化。结果表明:点带石斑鱼从初次摄食到PNR期仅2~3d,这个耐受饥饿的时间临界点发生在孵化后的第5天,即转入外源性营养的第3天。在饥饿条件下初孵仔鱼,5、8、10、15和20日龄仔鱼的全部死亡时间分别为7 d、3 d、4 d、3 d、4 d和6 d;半数死亡时间分别为5 d、1.5 d、2 d、1.5 d、2d和3.5d,说明5日龄和10日龄是点带石斑鱼早期发育中最为敏感的阶段。饥饿会延缓初孵仔鱼对油球的利用。随着饥饿时间的延长,仔鱼的生长与正常条件下仔鱼的生长差异显著(P<0.05)。饥饿仔鱼体长较短,头大且体瘦,长期饥饿后脑后部下陷;行为变化表现为表层集群游动觅食,游动缓慢,反应迟钝和静伏底。     

Comparative efficacy of trash fish versus compounded commercial feeds in cage aquaculture of Asian seabass (Lates calcarifer) (Bloch) and tiger grouper (Epinephelus fuscoguttatus) (Forsskål)

Trash fish/low valued fish is still the most commonly used feed in marine cage culture in Asia. Use of trash fish is controversial with regard to the sustainability of the farming practices, and related issues on environmental degradation. In this study, the results of farm based trials on Asian seabass (Lates calcarifer) and tiger grouper (Epinephelus fuscoguttatus) to evaluate the efficacies of the use of commercial pellet feed in comparison to trash fish/low valued fish in marine cages in Thailand are presented. In spite of the variations observed in each of the farm sites, the results indicate that the overall growth performances and fish survival rates between the two feed types were similar, for both species. However, better cost‐benefit and resource use were recorded for fish reared on trash fish/low valued fish. No significant differences were observed in water quality parameters, dissolved oxygen, transparency and ammonia, between sites where pellet or trash fish were used, inside and outside the cages and for both species. The results of the present study are discussed in the light of the current controversies on the use of trash fish/low valued fish in marine cage culture.     

Effects of alternative dietary oils on lipid metabolism and related gene expression in hybrid grouper (♀Epinephelus fuscoguttatus × ♂E. lanceolatu)

An 8‐week growth trial was conducted to evaluate effects of dietary oil sources on growth, enzymes activity and genes expression levels related to lipid metabolism of hybrid grouper (♀Epinephelus fuscoguttatus × ♂E. lanceolatu) juveniles. Seven iso‐lipid (97 g/kg of dry matter) and iso‐protein (503.5 g/kg of dry matter) experimental diets were formulated containing 50 g/kg fish oil (FO; acting as controls) or various vegetable oils (VOs): corn oil (CO), sunflower oil (SO), tea oil (TO), olive oil (OO), rice oil (RO) and mixed oil (MO; comprising equal amounts of these oils). Each diet was fed to triplicate groups of 40 fish for per repetition (15.09 ± 0.01 g) for 56 days. The results show that (a) alternative dietary oils had no significant effects on final weight compared with control group (p > .05); (b) compared with FO group, VOs significantly changed the contents of serum lipoproteins, cholesterol, triglycerides and the activity of liver lipid‐metabolizing enzymes (p < .05); (c) CO group had the least effect on the serum lipoproteins, triglycerides and cholesterol of grouper compared with control; the activity of liver lipid‐metabolizing enzymes in RO and control group was the closest; (d) the mRNA levels of Δ6 Fatty acid desaturase (Δ6Fad), hormone‐sensitive lipase (HSL) and lipoprotein lipase (LPL) were not significantly effected by lipid sources, but CO, TO, OO and MO significantly down‐regulated the expression of fatty acid synthetase (FAS) mRNA level in liver, while RO opposite (p < .05); (e) vegetable oil significantly up‐regulated peroxisome proliferator‐activated receptor α (PPARα) and peroxisome proliferator‐activated receptor β (PPARβ) mRNA levels, while TO and RO down‐regulated peroxisome proliferator‐activated receptor γ (PPARγ) mRNA levels (p < .05); and 6) MO significantly increased the mRNA levels of heart‐type fatty acid‐binding protein (H‐FABP) and adipocyte‐type fatty acid‐binding protein (A‐FABP) (p < .05), while other VOs had no effect on them (p > .05). In conclusion, dietary substitution of FO by VO in diet affected lipid metabolism of grouper, which may be regulated by PPARs.     

Capture and handling stress affects the endocrine and ovulatory response to exogenous hormone treatment in snapper, Pagrus auratus (Bloch & Schneider)

Sexually mature female hatchery‐reared snapper, Pagrus auratus (Bloch & Schneider) were captured from sea cages by handline and injected at first capture (control) or 24 h after capture, transport and subsequent confinement (delayed injection) with either saline, luteinizing hormone releasing hormone analogue, human chorionic gonadotropin, or 17α‐hydroxyprogesterone. Blood was sampled before hormone treatment and again after 168 h, and fish were checked daily for ovulation. Plasma levels of 17β‐estradiol (E2), testosterone (T), 17α, 20β dihydroxy‐4‐pregnen‐3‐one (17, 20βP) and cortisol were determined by radioimmunoassay. The ovulatory response was assessed from the proportion of fish ovulating, ovulation volume, egg quality and fertility. A delay in injection resulted in significantly lower plasma E2 and T levels in response to hormone treatment, smaller ovulation volumes, and poorer egg quality than in control fish. The results are consistent with the generally inhibitory effects of stress on reproduction in fish, and confirm the requirement to treat fish with hormones designed to induce ovulation, as soon as possible after capture and disturbance.     

Effects of fish oil with difference oxidation degree on growth performance and expression abundance of antioxidant and fat metabolism genes in orange spotted grouper,Epinephelus coioides

The present study was designed to assess the effects of fish oil with different oxidation degree on growth performance, serum biochemistry parameters and expressive abundance of oxidative stress and fat metabolism genes of orange spotted grouper Epinephelus coioides. The oxidized fish oil was conducted as follows: storage temperature: 4°C, ambient temperature (AT, [31.5 ± 3.5]°C); storage time: 45, 90, 135 days; antioxidant contents: 30 mg/kg (ethoxyquin [EQ]), 300 mg/kg Higher EQ (HEQ). According to the different treated conditions, 14 kinds of fish oil with different oxidation degree were obtained: T_F+EQ [positive control (fresh oil + EQ)], T_F (negative control [fresh oil]), T_4°C+45d+EQ, T_4°C+45d+HEQ, T_4°C+90d+EQ, T_4°C+90d+HEQ, T_4°C+135d+EQ, T_{4°C+135d+HEQ}, T_AT+45d+EQ, T_AT+45d+HEQ, T_AT+90d+EQ, T_AT+90d+HEQ, T_AT+135d+EQ, T_AT+135d+HEQ. Groupers were fed isonitrogenous and isolipidic diets containing 14 kinds of fish oil for 8 weeks, respectively. The results showed that survival, weight gain rate and thermal growth coefficient decreased as oxidation degree of dietary fish oil increased (p < 0.05). Higher serum total protein, triglyceride and glucose were observed with ascending oxidation degree of fish oil (p < 0.05). The genes expression levels of catalase, superoxide dismutase and glutathione peroxidase were up‐regulated with dietary oxidized level increasing (p < 0.05). In addition, the similar status also appeared in expression of peroxisome proliferator‐activated receptor gamma (PPARγ), hormone‐sensitive lipase (HSL) and fatty acid synthase (FAS) genes. In conclusion, the fish oil would show negative influence on the fish health until peroxide value and p‐anisidine value in oil exceed 12.96 meq/kg and 20.89. The best storage condition for fish oil is 4°C, 45 days and 30 mg/kg EQ which could keep fish oil available property to grouper.     

Molecular characterization and pathogenicity of a grouper iridovirus (GIV) isolated from yellow grouper, Epinephelus awoara (Temminck & Schlegel)

Molecular characterization was carried out on an iridovirus isolated from yellow grouper, Epinephelus awoara . The major capsid protein (MCP) gene was located, sequenced and compared with homologous genes from other iridoviruses. The nucleotide sequence is 1392 bases long and contains a single open reading frame beginning at an ATG codon from the 5' end and terminating at a TAA codon at the 3' end. The open reading frame encodes a protein of 463 amino acids with a predicted molecular weight of 50 272 Da. Pairwise amino acid alignments detected a high degree of sequence identity between grouper iridovirus (GIV) MCP and the homologous genes of other iridoviruses. The MCP gene of GIV was most similar to the MCP gene from frog virus 3 (FV3) with 70% nucleotide and 73% amino acid sequence identity. The predicted molecular weight of the protein of this gene is comparable with the apparent weight obtained by SDS–PAGE. Pathogenicity of the GIV was investigated in yellow grouper by intraperitoneal injection of 10⁷ and 10⁴ TCID₅₀ virus. Cumulative mortalities reached 100% within 11 and 25 days post-infection, respectively, while no grouper died in the control group. The molecular studies demonstrated that GIV is a member of the genus Ranavirus .     

Growth performance and disease resistance against Vibrio vulnificus infection of novel hybrid grouper (Epinephelus lanceolatus × Epinephelus fuscoguttatus)

Groupers are economically important for aquaculture in Thailand. A novel hybrid grouper (Epinephelus lanceolatus × Epinephelus fuscoguttatus) has been successful cross‐bred; therefore, the present work aimed to assess the hybrid's traits. The growth performance, strength and tolerance to a pathogenic bacterial infection of this hybrid were compared with its parent species, tiger grouper and giant grouper. The results of all measured growth parameters indicated that the hybrid strain grew fastest followed by giant and tiger grouper respectively. The expressions of the growth‐related genes, insulin‐like growth factor (IGF) I and II, were also analysed in fish muscle and liver which are the main target organs in fish growth regulation. Among tested species, similar expression patterns of IGF‐I and IGF‐II were detected in both organs. The levels of these genes in liver and muscle of hybrid and giant grouper were higher than those of tiger grouper comparable with the growth manner. After challenge with Vibrio vulnificus, the immunological parameters, clearance time of Vibrio in haemolymph and survival was measured to verify the fish immunity. Leucocyte number, lysozyme activity and the ability to eliminate the pathogen were very high in hybrid and giant grouper while these parameters were lower in tiger grouper. Correspondingly, the mortality rate of tiger grouper was higher than others and % survival at the end of observation time (15 days post challenge) was lowest in infected tiger grouper. Altogether, the results suggested that the hybrid grouper has desirable traits that will improve cultured grouper.     

Infection of captive Pagrus auratus (Bloch & Schneider) by the monogenean, Anoplodiscus cirrusspiralis Roubal, Armitage & Rohde (Anoplodiscidae) in Australia

Abstract The monogenean Anoplodiscus cirrtisspiralis infects the fins and nares of snapper, Pagrus auratus (Bloch & Schneider). The epidermis beneath the haptor in both microhabitats is eroded and the parasite attaches to the basement membrane by an adhesive secretion. Captive snapper suffered fin damage through high levels of infection by this parasite. Laboratory experiments showed A. cirrusspiralis to be adversely affected by reduced salinity and killed within 1 h by diluted sea water of < 5%0 salinity.     

Pathogenicity of the causative agent of viral nervous necrosis disease in striped jack, Pseudocaranx dentex (Bloch & Schneider)

Abstract. The pathogenicity of the agent causing viral nervous necrosis (VNN) of striped jack, Pseudocaranx dentex (Bloch & Schneider), was examined in striped jack and other selected marine fish species. Fish were exposed to purified striped jack nervous necrosis virus (SJNNV) (0·1–100 ng ml⁻¹) or homogenates of diseased striped jack larvae. Striped jack larvae (3·5 and 4·4 mm total length) were susceptible to the virus, but juveniles (78 mm) were not. The viral antigens were detected by indirect ELISA and the characteristic pathological changes, i.e. vacuolation in the retina and brain, were reproduced in the affected larvae. The infection was also established in healthy larvae by cohabitation with the diseased larvae. Larvae of red sea bream, Pagrus major Temminck & Schlegel, yellowtail, Seriola quinqueradiata Temminck & Schlegel, and goldstriped amberjack, Seriola lalandi Valenciennes, were not susceptible to SJNNV.     

Effect of temperature,hypoxia and disinfection with ozonated seawater during somitogenesis on muscular development of the trunk in larval seven‐band grouper,Epinephelus septemfasciatus (Thunberg)

The cause of abnormal musculature on both sides of the same myotome in artificially cultured seven‐band grouper Epinephelus septemfasciatus (Thunberg) larvae remains unclear. We evaluated the effect of three factors: changes in temperature (between 22 and 24°C), disinfection with ozonated seawater (0.3 mg TROs L^?1, 1 min), and hypoxia (22 or 14% saturation) during the early (20 h post‐fertilization, hpf) and mid‐ (24 hpf) somite stages on trunk muscular development in larval seven‐band grouper. The thermal regimes and disinfection with ozonated seawater had no effect on muscle development (P > 0.05, G‐test). In contrast, exposure to hypoxia during both the early and mid‐somite stages increased the incidence of abnormal musculature in the trunk (P < 0.01, G‐test). These larvae had abnormal musculature on both sides of the trunk. In addition, the number of abnormal myotomes differed among these larvae. Exposure in the early and mid‐somite stages resulted in the highest frequency of abnormalities in the 11th and 13th myotome respectively. Our results suggest that hypoxia during somitogenesis can lead to abnormal muscle development in the trunk of artificially cultured seven‐band grouper larva.     

Use of response surface methodology to study the combined effects of temperature and salinity on hatching and deformity of the hybrid grouper,Epinephelus fuscoguttatus (♀) × Epinephelus polyphekadion (♂)

This study seeks to identify the optimal combination of temperature and salinity for the embryo development of the hybrid groupers, Epinephelus fuscoguttatus (♀) × Epinephelus polyphekadion (♂), using the central composite design and response surface methodology. Results have shown that the low hatching rates of fertilized eggs and high deformity rates of larvae were the result of lower or higher temperature/salinity levels. The linear effects of temperature and salinity were significant in both hatching rates and deformity rates (p < .01). Similar results were observed in the quadratic effects of temperature and salinity (p < .01). Effects of temperature were positively related to salinity effects (p < .05). Salinity effects were more remarkable than temperature on hatching rates, but not the same on deformity rates. Model equations were established for temperature and salinity effects on hatching rates and deformity rates. A high coefficient of determination (R² > .97) was found for the adequacy and predictive capability of model equations, indicating that these models can be applied to prediction. The optimal combination of temperature and salinity was 27.58°C/30.94, which was derived from the statistical optimization approach. In this condition, the hatching rate was the highest at 85.73%, and the deformity rate was the lowest at 5.96%, with a high desirability function of 0.957. The results of this study can be applied to improve hatching and decrease deformity in future cultures of hybrid groupers.     

Ocular lesions associated with attachment of the parasitic copepod Ommatokoita elongata (Grant) to corneas of Greenland sharks, Somniosus microcephalus (Bloch & Schneider)

The eyes from six Greenland sharks, Somniosus microcephalus (Bloch & Schneider), infected with the parasitic copepod Ommatokoita elongata (Grant) were collected in the Arctic waters of Victor Bay, North-west Territories, Canada, for study. Transformed adult female copepods, one per eye, were firmly attached to the corneas by an anchoring structure (i.e. the bulla) and each bulla was associated with an opaque area on the cornea. Two eyes additionally had one O. elongata copepodid attached to the cornea via frontal filaments with bullas which were each associated with a small corneal opacity. In addition to opacities associated with copepods at the time of collection, there were several randomly distributed, small, round-to-irregular, full-thickness corneal opacities which were not associated with copepods. Two unfixed eye lenses grossly examined in the field were normal in appearance. Histologically, corneal epithelial ulceration and heterophilic keratitis, disruption, mineralization, and detachment of Bowman's membrane, thinning, disorganization, mineralization and fibrosis of the corneal substantia propria, and focal thinning and mineralization of Descement's membrane were observed. Mild heterophilic and mononuclear anterior uveitis was also present. Based on the present observations, it is concluded that parasitism by O. elongata could lead to severe vision impairment (possibly including blindness) in Greenland sharks. However, the otherwise healthy appearance of the infected sharks studied and the information contained in the literature suggests that O. elongata infections do not significantly debilitate the hosts.     

Effect of cage colour and light environment on the skin colour of Australian snapper Pagrus auratus (Bloch & Schneider, 1801)

A two‐factor experiment was performed to evaluate the effects of cage colour (black or white 0.5 m³ experiment cages) and light environment (natural sunlight or reduced level of natural sunlight) on the skin colour of darkened Australian snapper. Each treatment was replicated four times and each replicate cage was stocked with five snapper (mean weight=351 g). Snapper exposed to natural sunlight were held in experimental cages located in outdoor tanks. An approximately 70% reduction in natural sunlight (measured as PAR) was established by holding snapper in experimental cages that were housed inside a ‘shade‐house’ enclosure. The skin colour of anaesthetized fish was measured at stocking and after a 2‐, 7‐ and 14‐day exposure using a digital chroma‐meter (Minolta CR‐10) that quantified skin colour according to the L^*a^*b^* colour space. At the conclusion of the experiment, fish were killed in salt water ice slurry and post‐mortem skin colour was quantified after 0.75, 6 and 22 h respectively. In addition to these trials, an ad hoc market appraisal of chilled snapper (mean weight=409 g) that had been held in either white or in black cages was conducted at two local fish markets. Irrespective of the sampling time, skin lightness (L^*) was significantly affected by cage colour (P<0.05), with fish in white cages having much higher L^* values (L^*≈64) than fish held in black cages (L^*≈49). However, the value of L^* was not significantly affected by the light environment or the interaction between cage colour and the light environment. In general, the L^* values of anaesthetized snapper were sustained post mortem, but there were linear reductions in the a^* (red) and b^* (yellow) skin colour values of chilled snapper over time. According to the commercial buyers interviewed, chilled snapper that had been reared for a short period of time in white cages could demand a premium of 10–50% above the prices paid for similar‐sized snapper reared in black cages. Our results demonstrate that short‐term use of white cages can reduce the dark skin colour of farmed snapper, potentially improving the profitability of snapper farming.     

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.     

Changes in skin colour and cortisol response of Australian snapper Pagrus auratus (Bloch & Schneider, 1801) to different background colours

A two-factor experiment was carried out to investigate the change in skin colour and plasma cortisol response of cultured Australian snapper Pagrus auratus to a change in background colour. Snapper (mean weight=437 g) were held in black or white tanks and fed diets containing 39 mg unesterified astaxanthin kg⁻¹ for 49 days before being transferred from white tanks to black cages (WB) or black tanks to white cages (BW). Skin colour values [ L ^* (lightness), a ^* (redness) and b ^* (yellowness)] of all snapper were measured at stocking ( t =0 days) and from cages of fish randomly assigned to each sampling time at 0.25, 0.5, 1, 2, 3, 5 and 7 days. Plasma cortisol was measured in anaesthetized snapper following colour measurements at 0, 1 and 7 days. Fish from additional black-to-black (BB) and white-to-white (WW) control treatments were also sampled for colour and cortisol at those times. Rapid changes occurred in skin lightness ( L ^* values) after altering background colour with maximum change in L ^* values for BW and WB treatments occurring within 1 day. Skin redness ( a ^*) of BW snapper continued to steadily decrease over the 7 days ( a ^*=7.93 × e^{−0.051 × time}). Plasma cortisol concentrations were highest at stocking when fish were held at greater densities and were not affected by cage colour. The results of this study suggest that transferring dark coloured snapper to white cages for 1 day is sufficient to affect the greatest benefit in terms of producing light coloured fish while minimizing the reduction in favourable red skin colouration.