Accumulation of immunomodulatory laminaran [β(1,3)-D-glucan] in the spleen and kidney of Atlantic salmon, Salmo solar L.

Abstract. The tissue distribution of ³H- and FITC-laminaran in Atlantic salmon, Salmo salar L., was investigated after intravenous administration. Liquid scintillation counting and whole-body autoradiography revealed that the concentration of ³H-labelled laminaran in the spleen and anterior kidney was higher than in blood throughout the experimental period (1 h to 12 days). Renal excretion and intestinal exsorption were the main elimination pathways for laminaran. Microscopic examination revealed accumulation of FITC-labelled laminaran in macrophages in kidney and spleen. In addition, endothelial cells in the kidney, spleen, liver and intestine contained the immunomodulator. Thus, these experiments show that laminaran accumulates and is retained in immunologically relevant cells in the kidney and spleen.     

Intestinal absorption of immunomodulatory laminaran and derivatives in Atlantic salmon, Salmo salar L.

Abstract. Radiolabelled (¹²⁵I, ³H) immunomodulatory laminaran (isolated from Laminaria hyperborea ), a β(1,6)-branched β(1,3)-D-glucan and different radiolabelled sulphated analogues of laminaran were administered peranally to Atlantic salmon, Salmo salar L. Intestinal absorption and tissue distribution were examined by means of radioactive tracer techniques. The intestinal uptake was highest with native laminaran and laminaran with a low degree of sulphatation, while highly sulphated laminarans were poorly absorbed. The tissue distribution analysis revealed high amounts of radiolabelled compound in the liver, and anterior and posterior kidney, whereas the spleen contained low amounts. Peak serum and organ concentrations were reached about 30 min after administration. The results were confirmed by autoradiography of tissue sections from salmon after peranal administration of radiolabelled laminaran. Finally, the peranal administration of fluorescence labelled laminaran revealed epithelial supranuclear fluorescent vacuoles containing laminaran. It is concluded that native laminaran and slightly sulphated laminaran are absorbed from the posterior intestine and that they are distributed to tissues rich in immunocompetent cells. Thus, these compounds may have potential as immunomodulatory feed additives.     

Continuous exposure to infectious pancreatic necrosis virus during early life stages of rainbow trout, Oncorhynchus mykiss (Walbaum)

Rainbow trout, Oncorhynchus mykiss (Walbaum), were exposed continuously to infectious pancreatic necrosis virus (IPNV) at 0, 10¹, 10³ or 10⁵ plaque forming units (pfu) L⁻¹ of water to estimate the effects of chronic IPNV exposure on early life stages. Fish density averaged 35 fish L⁻¹ (low density) or 140 fish L⁻¹ (high density), and the tank flow rate was 250 mL⁻¹ min. Virus exposure began at 6 days before hatch and continued until fish were 44 days old. Cumulative per cent mortality, analysis of survival and hazard functions, and discrete-time event analysis were used to explore the patterns of survival and mortality. In eggs and fish exposed to IPNV, mortality significantly greater than in the 0 pfu L⁻¹ exposure did not occur until IPNV concentration was 10⁵ pfu L⁻¹ at low fish density and 10³ pfu IPNV L⁻¹ at high fish density. These results suggest that in the natural aquatic environment, where rainbow trout densities are likely to be considerably lower than in this study, mortality resulting from infection with IPNV will very likely not occur when ambient concentrations of virus are ≤10³ pfu IPNV L⁻¹. In aquaculture rearing units, trout density is likely to be as high or higher than the densities used in this study. Therefore, continuous inputs of virus at concentrations greater than 10¹ pfu L⁻¹ may result in IPN epidemics in aquaculture facilities.     

Intestinal absorption and tissue distribution of glucose and isoleucine in rainbow trout (Oncorhynchus mykiss)

The intestinal absorption and tissue distribution of D(U¹⁴C)-glucose (¹⁴C-glc), 3-O-methyl-D(U¹⁴C)-glucose (¹⁴C-OMG) and L(U¹⁴C)-isoleucine (¹⁴C-ile) were studied in rainbow trout, Oncorhynchus mykiss, using a forced-feeding technique. The appearance of radioactivity in faeces, blood, liver, white muscle and brain was monitored over 48 h. The recoveries of radioactivity 48 h postfeeding in the fish tissues studied were 36, 57 and 48% for ¹⁴C-glc, ¹⁴C-ile and ¹⁴C-OMG, respectively. White muscle and liver contained most radioactivity on the whole tissue basis. Concentrations of ¹⁴C-glc and its derivatives in the liver and the brain exceeded those of ¹⁴C-ile and its derivatives, but the reverse was found in the white muscle. These differences may reflect differences in the metabolism of glucose and isoleucine. All tissues studied showed some differences in the accumulation of ¹⁴C originating from ¹⁴C-OMG and ¹⁴C-glc. As ¹⁴C-OMG is a nonmetabolizable glucose analogue, these differences may be the result of glucose metabolism at tissue level.     

The effect of dietary phosphatidylcholine and its constituent fatty acids on microdiet ingestion and fatty acid absorption rate in gilthead sea bream, Sparus auratus, larvae

This study tested the effect of the level of dietary phosphatidylcholine (PC) and its constituent medium-chain fatty acids on microdiet ingestion (μg diet larva⁻¹ h⁻¹) and the absorption rate of the free fatty acid [¹⁴C]16:0 (pmole larva⁻¹ h⁻¹) in 15, 20, 21, 25, 26, 30 and 31-day-old gilthead sea bream, Sparus auratus L., larvae. Fish were fed four microdiets (A, B, C and D): microdiet A contained no phospholipid (PL), while microdiet B included 10 g kg⁻¹ Artemia nauplii PL (3.7 g kg⁻¹ PC). Microdiets C and D contained 10 g kg⁻¹ purified saturated PC dimyristoyl (C_14:0) and polyunsaturated PC dilinoleoyl (C_{18:2[cis]−9,12}), respectively.
Larvae from one or both of the PC microdiets demonstrated significantly higher ( P < 0.05) ingestion rates (μg diet larva⁻¹ h⁻¹) than the non-PL microdiet control in 15, 21, 22, 25 and 26-day-old larvae and the Artemia PL microdiet in 15, 22 and 26-day-old larvae. However, microdiet ingestion and fatty acid absorption rate appeared to be independent of the associated medium carbon chain saturated or polyunsaturated fatty acid moiety of the PC diets. Apparent absorption, as measured by the retention of radio-labelled [¹⁴C]16:0 following 8 h of non-labelled microdiet feeding, was possibly related to feeding.     

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.     

The infectivity by different routes of exposure and shedding rates of Aeromonas salmonicida subsp. salmonicida in Atlantic salmon, Salmo salar L., held in sea water

Abstract. The infectivity of the bacterial fish pathogen Aeromonas salmonicida subsp. salmonicida to Atlantic salmon, Salmo salar L., in sea water was investigated and found to be similar to that reported for fresh water. The minimal infective dose in short duration bath exposures (1–3 days) was 10⁴ colony-forming units (cfu) per ml, while prolonged exposure for three weeks, but not for 1 week, produced infection with 10² cfu/ml. Intragastric intubation of A. salmonicida established infection with doses of >10⁵ cfu. Release of bacteria from dead or morbid infected fish was monitored and found to be in the order of 10⁵–10⁸ cfu/fish/h. These results emphasize the importance of removing dead fish from farm sites.     

An experimental investigation on aspects of infectious salmon anaemia virus (ISAV) infection dynamics in seawater Atlantic salmon, Salmo salar L.

This study investigated infection dynamics of infectious salmon anaemia virus (ISAV) by conducting two experiments to examine minimum infective dose and viral shedding of ISAV. In terms of minimum infective dose, the high variability between replicate tanks and the relatively slow spread of infection through the population at 1 × 10¹ TCID₅₀ mL⁻¹ indicated this dose is approaching the minimum infective dose for ISAV in seawater salmon populations. A novel qPCR assay incorporating an influenza virus control standard with each seawater sample was developed that enabled the quantity of ISAV shed from infected populations to be estimated in values equivalent to viral titres. Viral shedding was first detected at 7 days post-challenge (5.8 × 10⁻² TCID₅₀ mL⁻¹ kg⁻¹) and rose to levels above the minimum infective dose (4.2 × 10¹ TCID₅₀ mL⁻¹ kg⁻¹) on day 11 post-challenge, 2 days before mortalities in ISAV inoculated fish started. These results clearly demonstrate that a large viral shedding event occurs before death. Viral titres peaked at 7.0 × 10¹ TCID₅₀ mL⁻¹ kg⁻¹ 15 days post-infection. These data provide important information relevant to the management of ISA.     

Spring viraemia of carp virus (SVCV): comparison of immunoperoxidase, fluorescent antibody and cell culture isolation techniques for detection of antigen

Abstract. The sensitivity of the immunoperoxidase (IP) and fluorescent antibody (FA) techniques applied to frozen sections of organs of carp infected with spring viraemia virus (SVCV) was similar, both in respect of the intensity of the reaction and in the detection rate of the antigen. Seven days after a waterborne infection both methods detected the antigen in the kidneys and to a lesser extent in the liver and spleen. Quantification of virus gave a titre of 10^2.8 to 10^5.5 TCID₅₀/g of kidney, whereas the agent could not be isolated from liver and spleen tissue. In contrast, at 24¹/₂ days post–infection the viral antigen could be readily detected in liver and spleen but only occasionally in the kidneys using the IP and FA techniques. At this time, liver and spleen showed infectivity titres of 10^3.8 to 10^7.2 TCID₅₀/g tissue, whereas the kidneys were found to be free of infective virus. It is concluded that using the IP and FA techniques SVCV can be detected most frequently in the kidneys from 7 to 14 days post–infection and in the liver and spleen from 14 to 24 days post-infection.     

Fertilization efficiency of cryopreserved sperm from striped catfish, Pangasius hypophthalmus (Sauvage)

The fertilization efficiency of cryopreserved sperm was compared with fresh sperm from striped catfish, Pangasius hypophthalmus . Of the two sets of experiments carried out, the first compared four sperm doses using fresh sperm and fresh eggs. The second experiment compared six concentrations of cryopreserved sperm ranging from 6.94 × 10⁷ to 6.94 × 10¹⁰ to fertilize 100 eggs per batch. Fertilization, hatch and survival rates were compared between cryopreserved and fresh sperm. The highest fertilization rate (53.75±1.62%) was achieved with a sperm dose of 6.94 × 10⁸. Increasing the sperm dose to 3.47 × 10⁹ did not increase the fertilization rate, indicating that the optimum sperm:egg ratio lies between 6.94 × 10⁶ and 3.47 × 10⁷ sperm per egg. Both highest (6.94 × 10¹⁰) and the lowest (6.94 × 10⁷) sperm doses resulted in lower fertilization rates (2.04% and 16.90% respectively). No significant differences were found among four fresh sperm doses compared. Mean hatch and survival rates resulting from fresh and cryopreserved sperm were similar. The experiment shows that while only 1.89 × 10⁶ fresh spermatozoa was required to fertilize a fresh egg, 6.94 × 10⁶ (or 3.67 times more) cryopreserved sperm was required to achieve the same level of fertilization. This provides important information for making decision to cryopreserve sperm for commercial and/or conservation purposes.     

Substrate specificity of carp hepatopancreatic cathepsin S for peptides and myofibrillar proteins

SUMMARY: From 250 g of carp hepatopancreas, 0.29 mg of the purified enzyme was obtained. The bond specificity of cathepsin S for α-neoendorphin and neurotensin was ⁶Arg-⁷Lys and ³Tyr-⁴Glu, respectively. The cleavage sites for insulin B-chain were estimated to be the bonds at ³Asn-⁴Gln, ⁶Leu-⁷Cys, ¹²Val-¹³Glu, ¹³Glu-¹⁴Ala, ¹⁶Tyr-¹⁷Leu, ²²Arg-²³Gly, ²⁴Phe-²⁵Phe and ²⁶Tyr-²⁷Thr. P2 position on these peptides were bulky and hydrophobic amino acid residues such as Phe or Leu, small amino acid residues such as Gly, Ala and Val were also accepted in these positions. Regarding the protein substrates, cathepsin S degraded carp α-actinin, actin, tropomyosin and troponins T and I although the proteolyzing speeds were distinct from one another.     

The potential of liposomes as a nutrient supplement in first-feeding marine fish larvae

The ingestion rate (ng liposome larva^–1 h^–1) of extruded [1–¹⁴C] palmitic acid-labelled liposomes containing physiological saline (PHS) or cod fish extract (CFE), was tested in 5-day-old gilthead seabream Sparus aurata and white grouper Epinephelus aeneus larvae. A follow-up study compared the assimilation of radioactive free fatty acid (FFA) label of these two liposome treatments into six phospholipid and neutral lipid fractions as well as the nonlipid fraction in 5-day-old seabream. In seabream larvae, there was a 50-fold ( P  < 0.05) increase in the net consumption rate when fed CFE liposomes (2305.8 ng liposome larva^–1 h^–1) compared with liposomes containing physiological saline (42.7 ng liposome larva^–1 h^–1). A similarly significant ( P  < 0.05) but less marked pattern was also observed in the grouper larvae where the CFE treatment larvae ingested 238.5 ng liposome larva^–1 h^–1 compared with 54.3 ng liposome larva^–1 h^–1 in larvae fed the PHS liposomes. In seabream larvae ingesting CFE and PHS liposomes, radioactivity was found in all larval fractions analysed. However, marked treatment differences ( P  > 0.05) in assimilation were found only in the triacylglycerol fraction (3.4 and 0.6 dpm larva^–1 h^–1, respectively) and nonlipid fraction (11.2 and 15 dpm larva^–1 h^–1, respectively).     

The enrichment and retention of ascorbic acid in rotifers fed microalgal diets

The enrichment and retention of ascorbic acid (AA) was investigated in rotifers Brachionus plicatilis fed on microalgae ( Nannochloropsis oculata and Isochrysis sp. (T.ISO)) and baker's yeast Saccharomyces cerevisiae . The concentrations of AA of the rotifer diets used in the study differed significantly: 4200 μg g⁻¹ of dry weight in Isochrysis sp. (T.ISO), 2600 μg g⁻¹ in N. oculata and only 77 μg g⁻¹ in S. cerevisiae . Rotifers contained 620 μg AA g⁻¹ prior to the experimental feeding. When subsequently fed for 3 h on microalgae at a ration of 0.13 mg dry microalgae per 10⁶ rotifers rapidly and efficiently increased their content of AA: Isochrysis sp.-fed rotifers contained 1600 μg AA g⁻¹ and N. oculata -fed rotifers contained 1100 μg AA g⁻¹. Concentrations were boosted by a further feeding of a second ration of algae at three times the initial feeding ration; 21 h later, Isochrysis sp.-fed rotifers contained 2500 μg AA g⁻¹ and N. oculata -fed rotifers contained 1700 μg AA g⁻¹. This represented a 180% and 310% increase in the pre-feeding vitamin concentrations in Isochrysis sp. and N. oculata -fed rotifers, respectively. There were no significant changes in AA concentration in rotifers fed a similar ration of yeast throughout the feeding period (520-620 μg AA g⁻¹). Rotifers retained AA during a subsequent 24 h non-feeding period, with no significant changes in the concentrations in any of the rotifer groups. The production of rotifers rich in AA may be particularly valuable for the culture of fish larvae that have a high requirement for the vitamin.     

Vaccination of Mixed and Full-Sib Families of Channel Catfish Ictalurus punctatus Against Enteric Septicemia of Catfish With a Live Attenuated Edwardsiella ictaluri Isolate (RE-33)

These studies were conducted to evaluate the efficacy of a live attenuated Edwardsiella ictaluri vaccine against enteric septicemia of catfish. In one study channel catfish fingerlings (72 d of age post hatch) were immersed for 30 min in water containing E. ictaluri RE-33 at dosages of 1 × 10⁶, 1 × 10⁷ and 2 × 10⁷ CFU/ML of water. No mortalities were observed following vaccination. Following exposure to virulent Edwardsiella ictaluri the cumulative mortality of fish vaccinated with dosages of at least 1 × 10⁷ CFU/mL were significantly lower than that of non-vacccinated fish in both laboratory and field challenges. Vaccination with 1 × 10⁶ CFU RE-33mL provided some protection during the laboratory challenge but failed to protect fish under field conditions. In a second study, vaccination of 6 full-sib families of channel catfish at a vaccine dosage of 1 × 10⁷ CFU/mL resulted in a relative percent survival among families ranging from 67.1 to 100%. Significant differences in mortality were found among families and between vaccinated and unvaccinated groups, but there was no family by vaccine interaction. Families with the highest mortality after vaccination were also shown to have the highest mortality without vaccination (r = 0.82; P = 0.04).     

Histopathology, electron microscopy and isolation of channel catfish virus in experimentally infected European catfish, Silurus glanis L.

Abstract Two groups of European catfish, Silurus glanis L., fingerlings were infected with channel catfish virus (CCV) by either intraperitoneal injection with 10⁵ TCID₅₀ of CCV, or bathing in water containing 10⁵ TCID₅₀ of CCV per 1·0 ml. The virus was isolated from spleen, intestine and brain of CCV-injected fish at day 1 and the titres ranged from 10^2·1 to 10^3·3 TCID₅₀/g. However, the tissue distribution of CCV was irregular and no virus was isolated after day 3 post-exposure. In CCV-bathed fish, the virus was isolated only from the liver of one specimen at day 3 post-exposure. No clinical signs of CCV disease developed in any of the fish. Specimens in each regime from all sampling periods showed some minor histopathological changes, but there were no differences between treatments. Lesions included oedema and focal haemorrhage in the liver and the spleen was congested. Electron micrographs of tissue samples showed the presence of a few virus particles around the nuclei of kidney, spleen and intestinal cells, and in or around a myelinated nerve within the optic lobes of infected fish during the first 4 days of infection.     

Genetic characterization of Portuguese brown trout (Salmo trutta L.) and comparison with other European populations

Abstract– Allozyme and other protein loci were examined to study the genetic structure of Portuguese brown trout ( Salmo trutta ) populations. A total of 247 individuals from three tributaries of the Lima hydrological basin and a hatchery, all located in northern Portugal, were analyzed. Four of 22 protein coding loci were found to be polymorphic: CK-A1^*, GPI-A2^*, MPI-2^* and TF^*. A new allelc at the latter locus was found in Atlantic populations. The data obtained for Portuguese brown trout were compared with published data for 14 European populations and three hatchery stocks. Six polymorphic loci (CK-A1^*, GPI-A2^*, GPI-B2^*, LDH-C^*, ME^* and MPI-2^*) were used in a cluster analysis. This showed the similarity of Portuguese natural populations and northern Iberian populations and that Portuguese hatchery fish have an autochthonous origin, distinct from that of other Atlantic hatchery stocks.     

Characterization of grouper nervous necrosis virus (GNNV)

Grouper nervous necrosis virus (GNNV) was isolated from moribund grouper larvae, Epinephelus sp., using a fish cell line GF-1. The present study describes the biochemical and biophysical properties of GNNV and the expression of GNNV in diseased grouper larvae. Viral protein was detectable in most of the GNNV-infected GF-1 cells by the fluorescent antibody technique (FAT) after 12 h post-infection (p.i.), although no cytopathic effect (CPE) appeared at that time. Clear CPE developed on the third day, and complete disintegration of the monolayer occurred over the subsequent two days. The infectivity of GNNV can be blocked following treatment at 60 °C for 1 h. GNNV was sensitive to pH 3 and pH 10–12 with a 4 log₁₀ drop in infectivity. Purified GNNV was analysed by SDS–PAGE, and then stained with periodic acid silver. The positive staining indicated that its two capsid proteins were glycoproteins. Genomic RNAs of GNNV were extracted from purified virions and analysed. The molecular weights of genomic RNAs were 1.02 × 10⁶ and 0.50 × 10⁶ Da. The T2 region of the coat protein gene of GNNV was amplified by polymerase chain reaction (PCR), and the multiple alignment of the T2 sequence of two GNNV isolates with four genotypes of fish nodaviruses revealed that these two isolates (GNNV9410 and GNNV9508) belong to the red-spotted grouper nervous necrosis virus (RGNNV) genotype. The tissue distribution of GNNV in naturally infected grouper larvae was investigated by in situ hybridization using a dig-labelled probe, which showed that GNNV was not only detected in the brain and retina, but also in the gill, skeletal muscle, liver, pyloric gland, intestine and blood cells in the heart.     

Two pathogens of Greenshell™ mussel larvae, Perna canaliculus: Vibrio splendidus and a V. coralliilyticus/neptunius-like isolate

Bacterial pathogens of Greenshell™ mussel (GSM) larvae can cause batch losses during hatchery production. Twenty-two isolates were screened using a larval bioassay. Two strains were identified as potential pathogens. Phenotypic identification of these strains revealed two non-reactive Gram-negative, oxidase positive rods. Sequencing of the 16S rRNA gene identified Vibrio splendidus and a V. coralliilyticus/neptunius -like isolate as pathogens of GSM larvae, with an ability to cause 83% and 75% larval mortality in vitro , respectively, at a concentration of 10² CFU mL⁻¹. Histopathology indicated that the route of infection was via the digestive system. Using healthy larvae as target hosts, Koch's postulates were confirmed for the two isolates. This is the first report on pathogens of GSM larvae.     

Growth and spawning of hatchery-reared Chinese white prawn Penaeus (Fenneropenaeus) chinensis released in the Ariake Sea, Japan

SUMMARY: Growth and reproduction of hatchery-reared Chinese white prawn Penaeus chinensis released in the Ariake Sea, Japan, were examined. Chinese white prawn grew rapidly, reaching a body length of 154 mm in males and 198 mm in females by November (219–229 days after hatching). Maximum body length of sampled individuals was 164 mm in males and 223 mm in females. Growth curve of the Chinese white prawn was fitted to the Pitcher and MacDonald's formula, L_t = 155.0{1 − e ^{2.925sin[2π(t − 16.151)/365] − 0.0623( t − 10.712)}} for males and the logistic curve, L_t = 200.3/[1 + e ^{(1.985–0.034 t )}] for females (where L_t is the body length t days after release and t is the number of days after release). Females reached sexual maturity in late February and spawning occurred until April. Minimum size at ripe and spawned stages was 189 mm and 193 mm body length, respectively.     

The growth rate of two species of microalgae used in shellfish hatcheries cultured under two light regimes

Abstract. The production potential of any shellfish hatchery depends on the capacity of its algal system. The hypothesis that microalgal cultures grown under 12:12h light:dark photoperiod may produce the same cell densities as those using constant light is tested.
Two species of marine microalgae used in shellfish hatcheries. Chaetoceros gracilis Schütt and Isochrysis galbana clone T-iso, were grown in cultures at 18°C, under continuous light (1.43 × 10⁷μ^-2day^-1) and 12:12h light:dark cycles (2.87 × 10⁷μE m^-2 day^-1). Both light regimes provided equal amounts of light per day. In the continuous light cultures the mean doublings per day for exponentially growing cells were 1.37 and 1.49 for C. gracilis and I. galbana respectively and for the 12:12h light:dark cycles were 1.47 and 1.56 respectively. After 14 days of growth, the numbers of cells per unit of volume showed no significant differences between the two light regimes. The results are discussed in terms of a review of other authors' findings and in terms of the usefulness of the continuous light method in producing algae to be used in shellfish hatcheries.