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.     

A bath challenge model for furunculosis in rainbow trout, Salmo gairdneri Richardson, and Atlantic salmon, Salmo salar L.

Abstract. A natural bath challenge method has been developed for furunculosis using Atlantic salmon, Salmo salar L., and rainbow trout, Salmo gairdneri Richardson. Fish were placed in an enclosed, continuously circulating tank system, supplemented with additional oxygen, the temperature raised gradually (overnight) to 15–16°C, a low dose of Aeromonas salmonicida (strain 184/186) introduced into the tank and the challenge maintained for 14 days. The challenge strain was characterized with respect to possible virulence factors and possessed an A-layer, ability to auto-agglutinate, haemagglutinate, adhere to Atlantic salmon cells and resist destruction by serum. No caseinase activity was detected. LD₅₀ for salmon using this method was 1.8×10³ cells per millilitre while trout had an LD₅₀ of 9.5×10⁴ cells per millilitre. Onset of the disease appeared to depend on fish size with larger trout (50 g) taking up to 10 days to show signs of the disease while mortalities in smaller trout (8.5g) commenced on day 1 post-challenge.     

Comparative sensitivity of carp, Cyprinus carpio L. and rainbow trout, Salmo gairdneri Richardson, to Renibacterium salmoninarum

Abstract. The pathogenicity of Renibacterium salmoninarum to carp, Cyprinus carpio L., and rainbow trout, Salmo gairdneri Richardson, was investigated. All carp injected with 4·8 × 10⁸ cells/fish, or 4·8 × 10⁷ cells/fish survived for 38 days. R. salmoninarum was isolated from all moribund fish, but not from the kidney of surviving fish, although R. salmoninarum antigen was detected in several of these fish by the dot blot assay. On the other hand, mortality in rainbow trout was 95% in the fish injected with 4·8 × 10⁸ cells/fish, and 15% in those which received 4·8 × 10⁷ cells/fish. R. salmoninarum antigen was detected by the dot blot assay in all surviving rainbow trout. The number of R. salmoninarum cells was immediately decreased by carp or rainbow trout serum, and the serum bactericidal activity of carp was higher than that of rainbow trout. Carp blood leucocytes had higher phagocytic activity than those of rainbow trout.     

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.     

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.     

Cultivation of Trypanosoma danilewskyi (Laveran & Mesnil, 1904) in serum-free medium and assessment of the course of infection in goldfish, Carassius auratus (L.)

Abstract. Purification and in vitro cultivation techniques were developed for the fish haemoflagellate, Trypanosoma danilewskyi. The parasites were isolated and purified from the peripheral blood of experimentally infected goldfish, using a combination of Ficolt-paque gradient centrifugation to remove fish red blood cells and in vitro incubation to remove the remaining fish leucocytes. A serum-free culture medium for T. danilewskyi supported both short- and long-term cultivation of the haemoflagellates. The serum-free medium is a mixture of reagents available commercially: Leibovitz's L-15 medium, Dulbecco's Modified Eagle Medium and Hank's balanced salt solution. The doubling time was calculated to be 44.4 × 7.8h. Typically, a two- to five-fold increase in the number of cultured parasites was observed on day 7 after subculture with 1 × 10⁶ and 5 × 10⁵ trypanosomes ml⁻¹, respectively. When administered to fish, the in vitro -derived parasites caused an infection and pathology whose characteristics were similar to those observed following infection with trypanosomes obtained from infected goldfish. The freshly isolated and in vitro -grown parasites were successfully cryoprescrvcd in the culture medium containing 10% glycerine at −80°C for at least 3 months. Although the viability of the parasite decreased by 40–50% after thawing, cryoprcserved parasites retained the ability to infect goldfish.
Correspondence: Dr M. Belosevic, Associate Professor, Department of Zoology, CW-312 Biological Sciences Building, University of Alberta, Edmonton, AB, Canada T6G 2E9.     

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.     

Effects of processing on physical characteristics of diets with distinct levels of carbohydrates and lipids: the outcomes on the growth of pacu (Piaractus mesopotamicus)

The use efficiency and feed conversion of extruded and pelletized diets were compared. Eight isoproteic diets (220 g kg⁻¹ digestible protein) were assayed for 90 days in a 2 × 2 × 2 multifactorial design with two carbohydrate levels (400 and 500 g kg⁻¹), two lipids levels (40 and 80 g kg⁻¹) and two diet processing (pelletization and extrusion) with three repetitions. The growth of Piaractus mesopotamicus fed with these diets and the quality control indices of diets were gauged. The density of extruded diets was lower as carbohydrate level was 400 g kg⁻¹ and lipid 40 g kg⁻¹. The interaction carbohydrate and diet processing presented higher leaching value for low carbohydrate level in extruded diet. Fish fed with extruded diets presented the best feed conversion and protein efficiency ratio. When high levels of carbohydrate and lipid are combined, the weight gain is impaired. The interaction between diet processing diet and lipid levels resulted in the best fish performance when pelletized diets with 40 g kg⁻¹ lipid or extruded diets with 80 g kg⁻¹ lipid were considered. The protein efficiency ratio increased with the increment of carbohydrates in the pelletized diets. The fish show low tolerance to lipids and a preference for carbohydrate when the lipid productive values are taken into account.     

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.     

Preliminary investigations on husbandry, nutrition and growth of glass eels and elvers, Anguilla anguilla L.

Abstract. Some effects of husbandry and feeding on growth of glass eels and elvers of the European eel. Anguilla anguilla L., were investigated. In the first 7 months after arrival the recorded mortality amounted to 34% of the initial population. due to escapes and possibly cannibalism the observed survival after 7 months was only 21% of the initial population.
Mean growth rate of the whole population was 7.1 g day⁻¹ kg^−0.8. Growth proved to be highly variable. Growth rate was clearly affected by feeding level but not by initial stocking density.
Maximum growth rate was approximately 8g day, ¹ kg^−0.8 with feed conversions of 1.7–2.0. An optimal feeding level of 7.5g day¹ kg^−0.8 resulted in feed conversions of 1.3–1.6.     

The susceptibility of salmonid fish to an atypical strain of Aeromonas salmonicida that infects goldfish, Carassius auratus (L.), in Australia

Abstract. An enzootic, Australian, atypical strain of Aeromonas salmonicida isolated from diseased goldfish, Carassius auratus (L.), was inoculated into Atlantic salmon, Salmo salar L., brown trout, S. trutta L., rainbow trout, S. gairdneri Richardson, and brook trout, Salvelinus fontinalis (Mitchill), fingerlings by intraperitoneal injection (i.p.) and by bath challenge, the latter with and without prior abrasion of skin. The 10-day LD₅₀ (i.p.) was estimated to be 7·4 × 10^-3 colony forming units (cfu) for Atlantic salmon, 3·0 × 10^-2 cfu for brown trout, 3·7 × 10² cfu for brook trout and 6·4 × 10³ cfu for rainbow trout. Brown, rainbow and brook trout succumbed to bath challenges with between 10⁵–10⁶ cfu/ml, developing ulcers of the skin and septicaemia. The organism was trasmitted from inoculated fish to five of 195 within-tank control fish via water and established a carrier state in one of 14 Atlantic salmon. It was concluded that the organism poses a significant threat to the salmonid farming industry and wild salmonid fisheries in Australia.     

An experimental study of the susceptibility of Atlantic salmon fry, Salmo salar L., to viral haemorrhagic septicaemia

Abstract. Infection trials using two serotypes of VHS viruses (type 1 and 23/75) demonstrated that Atlantic salmon fry were susceptible to the disease when injected intraperitoneally (i.p.) with 10³ pfu of virus/fish but resistant to infection by a bath method when exposed for 3 h in water containing 5 × 10⁴ pfu of virus/ml. In the i.p.-infected fish, mortality reached 78 and 67% within 13 days with VHSV₁ nad 23/75 serotypes, respectively. High virus yields were recovered from infected fish and virus shedding was demonstrated by the onset of VHS in rainbow trout kept in the outflow water from the aquaria containing infected salmon. Neither mortality nor virus shedding occurred in salmon infected by the water route but virus multiplication was demonstrated in 2 of 60 fish with VHSV₁ and 3 of 60 fish with virus 23/75. On day 79 post-infection the sera from surviving salmon of both i.p. and bath infection trials exhibited good neutralizing titres (around 1000) against the homologous viruses.     

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.     

Effects of Carbon Dioxide Exposure on Intensively Cultured Rainbow Trout Oncorhynchus mykiss: Physiological Responses and Fillet Attributes

Rainbow trout Oncorhynchus mykiss (261.6 × 24.7 g initial weight, mean × SEM) at 13.1 × 0.2 C were exposed for 94 d to one of three CO₂ treatments: control (22.1 × 2.8 mg/L), medium (34.5 × 3.8 mg/L), or high (48.7 × 4.4 mg/L). Trout were checked daily for survival, and fish were sampled at 0, 28, 56, and 84 d for physiological responses, growth, and fillet quality assessments. Trout were also challenged to a 15-min crowding stress at 93 d to assess their ability to initiate a stress response during hypercapnia. Chronically exposed trout showed nearly 100% survival through 84 d exposure (1 of 1,500 fish died). Growth and physiological results showed that increasing elevated CO₂, concentrations result in corresponding decreased growth rates and CO₂specific physiological parameters: The medium and high CO₂ treatments had significantly slower growth and subsequently smaller fish by 84 d. Exposed trout also showed significantly ( P < 0.05) decreased plasma chloride for medium and high CO₂ treatments compared to the control from 28 through 84 d. Decreased growth and smaller fish in the medium and high CO₂ treatments resulted in correspondingly smaller fresh and smoked fillet weights. Chronic CO₂ exposure did not result in notable changes in ultimate muscle pH. Exposure to 15-min crowding stress at 93 d resulted in significant changes in hematocrit, plasma cortisoI, glucose, and chloride for all treatment groups. CO₂-specific changes were detected in hematocrit, plasma cortisoI, and plasma chloride responses following the 15-min crowding stress.     

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).     

Underwater sound detection by cephalopod statocyst

Abstract:   The cephalopod receptor of particle motion was identified. In a previous study, it was suggested that statocysts served this function, but there was no direct supporting evidence, and epidermal hair cells had not been conclusively ruled out. Experiments on Octopus ocellatus were conducted using respiratory activity as an indicator of sound perception. Intact animals clearly responded to 141-Hz particle motion at particle accelerations below 1.3 × 10⁻³ m/s², and the mean perception threshold at this frequency was approximately 6.0 × 10⁻⁴ m/s². Specimens in which the statoliths had been surgically removed did not show any response for accelerations up to 3.9 × 10⁻³ m/s² at 141 Hz, which was approximately 16 dB greater than the mean perception threshold at this frequency. Specimens that had undergone a control operation in which the statoliths remained intact showed positive responses at 2.8 × 10⁻³ m/s² for the same frequency stimulus. This indicates that the statocyst, which is morphologically similar to the inner ear system in fish, is responsible for the observed responses to particle motion in O. ocellatus . This is the first direct evidence that cephalopods detect kinetic sound components using statocysts.     

The Induction of Tetraploidy in Salmonids

The induction of tetraploidy was attempted in three intraspecies crosses (autotetraploid) of sal-monid: chinook salmon ( Oncorhynchus tshawytscha ), coho salmon ( O. kisutch ) and rainbow trout ( Salmo gairdneri ), and in the interspecies hybrid (allotetraploid) coho × Atlantic salmon ( Salmo salar ).
Pressure treatments of 6.2 × 10⁴ kPa (9,000 psi) were applied for six minutes at intervals from 50% of the time to cleavage to beyond first cleavage. Analysis of eyed stage embryos via flow cytometry indicated two periods of tetraploid induction existed corresponding to 55–75% and 100–110% of the first cleavage interval (FCI).
The effects of different pressures and the times of application were analyzed in a study with rainbow trout. Treatments of 4.8 × 10⁴ kPa (7,000 psi) for eight minutes, and 5.5 and 6.2 × 10⁴ kPa (8,000 and 9,000 psi) for four minutes, were applied at 65, 78, and 100% FCI. Tetraploids were found in the 5.5 and 6.2 × 10⁴ kPa group treatments only when applied at 65% FCI.
The results indicate that, while tetraploidy is inducible in salmonids, further experimentation is necessary to discern the appropriate time and treatment for optimal tetraploid induction in each species.     

Migration of anadromous white-spotted charr <Emphasis Type="Italic">Salvelinus leucomaenis</Emphasis>, as determined by otolith strontium: calcium ratios

ABSTRACT:   The migratory history of anadromous white-spotted charr Salvelinus leucomaenis collected from Japanese coastal waters, was examined in terms of strontium (Sr) and calcium (Ca) uptake in the otolith, by means of wavelength dispersive X-ray spectrometry using an electron microprobe. Otolith Sr concentration or Sr : Ca ratios of anadromous S. leucomaenis , fluctuated strongly along the life history transect in accordance with the migration (habitat) pattern from sea to fresh water. The anadrmous S. leucomaenis showed phase L (low Sr : Ca ratio) from the core to the point 1000–2500 µm distant, averaging 1.3 × 10⁻³ to 2.7 × 10⁻³ and thereafter, the ratios increased sharply, being higher than 5.0 × 10⁻³ to 10.0 × 10⁻³. These findings indicated that otolith Sr : Ca ratios reflected individual life histories, enabling a sea habitat to be identified from a freshwater habitat in this species.     

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.