Further development of bithionol therapy as a treatment for amoebic gill disease in Atlantic salmon, Salmo salar L.

This study examined the efficacy of bithionol as a prophylactic or therapeutic oral treatment for Atlantic salmon (AS), Salmo salar , affected by amoebic gill disease (AGD). Furthermore, it explored the interaction of bithionol oral therapy with the current standard treatment (a freshwater bath for at least 3 h). The efficacy of three medicated feeds was determined in the trial by feeding AGD-affected AS at 1% body weight (BW) day⁻¹ either oil coated commercial feed (control) or prophylactic and therapeutic bithionol at 25 mg kg⁻¹ feed. Feeding commenced 2 weeks prior to exposure to Neoparamoeba spp. at 300 cells L⁻¹ and continued for 49 days post-exposure (PE). Bithionol when fed as a 2-week prophylactic or therapeutic treatment at 25 mg kg⁻¹ feed delayed the onset of AGD pathology and reduced the percentage of gill filaments with lesions. Administration of a 3-h freshwater bath at 28 days PE significantly reduced amoeba numbers to a similar level across all treatments; in contrast, gross gill score and percent lesioned filaments were reduced to different extents, the control having a significantly higher score than both bithionol treatments. Following the freshwater bath, clinical signs of AGD increased at a similar level across all treatments, albeit controls were significantly higher than the bithionol treatments immediately following freshwater treatment. This study demonstrated that bithionol at 25 mg kg⁻¹ feed, when fed as a 2-week prophylactic or a therapeutic treatment, delayed and reduced the intensity of AGD pathology and warrants further investigation as a treatment for AGD-affected AS.     

Furunculosis in Atlantic salmon (Salmo salar L.) is not readily controllable by bacteriophage therapy

The potential of bacteriophage therapy to control bacterial disease in farmed fish was tested using, as an example, furunculosis of Atlantic salmon, caused by Aeromonas salmonicida subsp. salmonicida.

In vivo testing with Atlantic salmon and rainbow trout (Oncorhynchus mykiss Walbaum) showed no adverse effects, with bacteriophage generally cleared within 96 h of administration by either intraperitoneal (i.p.) injection or oral in-feed.

Juvenile Atlantic salmon were administered a combination of bacteriophage O, R and B (1.9 × 10⁸ pfu fish^− 1) by i.p. injection, after they had been challenged with A. salmonicida subsp. salmonicida 78027, also by i.p. injection. The fish that were injected with bacteriophage immediately after challenge died at a significantly slower rate then those that were either not treated with bacteriophage, or treated 24 h post-challenge. However, the end result (100% mortality) was not affected.

In further experiments the effects of oral (1.88 × 10⁵ pfu g^− 1 fish^− 1 daily for 30 days), bath (1.04 × 10⁵ ml^− 1 daily for 30 days) and i.p. (6.25 × 10⁷ pfu fish^− 1) phage treatment to control furunculosis in experimentally infected Atlantic salmon were compared with antibiotherapy (treatment with 10 mg kg^− 1 bw^− 1 day^− 1 oxolinic acid for 10 days), using an indirect cohabitation challenge. No protection was offered by any of the bacteriophage treatments, compared to the positive challenge group, although significant protection was offered by the oxolinic acid treatment. Analysis of samples taken from the trials demonstrated that bacteriophage were correctly administered to the fish and, on occasion, were isolated from fish that had succumbed to furunculosis. It was also shown that bacteriophage resistant A. salmonicida subsp. salmonicida isolates could be recovered from mortalities in all the treatment groups.

The results suggest that, although there were no safety problems associated with the approach, furunculosis in Atlantic salmon is not readily controllable by application of bacteriophage.     

In vitro survival and the effect of water chemistry and oxidative chemical treatments on isolated gill amoebae from AGD-affected Atlantic salmon

Amoebic gill disease (AGD) of cultured salmonids in Tasmania is caused by the amphizoic parasitic amoeba Neoparamoeba pemaquidensis. The freshwater tolerance of amoebae isolated from the gills of AGD-affected salmon (predominantly N. pemaquidensis) was tested in vitro using a trypan blue exclusion assay. Amoebae exposed to water containing high concentrations of Ca²⁺ or Mg²⁺ (200 mg l⁻¹) showed high levels of survival up to 3 h of exposure. Exposure to water containing elevated Na⁺, choline chloride or water at different pH all had no significant survival of amoebae. Exposure of amoebae to different concentrations of chlorine dioxide, chloramine-T or hydrogen peroxide in artificially hard water demonstrated that chloramine-T and hydrogen peroxide were the most efficacious at killing amoebae in vitro. This work suggests that the hardness of freshwater may be an important factor for the survival of marine amoebae (predominantly N. pemaquidensis) on the gills of AGD-affected salmon and have significant implications with regard to the efficacy of freshwater bathing practices for the control of AGD on farms. Additionally, chloramine-T and hydrogen peroxide appear to be efficacious at killing marine gill amoebae in vitro and may be useful for the control of AGD in farmed Atlantic salmon.     

Metabolic scope, swimming performance and the effects of hypoxia in the mulloway, Argyrosomus japonicus (Pisces: Sciaenidae)

The culture of the mulloway (Argyrosomus japonicus), like many other Sciaenidae fishes, is rapidly growing. However there is no information on their metabolic physiology. In this study, the effects of various hypoxia levels on the swimming performance and metabolic scope of juvenile mulloway (0.34 ± 0.01 kg, mean ± SE, n = 30) was investigated (water temperature = 22 °C). In normoxic conditions (dissolved oxygen = 6.85 mg l^− 1), mulloway oxygen consumption rate (M·o₂) increased exponentially with swimming speed to a maximum velocity (U_crit) of 1.7 ± < 0.1 body lengths s^− 1 (BL s^− 1) (n = 6). Mulloway standard metabolic rate (SMR) was typical for non-tuna fishes (73 ± 8 mg kg^− 1 h^− 1) and they had a moderate scope for aerobic metabolism (5 times the SMR). Mulloway minimum gross cost of transport (GCOT_min, 0.14 ± 0.01 mg kg^− 1 m^− 1) and optimum swimming velocity (U_opt, 1.3 ± 0.2 BL s^− 1) were comparable to many other body and caudal fin swimming fish species. Energy expenditure was minimum when swimming between 0.3 and 0.5 BL s^− 1. The critical dissolved oxygen level was 1.80 mg l^− 1 for mulloway swimming at 0.9 BL s^− 1. This reveals that mulloway are well adapted to hypoxia, which is probably adaptive from their natural early life history within estuaries. In all levels of hypoxia (75% saturation = 5.23, 50% = 3.64, and 25% = 1 .86 mg l^− 1), M·o₂ increased linearly with swimming speed and active metabolic rate (AMR) was reduced (218 ± 17, 202 ± 14 and 175 ± 10 mg kg^− 1 h^− 1 for 75%, 50% and 25% saturation respectively). However, U_crit was only reduced at 50% and 25% saturation (1.4 ± < 0.1 and 1.4 ± < 0.1 BL s^− 1 respectively). This demonstrates that although the metabolic capacity of mulloway is reduced in mild hypoxia (75% saturation) they are able to compensate to maintain swimming performance. GCOT_min (0.09 ± 0.01 mg kg^− 1 m^− 1) and U_opt (0.8 ± 0.1 BL s^− 1) were significantly reduced at 25% dissolved oxygen saturation. As mulloway metabolic scope was significantly reduced at all hypoxia levels, it suggests that even mild hypoxia may reduce growth productivity.     

Energy and nitrogen partitioning in 250 g Atlantic cod (Gadus morhua L.) given graded levels of feed with different protein and lipid content

The effects of feed intake level on energy and nitrogen partitioning were studied in juvenile Atlantic cod (250 g) fed two fish meal based diets differing in protein and lipid content (54:31 and 65:16) at 10 °C. Replicate groups of cod were feed deprived for 32 days or fed one of the two diets at 25, 50, 75 or 100% of group satiation for 60 days. Feed intake and oxygen consumption were measured daily and weights and chemical composition of carcass, liver, viscera and whole body were measured at start and end. Diet digestibilities were assessed in a separate experiment.

The whole body and carcass growth rates at a given feed intake did not differ between dietary groups, but the liver grew faster in the fish fed the low protein diet, resulting in higher hepatosomatic indices at the end of the experiment in the groups fed this diet.

The efficiency of utilisation of digestible nitrogen for growth (k_DNg) was higher for the low protein diet (0.73 ± 0.02) than for the high protein diet (0.53 ± 0.05), resulting in higher nitrogen retention at a given nitrogen intake. No difference in percentage nitrogen retention was seen in full-fed fish however (31.2 ± 2.5 and 28.4 ± 1.6% for the low protein and high protein diets, respectively). This can be explained by higher nitrogen intake in the fish fed the high protein diet, resulting in a smaller proportion of the intake being used for maintenance.

There was no difference in energy utilisation between dietary groups. The digestible energy requirement for maintenance (DE_maint) was 53.8 ± 0.9 kJ kg^− 1 d^− 1 (42.3 ± 0.7 kJ kg^− 0.8 d^− 1) and the utilisation efficiency for growth (k_DEg) was 0.80 ± 0.02. The energy retention in full-fed fish was 31.3 ± 3.5 and 31.7 ± 1.0% for the low protein and high protein diets, respectively. The deposited energy was distributed in approximately equal proportions in the liver and carcass, whereas viscera accounted for a minor proportion. At a given energy intake, the fish fed the high protein diet deposited more energy in the carcass and less in the liver than did those fed the low protein diet.     

Growth efficiency of juvenile barramundi, Lates calcarifer, at high temperatures

Temperature is recognized to be the most important environmental factor affecting growth in fish. Barramundi are cultured over a wide range of temperatures some of which approach the upper thermal tolerance for this species. A growth trial was conducted on juvenile barramundi to examine the effects of high temperatures ranging from the minimum optimal temperature (27 °C) for growth efficiency to the extreme upper thermal limits (39 °C) for feed intake, growth and growth efficiency. Juveniles (4.87 ± 0.32 g) were held at four different temperatures 27, 33, 36 and 39 °C and fed twice daily to satiation (503.5 g kg^− 1 crude protein, 182.5 g kg^− 1 lipid, 150.1 g kg^− 1 ash, 20.52 GE MJ kg^− 1). Feed intake (g·day^− 1) and SGR (%·day^− 1) increased with increasing temperature up to 36 °C. At 39 °C feed intake, growth, feed efficiency ratio, protein efficiency ratio and productive energy value were significantly lower than at the other temperatures. This demonstrates that growth was optimized at temperatures from 27 to 36 °C and that barramundi have a much wider range for maximum growth efficiency than previously thought.     

Effect of low water temperature on viral replication of white spot syndrome virus in Procambarus clarkii

This study evaluated the effect of low water temperature (10 ± 1 °C) on viral infection and replication of white spot syndrome virus (WSSV) in crayfish, Procambarus clarkii, under standardized conditions. Crayfish were (i) maintained at 24 ± 1 °C before challenge and 10 ± 1 °C afterwards, or (ii) maintained at 10 ± 1 °C before challenge and 24 ± 1 °C afterwards. No mortality was observed when crayfish were held at 10 ± 1 °C after challenge, but mortality reached 100% when they were transferred to 24 ± 1 °C. Competitive PCR showed that viral levels at 10 ± 1 °C rose from 10⁶ to 10⁸ copies/mg of gill tissues, while at 24 ± 1 °C levels increased from 10⁶ to 10¹⁰ copies/mg of gill tissues during the same time interval. These results showed that a low water temperature of 10 ± 1 °C could reduce viral replication when compared to 24 ± 1 °C but could not prevent it.     

Feed intake, growth and feed utilization of offspring from wild and selected Atlantic salmon (Salmo salar)

The aim was to assess whether selection for increased growth rate in Atlantic salmon (Salmo salar) is associated with increased feed intake and/or better feed utilization. Growth responses of offspring from selected and wild lines of Atlantic salmon (initial weight 814±14 g and 533±12 g, respectively) were tested in a 14-week experiment. Selected and wild salmon increased body weight by 79 and 39%, respectively, during the experiment. Relative feed intake (DFI), thermal growth coefficient (TGC) and feed efficiency ratio (FER) were significantly higher in the selected (DFI: 0.67% BW d⁻¹, TGC: 2.96×10⁻³ and FER: 1.16), than in the wild, line (DFI: 0.48% BW d⁻¹, TGC: 1.39×10⁻³ and FER: 0.93). FER was positively correlated with mean growth rate (r=0.90, n=6, P<0.05), and differences between the lines indicated a 4.6% increase in FER per generation of selection. Fish of the selected line had a significantly lower intake of protein and energy per kilogram gain, so the higher growth rate of the selected line was the result of both greater feed consumption and more efficient feed utilization for growth. This implies that selection for increased growth rate in Atlantic salmon may improve both of these traits.     

Physiological changes in rainbow trout held under crowded conditions and fed diets with different levels of vitamins E and C and highly unsaturated fatty acids (HUFA)

Rainbow trout (Oncorhynchus mykiss) maintained in crowded (100 kg m^− 3) and uncrowded (20 kg m^− 3) conditions were fed 42 days with five experimental diets having different levels of vitamin E (25.6 and 275.6 mg kg diet^− 1), C (0 and 1000 mg kg diet^− 1) and HUFA (highly unsaturated fatty acids, 12.5 and 320.5 g kg diet^− 1): −E−HUFA, −E+HUFA, +E−HUFA, +E+HUFA, −C+E+HUFA. Cortisol, plasma metabolites, tissue glycogen, fish composition, and tissue fatty-acid profile were evaluated at the end of the experimental period. In general, no changes in cortisol levels were associated with crowding, although +E+HUFA and −C+E+HUFA fish showed higher levels (mean ± SE, 55.5 ± 11.1 and 78.0 ± 11.3 ng ml^− 1) as a consequence of a possible interaction between chronic crowding and diet composition. Protein and glucose con-centration in plasma displayed no effect of crowding, but liver glycogen showed a general tendency to decrease in −E−HUFA, −E+HUFA, +E−HUFA, +E+HUFA, −C+E+HUFA crowded groups (70.2 ± 2.1, 52.1 ± 2.5, 73.4 ± 7.4, 91.7 ± 3.3, 74.2 ± 8.4 mg g^− 1 tissue, respectively) compared to uncrowded groups (108.9 ± 14.2, 82.7 ± 8.8, 92.4 ± 10.7, 99.1 ± 10.0, 103.5 ± 15.6 mg g^− 1 tissue, respectively), thus proving significant in −E+HUFA fish. Variations in total lipids, triglycerides, total cholesterol and HDL as well as LDL cholesterol in plasma were manifested under crowding conditions, displaying a certain influence of vitamin E and HUFA dietary content. Final body composition, in general, showed no change attributable to fish density, but some differences associated with diet composition were found in lipid and moisture percentages of crowded fish. Liver and muscle fatty-acid profile revealed a clear effect of the dietary lipid source that was more evident in muscle than in liver at normal fish density, and in some cases this effect was modulated by dietary vitamin E and C content and fish-culture conditions.     

Safety and efficacy of emamectin benzoate administered in-feed to Atlantic salmon, Salmo salar L., smolts in freshwater, as a preventative treatment against infestations of sea lice, Lepeophtheirus salmonis (Krøyer)

The safety and efficacy of emamectin benzoate, administered in-feed to Atlantic salmon smolts, Salmo salar L., held in freshwater, was evaluated as a preventative treatment against sea lice, Lepeophtheirus salmonis, following transfer of fish to seawater.

In the safety study, salmon smolts held in freshwater were fed with diets containing emamectin benzoate at nominal doses of 0 (control), 50 (recommended dose) and 250 (5× recommended dose) μg kg⁻¹ fish day⁻¹ for 7 days (days 0–6). Actual dose rates, based on measured concentrations of emamectin benzoate in feed, differences in fish weight, and feed consumed, were 0, 54, and 272 μg kg⁻¹ day⁻¹, respectively. On day 9, fish were transferred to seawater and observed for 14 days. No differences in feeding response, coordination, behaviour, gross and histological appearance were observed between control fish and those that received 54 μg kg⁻¹ day⁻¹. Among smolts that received 272 μg kg⁻¹ day⁻¹, approximately 50% exhibited darker coloration, and one fish (1%) exhibited uncoordinated swimming behaviour. No pathognomonic signs of emamectin benzoate toxicity were identified.

In the efficacy study, smolts held in freshwater were fed an unmedicated ration (control group) or emamectin benzoate at 50 μg kg⁻¹ day⁻¹ (treated group) for 7 days (days 0–6). On day 9, fish were re-distributed to eight seawater tanks, each holding 30 control and 30 treated fish. On days 28, 56, 77 and 109, respectively, control and treated fish in two tanks were challenged with L. salmonis copepodites. When lice in each group reached chalimus stage IV, fish were sampled and the numbers of lice were recorded. Fish challenged at day 109 were sampled for the second time when lice were at the adult stage. Efficacy was calculated as the reduction in the mean number of lice on treated fish relative to the mean on control fish. Treatment with emamectin benzoate resulted in an efficacy of 85.0–99.8% in fish challenged at days 28–77, from the start of treatment, and lice counts were significantly lower (P<0.001) on treated fish than on controls. When fish challenged at day 109 were sampled at day 128, efficacy was 44.3%, but survival of chalimus to adult lice on treated fish was lower, and at day 159, efficacy had increased to 73%. These results demonstrate that treatment of salmon smolts with emamectin benzoate in freshwater was well tolerated and highly effective in preventing sea lice infestation following transfer of fish to seawater.     

Effects of body weight, water temperature and ration size on ammonia excretion by the areolated grouper (Epinephelus areolatus) and mangrove snapper (Lutjanus argentimaculatus)

The effects of body weight, water temperature and ration size on ammonia excretion rates of the areolated grouper Epinephelus areolatus and the mangrove snapper Lutjanus argentimaculatus were investigated. Under given experimental conditions, L. argentimaculatus had a higher weight-specific ammonia excretion rate than E. areolatus. Weight-specific ammonia excretion rates of fasted individuals of both species showed an inverse relationship with body weight (W, g wet wt.), but a positive relationship with water temperature (t, °C). The relationships for total ammonia nitrogen (TAN) were: E. areolatus: TAN (mg N kg⁻¹ d⁻¹)=21.4·exp^0.11t·W^−0.43 (r²=0.919, n=60); L. argentimaculatus: TAN (mg N kg⁻¹ d⁻¹)=121.5·exp^0.12t·W^−0.55 (r²=0.931, n=60). Following feeding, the weight-specific ammonia excretion rate of E. areolatus increased, peaked at 2 to 12 h (depending on temperature), and returned to pre-feeding levels within 24 h. A similar pattern was observed for L. argentimaculatus, with a peak of TAN excretion being found 6 to 12 h after feeding. Stepwise multiple regression analysis indicated that weight-specific TAN excretion rates of both species increased with increasing temperature and ration (R, percent body wt. d⁻¹): E. areolatus: TAN (mg N kg⁻¹ d⁻¹)=22.8·t−28.8·R−378.2 (r²=0.832, n=24); L. argentimaculatus: TAN (mg N kg⁻¹ d⁻¹)=22.9·t−25.4·R−216.4 (r²=0.611, n=24). The effect of body weight on weight-specific postprandial TAN excretion was not significant in either species (p>0.05). This study provides empirical data for estimating ammonia excretion of these two species under varying conditions. This has application for culture management.     

Impact of starvation and handling stress on rigor development and quality of Atlantic salmon (Salmon salar L)

Farmed Atlantic salmon (Salmo salar L) were fed to satiation or starved for 35 d, and subjected to acute pre-slaughter stress or careful handling before slaughtering. Rigor development, post-mortem energy metabolism and quality variations were analysed during 72 h cold storage. The body weight averaged 2.9 and 3.5 kg of the fed and starved salmon, respectively. The starved salmon had lower condition factor (0.99 vs. 1.11) and higher slaughter yield (92.6 vs. 87.5%), but the fat content was similar (11.8–12.9%) in the fed and starved salmon. The initial glycogen level was lower in the starved salmon (21 vs. 29 μmol g^− 1), whereas initial ATP levels (6.4–6.6 μmol g^− 1) were not affected by starvation. Pre-slaughter stress accelerated rigor development, stimulated lactate formation through post-mortem glycolysis, and increased the breakdown of ATP and CP. The effect of stress on rigor development was less pronounced in starved salmon, and a significant interaction was observed between nutritional status and stress. Rigor development was closely correlated to ATP breakdown, especially in fillets. Acute stress accelerated flesh softening during ice storage. After 72 h storage, the fillets of the starved salmon exposed to careful slaughter handling had the significantly firmest texture, whereas the most intense fillet colour was observed in the starved/stressed salmon. Liquid leakage from the muscle during cold storage was lower in the starved salmon, but pre-slaughter stress had no significant impact. It is concluded that a starvation period of five weeks can improve the resistant to acute stress prior to slaughtering of Atlantic salmon that in turn hampers rigor development.     

Dopamine modulates the physiological response of the tiger shrimp Penaeus monodon

Levels of glucose, lactate, pO₂, pCO₂, HCO₃⁻, TCO₂, Na⁺, K⁺, Cl⁻, protein, and oxyhemocyanin in the hemolymph and its osmolality and pH were measured when tiger shrimp, Penaeus monodon (13.5 ± 1.5 g body weight), were individually injected with saline or dopamine at 10^− 8, 10^− 7, or 10^− 6 mol shrimp^− 1. Results showed that hemolymph glucose, lactate, pCO₂, HCO₃⁻, and TCO₂ values increased from 2 to 4 h; hemolymph osmolality, Na⁺, and total protein had increased at 2 h; and hemolymph K⁺ decreased from 2 to 8 h after the dopamine injection. All physiological parameters returned to the control values 4–16 h after receiving dopamine. The dopamine injection also significantly decreased the oxyhemocyanin/protein ratio of P. monodon which occurred at 2 h, resulting from an elevation of hemolymph protein and a slight decrease of oxyhemocyanin. These results suggest that stress-inducing dopamine caused a transient period of modulation of energy metabolism, osmoregulation, respiration, and the acid–base balance in P. monodon in adapting to this environmental stress.     

Substitution of Chaetoceros muelleri by Spirulina platensis meal in diets for Litopenaeus schmitti larvae

The nutritional response of Litopenaeus schmitti larvae to substitution of Chaetoceros muelleri by Spirulina platensis meal (SPM) was evaluated. The substitution levels (S) were 0%, 25%, 50%, 75% and 100%, dry weight basis. Final larval length (FL) ranged from 1.98 to 3.16 mm for the different substitution levels. There was a significant relationship between S and FL, described by the following quadratic equation: FL = 2.853 + 0.01598S − 0.000233S². The substitution level (S) yielding maximum FL was 34.2%. Development index (DI) values ranged from 2.84 to 3.93 and were dependent on substitution level. The corresponding equation was DI = 3.799 + 0.00945S − 0.000189S² (P < 0.01). Maximum DI was obtained at 25.0% substitution. Survival was high (82–87%) and no significant differences were found between treatments. Protein digestibility of either microalgae was high, with 92% for SPM and 94% for C. muelleri, with no significant differences between them. The results in this study indicate that an adequate balance of nutrients in relation to the requirements of the species is critical. To simultaneously improve FL and DI, a 30% substitution of C. muelleri by SPM is suggested. This is equivalent to feeding 0.15 mg larvae^− 1 day^− 1 dry weight basis of a 70% C. muelleri/30% SPM diet, representing 0.078 mg protein larvae^− 1 day^− 1, 0.026 mg lipids larvae^− 1 day^− 1 and 2.732 J larvae^− 1 day^− 1.     

Production of salmonid amoebic gill disease by exposure to Paramoeba sp. harvested from the gills of infected fish

Atlantic salmon, S almo salar L., were exposed to different concentrations of Paramoeba sp. The lowest concentration which induced amoebic gill disease (AGD) was 230 Paramoeba sp. L^–1 and the severity of infection increased with increasing concentration. The concentration of Paramoeba sp. positively correlated with the number of gill lesions ( R ² > 0.7). This study provides evidence that Paramoeba sp. is the causative agent of AGD and describes an experimental model that enables the severity of the induced disease to be controlled.     

Serotonin induces ovarian maturation in giant freshwater prawn broodstock, Macrobrachium rosenbergii de Man

This study investigated the effects of serotonin (5-hydroxytryptamine or 5HT) on ovarian development in Macrobrachium rosenbergii de Man. Adult female prawns at the ovarian stage I (spent) were injected with 5HT at 1, 5, 10, 20 and 50 μg g^− 1 body weight (BW) intramuscularly on days 0, 5 and 10, and sacrificed on day 15. The doses as related to the effect could be categorized into three levels: low (1 and 5 μg g^− 1 BW of 5HT), medium (10 and 20 μg g^− 1 BW of 5HT) and high (50 μg g^− 1 BW of 5HT). The low-dose, especially at 1 μg g^− 1 BW, caused prawns to exhibit a significant increase in ovarian index (ovarian weight/body weight × 100) (5.79 ± 0.09%) as compared to the control (1.49%). The ovaries of most of these prawns could develop to stage IV (mature) and contained synchronously mature oocytes while most of the control ovaries remained at stage I and II (proliferative), and contained only oogonia to previtellogenic (Oc1, Oc2) and early vitellogenic oocytes (Oc3). The medium- and high-dose treated prawns exhibited ovaries that could reach stages III and IV and contained various types of oocytes of different maturity. Pretreatment with 5HT receptor antagonist, cyproheptadine (CYP), at 10 μg g^− 1 BW before 5HT injection significantly suppressed the effect of 5HT. Intramuscular injection of the 5HT-primed thoracic ganglion culture medium into CYP-pretreated prawns resulted in the increase of ovarian index about 5–6 times more than in the control, and in the groups injected with 5HT-primed media from muscle strip, eyestalk and brain. The ovaries of most prawn could develop up to stage IV and contained synchronously developed vitellogenic (Oc4) and mature oocytes (Oc5). These findings suggest that 5HT indirectly induces ovarian development and oocytes maturation in M. rosenbergii, probably via a putative ovarian stimulating factor released from the thoracic ganglia.     

An improved enzyme preparation for rapid mass production of protoplasts as seed stock for aquaculture of macrophytic marine green algae

Preparation of protoplasts and their subsequent applications for both basic and applied research of marine macroalgae remains largely under developed due to lack of development of reliable methods with consistent yields of viable protoplasts. An improved enzyme preparation with a single commercial enzyme, e.g. 2% Cellulase Onozuka R-10 in 1% NaCl solution, was developed to produce protoplasts rapidly from different green algal genera of Ulva, Enteromorpha and Monostroma. The simple dissolution of enzyme powder in 1% NaCl resulted in exclusion of 2% Macerozyme R-10 from the mixture consisting of 2% Cellulase Onozuka R-10 with 3% NaCl earlier reported as superior for the same algae. Optimal conditions for the isolation of maximum yields of viable protoplasts were found to be with 2% Cellulase Onozuka R-10 incubated at 20 °C for 2 h in 1% NaCl solution with 0.8 M mannitol adjusted to pH 6.0. The protoplast yield with optimized enzyme mixture was as high as 102.8 × 10⁶ cells g^− 1 f. wt for M. oxyspermum while it was in the range of 74.4–88.6 × 10⁶ cells g^− 1 f. wt thallus for seven species of Ulva, and 82.5–95.4 × 10⁶ cells g^− 1 f. wt for three species of Enteromorpha. The regeneration rate of protoplasts isolated using this method ranged from 89 to 92% with normal morphogenesis. The seeding of nylon threads with isolated protoplasts of M. oxyspermum was successful and after 3–4 weeks the entire frame with nylon threads became thick green in color with tiny germlings in laboratory culture. Thus, the method described in the present study allow for rapid mass production of viable protoplasts that could be potentially used as a source for seed material for mariculture and for other applied phycological research.     

Pathology of experimental amoebic gill disease in Atlantic salmon, Salmo salar L., and the effect of pre-maintenance of fish in sea water on the infection

Atlantic salmon were exposed to amoebic gill disease (AGD) immediately following their acclimatization to sea water (group 1), or following a 2 week period of maintenance in sea water (group 2). Three fish from each group were sampled on days 0, 1, 2, 4, 7, 14 and 28 post-infection. Characteristic gill lesions began to occur between days 2 and 4, and dramatically increased by day 7. The number of gill lesions on fish from group 2 was significantly higher than on fish from group 1 on days 7 and 14 ( P <0.001), but the two groups did not differ in any other parameter. Histologically, Paramoeba sp., the aetiological agent of AGD, could be seen on the gills of fish as soon as 1 day post-exposure, attached to healthy-appearing gills. Gill pathology in the form of hyperplasia and lamellar fusion followed shortly. AGD infection was accompanied by a significant increase in the number of gill mucous cells ( P =0.002). Different methods for the diagnosis of AGD are discussed.     

Evidences of the intertidal red alga Grateloupia turuturu in turning Vibrio parahaemolyticus into non-culturable state in the presence of light

Grateloupia turuturu, previously known as Grateloupia doryphora, has been widely reported to be an invasive algal species. There are no studies to relate the impact of its existence on its surrounding environment. In this paper, we present our results to show that about 70% of individuals collected from the field could turn Vibrio parahaemolyticus into non-culturable state on both selective (TCBS) and non-selective (2216E) culture medium in 24 h in the presence of light in live algal culture. Total bacteria counts on TCBS and 2216E plates dropped from the initial 565 (± 174) and 1192 (± 60) cfu ml^− 1 respectively to zero in 24 h. This effect disappeared when the alga was grown in darkness. The same effect was not found in two other intertidal macroalgae Laminaria japonica and Palmaria palmata. Further tests showed that the settlement ability of bacteria in seawater was impaired significantly in the presence of this alga in comparison with three other algal species.     

Seeding nets with neutral spores of the red alga Porphyra umbilicalis (L.) Kützing for use in integrated multi-trophic aquaculture (IMTA)

Nets in traditional Porphyra mariculture are seeded with conchospores derived from the conchocelis phase, and spend a nursery period in culture tanks or calm coastal waters until they reach several centimeters in length. Some species of Porphyra can regenerate the foliose phase directly through asexual reproduction, which suggests that the time, infrastructure, and costs associated with conchocelis culture might be avoided by seeding nets with asexual spores. Here, we present work from a short-term mariculture study using nets seeded with asexual spores (neutral spores) of a native Maine species of Porphyra. Porphyra umbilicalis (L.) Kützing was selected for this proof of concept research because of its reproductive biology, abundance across seasons in Maine, and evidence of its promise as a mariculture crop. We studied the maturation, release, and germination of the neutral spores to develop an appropriate seeding protocol for nets, followed by development of a nursery raceway to provide an easily manipulated environment for the seeded nets. Neutral spores were produced throughout the year on the central Maine coast; however, there was a temporal variability in the number and survival of released neutral spores, depending upon thallus position in the intertidal zone. Small thalli were strictly vegetative, but most thalli reproduced by neutral spores; sexual reproduction was absent. Neutral spores germinated quickly at 10 and 15 °C, but germination was delayed at 5 °C. Unlike some algal zygotes and spores, neutral spores of P. umbilicalis required light to germinate; however, irradiances of 25 and 100 μmol photons m^− 2 s^− 1 were equally sufficient for germination. Rafts of seeded nets were deployed in Cobscook Bay, Maine, at two distances from salmon aquaculture pens and at a control site on a nearby, fallow aquaculture site (no salmon). There was no difference in nitrogen content of harvested thalli; however, both the density and the surface area of harvested thalli were different among the sites. The possible causes of these differences are discussed in the context of potential use of P. umbilicalis in IMTA.