Perception of light intensity by Haliotis discus discus based on locomotor activity patterns

ABSTRACT:   An apparatus to measure the locomotor activity of aquatic benthic organisms at variable low light levels was developed and the diurnal behavioral pattern of the abalone Haliotis discus discus was measured at various low light intensities. During the experiment, abalone were exposed to 12 h light–dark cycles of complete darkness, 0 µmol/m²/s throughout the 12 h dark cycle and, during periods I (days 1–8) and III (days 19–26), the 12 h light cycles were set at 10 µmol/m²/s. During period II (days 10–17), abalone were exposed to a light level during the 12 h light cycles of 1 × 10⁻⁵, 1 × 10⁻⁶, 1 × 10⁻⁷ or 1 × 10⁻⁸ µmol/m²/s and the changes in locomotor activity assessed. At daytime levels of 1 × 10⁻⁵ µmol/m²/s, typical behavioral patterns were observed of high locomotory activity during the night-time cycle. However, at lower light intensities, the distinction between day and night activity patterns became less clear and, at intensities lower than 1 × 10⁻⁷ µmol/m²/s, the difference between activity during the light and dark cycles became negligible. Based on this, we conclude that the threshold of light level perception in relation to locomotor activity is approximately 1 × 10⁻⁷ µmol/m²/s. The significance of these results in relation to the entrainment of behavior in abalone is discussed.     

Effects of temperature, salinity and irradiance on the growth of the toxic dinoflagellate Gymnodinium catenatum (Dinophyceae) isolated from Hiroshima Bay, Japan

ABSTRACT: Temperature and salinity ranges in which Gymnodinium catenatum (Hiroshima Bay strain) showed specific growth rates higher than 0.2/day were approximately 20–30°C and 20–32. The specific growth rate (μ), expressed as a polynomial equation as functions of temperature ( T ; °C) and salinity ( S ) were μ = (−6.84 × 10⁻⁴ T ² + 0.0354 T – 0.213) × (−1.03 × 10⁻³ S ² + 0.0579 S – 0.548)/0.31; the maximum growth rate (0.31/day) was obtained at 25°C and 30. From a comparison with field data recording temperature, salinity and light intensity, this species may be expected to bloom from summer to autumn in Hiroshima Bay.     

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.     

Cryopreservation of YamúBrycon siebenthalae Milt

The yamú Brycon siebenthalae is an endemic fish of the Orinoco river basin, but wild stocks are decreasing because of the disruption of their habitat. We evaluated a protocol for the cryopreservation of yamú sperm to contribute to the preservation of this endangered genetic resource. Milt was mixed with a cryoprotectant medium (5.5% glucose, 12% egg yolk, and 5%, 10%, or 15% dimethyl sulfoxide - DMSO) in a ratio 1:4 (milt:medium), stored in 0.5-mL French straws, frozen in nitrogen liquid vapor (-76 C), then immersed and stored in liquid nitrogen for 10 d or 12 mo. Motility of thawed spermatozoa was higher ( P < 0.001) in 10% DMSO medium than 5% DMSO or 15% DMSO mediums; but lower than the control ( P < 0.001). With sperm cryopreserved, the highest level of fertilization was achieved with 10% DMSO ( P < 0.001) after 10 d or 12 mo of cryopreservation. Fertilization of eggs inseminated with 6.4 × 10⁹ spermatozoa per g of eggs was higher ( P <0.05) than with 1.6 × 10⁹ spermatozoa per g of eggs. There was no difference (P > 0.05) in fertilization between insemination doses of 3.2 × 10⁹ and 6.4 × 10⁹ spermatozoa per g of eggs. Cryopreservation of yamu milt can be performed successfully with a simple medium combined with 10% of DMSO as cryoprotectant. The highest level of fertility was achieved using between 3 × 10⁹ and 6 × 10⁹ spermatozoa per g of fresh eggs.     

Quantitative real time PCR for the measurement of white spot syndrome virus in shrimp

Quantitative real time PCR, recently developed in molecular biology, is applied in this paper to quantify the white spot syndrome virus (WSSV) in infected shrimp tissue. The WSSV content in moribund shrimp of all species tested ( Penaeus stylirostris, P. monodon, P. vannamei ) ranged from 2.0 × 10⁴ to 9.0 × 10¹⁰ WSSV copies μg^–1 of total DNA ( n =26). In whole moribund post-larvae, 4.3 × 10⁹ WSSV copies μg^–1 of DNA were detected which is equivalent to 5.7 × 10¹⁰ WSSV copies g^–1 of post-larvae. The comparison of WSSV content between different tissues showed that muscle and hepatopancreas tissues contained 10 times less virus than gills, pleopods and haemolymph. With inocula of known virus content, bioassays by immersion challenge showed that a minimum of five logs of WSSV copies was necessary to establish disease in the challenged shrimp. In contrast, five logs of WSSV copies injected into shrimp muscle produced a LT-50 of 52 h. This real time polymerase chain reaction (PCR) technique is sensitive (four copies), specific (negative with DNA from shrimp baculoviruses and parvoviruses), dynamic (seven logs) and easy to perform (96 tests in <4 h).     

The specific detection of infectious pancreatic necrosis virus in infected cells and fish by the immuno dot blot method

Abstract. The immuno dot blot technique is a rapid, sensitive and quantitative method for the diagnosis of infectious pancreatic necrosis virus ( ipnv ) in both infected tissue culture lysates and tissue homogenates of fish. Uninfected tissue culture lysates and fish homogenates do not react in the peroxidase dot blot system after appropriate treatment. The immuno dot blot method takes 4h to complete, and the dot blot membrane can be stored in the dark for a long period. The viral proteins in the infected tissue culture cells could be detected at 3h post-infection if an initial high MOI was used. The minimum dose of ipnv-ab strain which could be detected using homologous serum was 2·5 ng purified ipnv protein/dot. The smallest infectious dose of VR-299, SP, AB, EVE and CY-1 ipnv which could be detected by the immuno dot blot system was 10⁴ to 10⁵ TCID₅₀. When one μg of purified ipnv was used, a 1·6 × 10⁴ dilution of rabbit anti-AB antisera could be detected.     

Influence of photoperiod, frequency of stripping and presence of females on sperm output in turbot, Scophthalmus maximus (L.)

Abstract. The effect of four environmental conditions was investigated upon sperm output in turbot, Scophthalmus maximus (L.), submitted to three different rhythms of stripping. Males kept under a natural light cycle and under a 6-month contracted light programme released a similar sperm output in terms of total volume of semen produced per fish during the experimental period (4·9 ± 0·9ml), mean sperm concentration (29·4 ± 2·8 × 10⁹ spermatozoa/ml) and total sperm number (163·2 ± 40·5 × 10⁹ spermatozoa). Attempts to stimulate spermiation for a second time just after the end of the natural reproduction period resulted in the release of low sperm output (total volume of semen: 1·6 ± 0·4 ml; mean sperm motility: 2 min 36s ± 0 min 47s; mean sperm concentration: 47·6 ± 10·2 × 10⁹ spermatozoa/ml; total sperm number: 84·5 ± 25·3 × 10⁹ spermatozoa). Stripping frequency had no effect on total volume of semen, mean sperm motility and total sperm number. Monthly collection did not modify sperm samples in relation to stripping rank. However, decreasing volume, motility and sperm concentration were observed when males were stripped fortnightly and weekly. During the natural spawning period, the presence of females in the tank enhanced mean sperm motility (from 3 min 27s + 0 min 52s to 6 min 38s ± 1 min 38s).     

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.     

Effect of different light regimes on the docosahexaenoic acid (DHA) content of Isochrysis aff. galbana (clone T-ISO)

The culture of Isochrysis aff. galbana Green (T-ISO) in hatcheries is promoted for its high docosahexaenoic acid (DHA) content which favours the successful rearing of aquatic larvae. In this paper we identified the need for well-defined culture conditions of microalgae if sufficient DHA content is the final goal. Using photoperiods of 24:0 h, 12:12 h, and 16:8 h L/D combined with photon flux densities (PFDs) of 110, 220, and 460E m-2 s-1, batch cultures of T-ISO were sampled for DHA content in the exponential as well as the post-exponential (light limited) growth phase. The various light regimes resulted in differences of DHA content of T-ISO revealing influences and strong interactions among the three variables studied. For fast and economic production of T-ISO cells rich in DHA it is recommended to harvest batch cultures at the end of the exponential phase using high photon flux densities (PFDs) under 12:12 h L/D cycles. The same regime or lower PFD under 24:0 h L/D seem to be sufficient for a continuous culture optimization scheme. In any case a strict following of culture protocols is very important since there is evidence for coexistence of different clones within the parental cultures leading to different results depending on which one prevails.     

Uptake and Clearance of Edwardsiella ictaluri in the Peripheral Blood of Channel Catfish Ictalurus punctatus Fingerlings during Immersion Challenge

Uptake and clearance of Edwardsiella ictaluri in the peripheral blood of channel catfish Ictalurus punctatus fingerlings were monitored for 216 h after exposure to E. ictaluri for 4 h and 8 h under static conditions. Most fish exposed to E. ictaluri developed bacteriemia 24 h post-exposure, and by 72 h post-exposure E. ictaluri was recovered from all the blood of all exposed fish. The number of E. ictaluri colony forming units (CFU) in the blood of moribund fish ranged between 1.7 × 10³ to 1.6 × 10⁵ CFU/50 μL whole blood. Clearance of bacteria from the blood was observed by 216 h post-exposure and all fish surviving bacterial exposure developed agglutinating antibody against E. ictaluri . The pathogenesis of the infection was accompanied by the shedding of viable E. ictaluri into the water which may serve as a mechanism by which fish to fish transmission occurs.     

Preliminary investigations on the effects of dietary lipid on the spawning performance and egg quality of black sea bass Centropristis striata L

Adult black sea bass Centropristis striata broodstock ( N =162) were fed three different dietary treatments: two commercially prepared diets with 45% protein and two different lipid levels (12% and 20%) (diets 1 and 2), and a diet of frozen Atlantic silversides Menidia menidia (SS, diet 3). Broodstock were held under controlled photothermal conditions and induced to spawn with an LHRHa pellet (72 μg kg⁻¹ bw). Dietary lipid had pronounced effects on spawning performance and egg quality. Diet 3 (SS) produced a significantly ( P <0.05) higher fertilization success (22.4%) than diets 1 (0.6%) and 2 (4.8%). The hatching success of fertilized eggs was similar in all diets (range=40–58.6%), but only two spawns from diet 1 (12% lipid) yielded viable yolk-sac larvae (YSL). Diet 3 (SS) also produced significantly more YSL per female (21.8 × 10³) than the diet 1 (0.3 × 10³). Eggs from diet 3 (SS) contained a significantly greater proportion of n-3 series fatty acids, with docosahexaenoic acid (DHA) as the largest fraction. Eggs from commercially prepared dietary treatments contained significantly more n-6 fatty acids. The poor spawning performance of fish fed diet 1 (12% lipid) may be related to higher levels of linoleic acid and lower levels of DHA in the diet.     

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).     

Analysis of bacterial communities in <Emphasis Type="Italic">Nannochloropsis</Emphasis> sp. cultures used for larval fish production

ABSTRACT:   Phytoplankton used in fish hatcheries is mass-cultured in the open air and usually contains large numbers of bacteria. In commercial fish production, the phytoplankton cultures are usually added into the larval rearing tanks; however, the numbers and types of bacteria introduced into the rearing tanks simultaneously are unknown. In this study, the bacterial community structures in Nannochloropsis sp. cultures were analyzed by using fluorescence in situ hybridization (FISH). A direct viable count (DVC)-FISH analysis was also performed as DVC is useful for the detection of actively growing cells. Total numbers of bacteria in Nannochloropsis sp. cultures ranged from 7.72 × 10⁵−2.39 × 10⁶ cells/mL. High proportions of the total bacteria (31.6–53.6%) in the Nannochloropsis sp. cultures showed growth potential. DVC-FISH analysis revealed that α-proteobacteria and the Cytophaga – Flavobacterium cluster were abundant in the bacterial community of actively growing cells. Thus, the high growth potentials of the distinct bacterial communities in Nannochloropsis sp. culture must influence the bacterial communities in larval rearing tanks.     

Temporal variation of seasonality of egg production and the spawning biomass of Pacific anchovy, Engraulis japonicus, in the southern waters of Korea in 1983–1994

Embryonic mortality, egg production and the spawning stock biomass of Pacific anchovy, Engraulis japonicus , off Southern Korea during 1983–1994, and their biological response to oceanographic features in spring and summer, were analysed. The instantaneous mortality rate (IMR) of embryonic stages decreased in spring and increased in summer, with a range of 0.33–1.23 day^–1 in spring and 0.78–1.69 day^–1 in summer. Egg production in summer was three times that during spring and production was low in the late 1980s. Mean lengths of yolk-sac larvae and adult females were greater in spring than in summer, whereas spawning fraction and spawning stock ratio (spawning biomass:adult biomass) were lower in spring than summer. Estimated mean spawning stock biomass ranged from 141 × 10³ to 380 × 10³ MT in spring and from 221 × 10³ to 557 × 10³ MT in summer. Statistically, the seasonal and long-term trends of embryonic mortality, egg production and spawning stock biomass of Pacific anchovy can be explained largely by spring warming, summer cooling and by less abundant zooplankton in the late 1980s.     

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.     

Enrichment of Rotifers Brachionus plicatilis with Eicosapentaenoic Acid and Docosahexaenoic Acid Produced by Bacteria

Abstract— Two bacterial strains, rich in either eicosapentaenoic acid [EPA, 20:5(n-3)] ( Shewanella gel-idimarina ACAM 456) or docosahexaenoic acid [DHA, 22:6(n-3)] ( Colwellia psychroeryrhrus ACAM 605) were tested for their ability to enrich rotifers Erachionus plicatilis in these polyunsaturated fatty acids. Rotifers were exposed for 24 h to each bacterial strain and to a mixture of the two strains. They were then harvested and their fatty acid compositions were analysed and compared to those of rotifers that had been either starved or fed yeast Saccharomyces cerevisiae or microalgae Tetraselmis suecica in 2-L glass flasks. Exposure to 1.4 × 10⁹ cells/ml of the EPA-producing bacterium only resulted in rotifer EPA levels increasing from 0.1% to 1.2% of total dry weight (%dw). Similarly, following exposure to 1.0 × 10⁹ cells/mL of the DHA-producing bacterium only, rotifer DHA levels increased from below detection to 0.1% dw. When exposed to a mixture of the two bacterial strains, containing 7.0 × 10⁸ celldml of the EPA producer and 5.0 × 10⁸ cells/mL of the DHA producer, the rotifers'final EPA and DHA levels were 0.5% dw and 0.3% dw respectively. Although feeding strategies need refining, these results show, for the first time, that rotifers can be enriched with DHA from bacteria, and that rotifers can be enriched simultaneously with both DHA and EPA from different bacterial strains.     

Serratia marcescens: a potential pathogen for fish

Abstract. Characterization of a red pigmented enterobacterium isolated from natural population of white perch, Morone americanus (Gmelin), during the course of a bacteriological survey in the Back River (Baltimore, Maryland, USA) indicated that the bacterium belonged to the species Serratia marcescents. The virulence properties of this isolate (RB 469), studied in comparison with the reference strain of S. marcescens ATCC 1800 and S. plymuthica K1R, revealed that all the strains were highly pathogenic for fish with LD₅₀s raging from 5 × 10³ to 1 × 10⁵. Similarly, te extracellular products (ECP) from the three isolates were lethal for fish (LD₅₀ ranging from 0·22 to 4·8 μg protein g^-1 fish). However, only ECP from strains with strong proteolytic activity (the white perch isolate and S. plymuthica ) were cytotoxic for both in fish and homoiothermic cell cultures and both activities were completely destroyed by heating at 100°C for 10min. In contrast, only the two S. marcescens strains which possessed phospholipase active were pathogenic for mice and produced enterotoxins. None of the Serratia strains displayed dermonecrotic factor in rabbits. All these lindings indicate that a direct relationship between eytotoxicity and virulence cannot be established.     

The Potential Use of Mangrove Forests as Nitrogen Sinks of Shrimp Aquaculture Pond Effluents: The Role of Denitrification

A generalized nitrogen budget was constructed to evaluate the potential role of mangrove sediments as a sink for dissolved inorganic nitrogen (DIN) in shrimp pond effluents. DIN concentrations were measured in pond effluents from three semi-intensive shrimp ponds along the Caribbean coast of Colombia between 1994–1995. Mean NH₄⁺ concentrations in the discharge water for all farms were significantly higher (67 × 12 μg/L) than in the adjacent estuaries (33 × 8 μg/L). Average NH₄⁺ concentrations in the pond discharge over all growout cycles were similar, representing an approximate doubling in relation to estuarine water concentrations. In contrast, NO₂^-+ NO₃^- concentrations were similar in both pond effluent and estuarine waters. Dissolved inorganic nitrogen loading of the ponds was similar. The estimated reduction of DIN in pond effluents by preliminary diversion of outflow to mangrove wetlands rather than directly to estuarine waters would be × 190 mg N/m² per d. Based on this nitrogen loss and depending upon the enrichment rate, between 0.04 to 0.12 ha of mangrove forest is required to completely remove the DIN load from effluents produced by a 1-ha pond.     

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.