Effects of hypoxia on lysozyme activity and antioxidant defences in the kidney and spleen of Carassius auratus

Assessing the responses of lysozyme activity and antioxidant defences to hypoxia is important to understand the adaptation and tolerance strategies of fish under hypoxia. This study investigated the effects of different dissolved oxygen (DO) levels on Carassius auratus, a natural triploid fish, from Qihe River. The content of malondialdehyde (MDA) and the activities of lysozyme and antioxidant enzymes were measured in the kidney and spleen after hypoxic exposure. At the DO concentrations of 1 and 2 mg L^?1, the activities of antioxidant enzymes and lysozyme significantly decreased and the content of MDA significantly increased (P < 0.01). This result suggests that hypoxia decreased antioxidant enzyme and lysozyme activities and caused MDA accumulation in a concentration‐dependent manner. The DO level of 4 mg L^?1 increased the activities of superoxide dismutase and catalase in the kidney and the activities of glutathione peroxidase and catalase in the spleen (P < 0.05). This result implies that slight hypoxic stress can enhance antioxidant defences to alleviate the damage of oxidative stress. The reduced activities of antioxidant enzymes and the accumulation of MDA at the DO level of ≤2 mg L^?1 implied the decrease in antioxidative ability and the occurrence of oxidative stress. The decrement in lysozyme activity indicated that the antibacterial ability was weakened to some degree. Therefore, hypoxic stress at DO levels ≤2 mg L^?1 should be removed by aeration to avoid the oxidative damage resulting from the reduced antioxidative ability and prevent the outbreak of diseases caused by weakened antibacterial effects.     

Acute ammonia toxicity and gill morphological changes of Japanese flounder Paralichthys olivaceus in normal versus supersaturated oxygen

Ammonia toxicity and morphological changes in gills of juvenile Japanese flounder Paralichthys olivaceus (5.76 ± 0.12 g) were investigated when fish were separately exposed to normal dissolved oxygen (DO) at 6.5 ± 0.5 mg L^?1 and supersaturated oxygen at 16.0 ± 2.0 mg L^?1 at different ammonia concentrations. Under normal oxygen, ammonia concentrations were tested from 0.04 (control) to 93.3 mg L^?1 total ammonia nitrogen (TAN), whereas under oxygen supersaturation, ammonia concentrations ranged from 0.04 (control) to 226.7 mg L^?1 TAN in the trial. After exposure to ammonia for 96 h, the ammonia LC₅₀ for fish was 62.48 mg L^?1 TAN (0.50 mg L^?1 NH₃–N) at normal oxygen and 160.71 mg L^?1 TAN (0.65 mg L^?1 NH₃–N) at oxygen supersaturation. Light microscopic observations confirmed that gill damage in normal oxygen was more profound than in oxygen supersaturation when fish were exposed to the same level of TAN (93.3 mg L^?1). Furthermore, electron microscopic scanning also showed more crimple, retraction and fibrosis on the secondary lamella surface in fish exposed to normal oxygen than those in fish exposed to supersaturated oxygen at the same TAN (93.3 mg L^?1). This study suggests that supersaturated oxygen can increase ammonia tolerance in Japanese flounder through reducing gill damage by ammonia, which partially explains the merit of using pure oxygen injection in intensive fish farming.     

Effect of dietary lysozyme on growth,immune response,intestine microbiota,intestine morphology and resistance to Aeromonas hydrophilia in gibel carp (Carassius auratus gibelio)

A 75 days experiment was conducted in a flow‐through system on juvenile gibel carp (Carassius auratus gibelio) (3.43 ± 0.01 g) to evaluate the effects of dietary lysozyme on growth performance, intestine morphology, microbiota and immune response. Four isonitrogenous (crude protein: 367 g kg^?1) isolipid (62 g kg^?1) and isocaloric (gross energy: 17.92 kJ g^?1) diets were formulated to contain 0, 100, 500 and 1000 mg kg^?1 lysozyme, respectively. The results showed that specific growth rate (SGR) and feed efficiency (FE) increased at 1000 mg kg^?1 lysozyme. Blood leucocyte phagocytic activity (PA) and serum lysozyme (LZM) decreased with dietary lysozyme on day 25, 50 and 75. There were no significant differences in alternative complement pathway (ACP), respiratory burst (ROS), serum superoxide dismutase (SOD), glutathione peroxidase (GSHpx) or malonaldehyde (MDA). After Aeromonas hydrophilia challenge, higher survival was obtained at 500 mg kg^?1 group. PA, ROS, SOD, LZM and ACP increased with increasing dietary lysozyme, while MDA reversed. Goblet cells in mid‐intestine and microvilli height in distal intestine increased with dietary lysozyme on day 75. Dietary lysozyme reduced the diversity of intestine microbiota. In conclusion, oral administration of 500 mg kg^?1 dietary lysozyme for 75 days is recommended for the survival of gibel carp and 1000 mg kg^?1 dietary lysozyme for fast growth.     

Preliminary success using hydrogen peroxide to treat Atlantic salmon,Salmo salar L., affected with experimentally induced amoebic gill disease (AGD)

Currently, the only effective and commercially used treatment for amoebic gill disease (AGD) in farmed Tasmanian Atlantic salmon is freshwater bathing. Hydrogen peroxide (H₂O₂), commonly used throughout the aquaculture industry for a range of topical skin and gill infections, was trialled in vitro and in vivo to ascertain its potential as an alternative treatment against AGD. Under in vitro conditions, trophozoites of Neoparamoeba perurans were exposed to three concentrations of H₂O₂ in sea water (500, 1000 and 1500 mg L^?1) over four durations (10, 20, 30 and 60 min) each at two temperatures (12 and 18 °C). Trophozoite viability was assessed immediately post‐exposure and after 24 h. A concentration/duration combination of 1000 mg L^?1 for >10 min demonstrated potent amoebicidal activity. Subsequently, Atlantic salmon mildly affected with experimentally induced AGD were treated with H₂O₂ at 12 and 18 °C for 15 min at 1250 mg L^?1 and their re‐infection rate was compared to freshwater‐treated fish over 21 days. Significant differences in the percentage of filaments affected with hyperplastic lesions (in association with amoebae) and plasma osmolality were noted between treatment groups immediately post‐bath. However, the results were largely equivocal in terms of disease resolution over a 3‐week period following treatment. These data suggest that H₂O₂ treatment in sea water successfully ameliorated a clinically light case of AGD under laboratory conditions.     

Epidemiological cut‐off values for Flavobacterium psychrophilum MIC data generated by a standard test protocol

Epidemiological cut‐off values were developed for application to antibiotic susceptibility data for Flavobacterium psychrophilum generated by standard CLSI test protocols. The MIC values for ten antibiotic agents against Flavobacterium psychrophilum were determined in two laboratories. For five antibiotics, the data sets were of sufficient quality and quantity to allow the setting of valid epidemiological cut‐off values. For these agents, the cut‐off values, calculated by the application of the statistically based normalized resistance interpretation method, were ≤16 mg L^?1 for erythromycin, ≤2 mg L^?1 for florfenicol, ≤0.025 mg L^?1 for oxolinic acid (OXO), ≤0.125 mg L^?1 for oxytetracycline and ≤20 (1/19) mg L^?1 for trimethoprim/sulphamethoxazole. For ampicillin and amoxicillin, the majority of putative wild‐type observations were ‘off scale’, and therefore, statistically valid cut‐off values could not be calculated. For ormetoprim/sulphadimethoxine, the data were excessively diverse and a valid cut‐off could not be determined. For flumequine, the putative wild‐type data were extremely skewed, and for enrofloxacin, there was inadequate separation in the MIC values for putative wild‐type and non‐wild‐type strains. It is argued that the adoption of OXO as a class representative for the quinolone group would be a valid method of determining susceptibilities to these agents.     

The efficacy of MS‐222 as anaesthetic agent in four freshwater aquarium fish species

The efficacy of anaesthetic tricaine methanesulfonate (MS‐222) was evaluated in four freshwater aquarium fish species, Zebrafish (Danio rerio), Guppy (Poecilia reticulata), Discu (Symphysodon discus) and Green swordtail (Xiphophorus helleri). The correct dose of anaesthetic should induce the plane 4 of anaesthesia in less than 180 s, recovery in less than 300 s and must survive when exposed during 30 min to anaesthetic. Fishes were exposed to six concentrations of anaesthetic (75, 100, 125, 150, 200 and 250 mg L^?1) and the time of fish reaching plane 4 of anaesthesia, post exposure recovery, and the percentage of survival when fish were subject to 30 min in the anaesthetic were recorded. The optimal doses varied according to the species: D. rerio – 75, 100 and 125 mg L^?1, P. reticulata – 125, 150 and 200 mg L^?1, S. discus – 75 and 100 mg L^?1 and X. helleri – 125 and 150 mg L^?1. The induction time generally decreased significantly with increasing concentration of MS‐222 for all of the species evaluated. The recovery time had a tendency to increase with the increase of the MS‐222 concentration for D. rerio, P. reticulata and S. discus. On the other hand, X. helleri recovery time decreased with the increase of MS‐222 concentration. MS‐222 proved to be effective in anaesthesia for all the freshwater ornamental species studied. The main results clearly show that the optimal dose to anesthetize is fish species dependent and it is completely wrong to extrapolate optimal anaesthetic concentrations between different species.     

Acute and chronic effects of aqueous ammonia on marbled spinefoot rabbitfish,Siganus rivulatus (Forsskål 1775)

Ammonia is a metabolite of aquatic organisms which might reach deleterious levels in intensive fish farms. The aim of the present study was to determine median lethal concentrations (96‐h LC₅₀) of total ammonia nitrogen (TA‐N) on marbled spinefoot rabbitfish (Siganus rivulatus) and chronic effects of TA‐N on survival, growth and behaviour of juvenile rabbitfish over a 50 day period. In the first experiment, fish were exposed to 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 mg L^?1 TA‐N for 96 h and survival evaluated. In the second experiment, 12 fish were stocked per 50‐L tank and treated with one of 0, 2, 4, 6, 8, 10 and 12 mg L^?1 TA‐N with three replicate tanks per treatment. Survival and growth were determined and histopathological alterations of gills due to chronic ammonia exposure were studied by light and electron microscopy. The 96‐h LC₅₀ values were 16–18 mg L^?1 TA‐N. In the chronic exposure experiment, fish reared in water with 0 mg L^?1 TA‐N had 100% survival and had 50% weight increase in 50 days. Fish at 2 and 4 mg L^?1 TA‐N all died whilst fish in 6, 8, 10 and 12 mg L^?1 TA‐N survived and grew albeit less than in treatment 0 mg L^?1. Gills from ammonia treated fish displayed severe histological and ultrastructural alterations including hyperplasia, hypertrophy and fusion of secondary lamellae, aneurysms and presence of pleomorphic altered cells. Chronic exposure to ammonia is deleterious to marbled spinefoot rabbitfish and low concentrations of ammonia appear to kill the fish in <50 days whilst fish can survive for more than 50 days at concentrations between 6 and 12 mg L^?1 TA‐N.     

Silver catfish Rhamdia quelen immersion anaesthesia with essential oil of Aloysia triphylla (L'Hérit) Britton or tricaine methanesulfonate: effect on stress response and antioxidant status

Responses to anaesthesia with essential oil (EO) of Aloysia triphylla (135 and 180 mg L^?1) and tricaine methanesulfonate (MS222) (150 and 300 mg L^?1) were assessed in silver catfish. Exposure to the anaesthetics elicited a stress response in the species. In the case of MS222, it was displayed as a release of cortisol into bloodstream, elevation in hematocrit and plasma ion loss. The EO presented cortisol‐blocking properties, but increased haematocrit and disturbances of hydromineral balance were observed. Liver antioxidant/oxidant status of EO and MS222‐anaesthetized silver catfish was also estimated. The synthetic anaesthetic induced lipoperoxidation, notwithstanding increased catalase contents, whereas the naturally occurring product was capable of preventing the formation of lipid peroxides, possibly due to combined actions of catalase and glutathione‐S‐transferase. Anaesthetic efficacy was also tested via induction and recovery times. Overall, the promising results obtained for the physiological parameters of the EO‐treated fish counterbalanced the slight prolonged induction time observed for 180 mg L^?1. As for 135 mg L^?1, both induction and recovery times were lengthy; despite that, the EO was able to promote oxidative protection and mitigate stress. None of the MS222 concentrations prompted such responses concomitantly.     

Effect of different total suspended solids levels on a Litopenaeus vannamei (Boone, 1931) BFT culture system during biofloc formation

In a Biofloc Technology System (BFT), there is constant biofloc formation and suspended solids accumulation, leading to effects on water quality parameters that may affect the growth performance of cultured shrimp. This study aimed to analyse during biofloc formation the effect of different total suspended solids (TSS) levels on water quality and the growth performance of Litopenaeus vannamei shrimp in a BFT system. A 42‐day trial was conducted with treatments of three ranges of TSS: 100–300 mg L^?1 as low (TL), 300–600 as medium (TM) and 600–1000 as high (TH). The initial concentrations of 100 (TL), 300 (TM) and 600 mg L^?1 (TH) were achieved by fertilization before starting the experiment. Litopenaeus  vannamei juveniles with an average weight of 4.54 ± 1.19 g were stocked at a density of 372 shrimp m^?3. Physical and chemical water parameters and shrimp growth performance were analysed. After 6 weeks, TSS mean concentrations were 306.37, 532.43 and 745.2 mg L^?1 for, respectively, TL, TM and TH treatments. Significant differences (P < 0.05) were observed in TSS, settleable solids, pH, alkalinity and nitrite, especially between the TL and TH treatments. Similarly, differences (P < 0.05) were observed in the growth performance parameters, specifically final weight, survival, feed conversion and productivity. The water quality parameters at lower range of total suspended solids concentration (TL) treatment resulted in a better performance of L. vannamei in the BFT system. The maintenance at range of 100–300 mg L^?1 TSS is thus important to the success of shrimp culture.     

Dose‐dependent protective effects of dietary selenium on abalone Haliotis discus hannai Ino against the toxicity of waterborne copper

This study was designed to assess the protective effects of dietary selenium (Se) on abalone Haliotis discus hannai Ino against the toxicity of waterborne copper (Cu). A 60‐day feeding trial was conducted in a static water system for abalone (initial weight: 3.17 ± 0.01 g) exposed to 0.02 mg L^?1 of waterborne Cu. The animals were fed one of the three experimental diets with 0.10, 1.31 and 4.20 mg kg^?1 of Se from Na₂SeO₃·5H₂O respectively. Results showed that the abalone fed 1.31 mg kg^?1 of dietary Se had the lowest Cu concentration in shell, muscle, mantle, gill, hepatopancreas and serum. Meanwhile, the significant lowest contents of malondiadehyde and protein carbonyl in hepatopancreas were also found in the treatment with 1.31 mg kg^?1 of dietary Se (P < 0.05). In addition, this treatment had significant higher glutathione content and thioredoxin reductase activity in hepatopancreas (P < 0.05). However, the activity of Se‐dependent glutathione peroxidase (Se‐GPx) was significantly decreased in the treatment with 4.20 mg kg^?1 of dietary Se (P < 0.05). In this treatment, the protein carbonyl content in hepatopancreas was significantly higher than that in the group with 1.31 mg kg^?1 of dietary Se (P < 0.05). In conclusion, in terms of anti‐oxidation and Cu accumulation, the protective effects of dietary Se on abalone against waterborne Cu were dose‐dependent. The 1.31 mg kg^?1 of dietary Se had this effect, but not 4.20 mg kg^?1 of dietary Se. Moreover, the latter increased the oxidative stress in abalone exposed to the waterborne Cu.     

Effectiveness of copper sulphate,potassium permanganate and peracetic acid to reduce mortality and infestation of Ichthyobodo necator in channel catfish Ictalurus punctatus (Rafinesque 1818)

Ichthyobodo necator is a single‐celled biflagellate parasite, which in high density can cause significant mortality in young fish. Copper sulphate (CuSO₄), potassium permanganate (KMnO₄) and peracetic acid (PAA) were evaluated for effectiveness against ichthyobodosis. Treatments were: untreated control, 2.1 mg L^?1CuSO₄, 3.0 mg L^?1 KMnO₄, 1.5 mg L^?1 PAA and 3.0 mg L^?1 PAA, and were applied to flow‐through tanks on three consecutive days. The study was designed to simulate the flow‐through systems utilized in the commercial rearing of juvenile channel catfish (Ictalurus punctatus). Mortality was monitored daily to compare survival rate among treatments. Parasite intensity was assessed pre chemical exposure and 20–24 h after the third application to determine effectiveness of the treatment. An assessment was also done 7 days post application to investigate possible reoccurrence. Copper sulphate, KMnO₄ and PAA (3.0 mg L^?1) significantly reduced the infestation rate of I. necator. Copper sulphate significantly improved the survival of I. necator infested channel catfish after three flow‐through applications compared with the untreated control. The 3.0 mg L^?1 PAA resulted in significantly lower survival than the untreated control, the 1.5 mg L^?1 PAA and the KMnO₄ were not statistically different from the untreated control.     

Gill damage to juvenile orange‐spotted grouper Epinephelus coioides (Hamilton, 1822) following exposure to suspended sediments

Juvenile Epinephelus coioides were exposed to three nominal concentrations (8, 32, 128 mg wet sediments L^?1) of suspended sediments (SS) from Port Shelter (PS), Mirs Bay (MB) and Victoria Harbour (VH) for 10 and 30 days using semi‐static system. Sediments from VH contained higher concentrations of Cu and polycyclic aromatic hydrocarbons (PAHs) than sediments from PS and MB. Gill damages including lamellar blood sinus dilation and vascular congestion were prevalent after just 10 days of exposure to SS. Fish exposed to SS at the highest concentration of 128 mg L^?1 showed higher incidence of lamellar aneurism. Hyperplasia in the base of lamellae was recorded in fish that had been exposed to contaminated SS from VH. Significant increases in the density of chloride cells and mucous cells were found on the gills of fish that had been exposed to 128 mg L^?1 of SS from PS. Clogging of gills by SS produced hypoxic‐like responses in fish. Polluted sediments from VH produced addictive or synergistic effects between SS and chemical contaminants on fish.     

Acute toxicity of ammonia in Pacu fish (Piaractus mesopotamicus,Holmberg, 1887) at different temperatures levels

Piaractus mesopotamicus juveniles (total length 12 ± 0.5 mm) were exposed to different concentrations of ammonia‐N (un‐ionized plus ionized ammonia as nitrogen), using the static renewal method at different temperature levels (15, 20 and 25°C) at pH 7. The 24, 48, 72, 96 h LC₅₀ values of ammonia‐N in P. mesopotamicus juveniles were 5.32, 4.19, 3.79 and 2.85 mg L^?1 at 15°C; 4.81, 3.97, 3.25 and 2.50 mg L^?1 at 20°C; and 4.16, 3.79, 2.58 and 1.97 mg L^?1 at 25°C respectively. The 24, 48, 72, 96 h LC₅₀ values of NH₃‐N (un‐ionized ammonia as nitrogen) were 0.018, 0.014, 0.013, 0.009 mg L^?1 at 15°C temperature; 0.023, 0.019, 0.016 and 0.012 mg L^?1 at 20°C; 0.029, 0.026, 0.018 and 0.014 mg L^?1 at 25°C. The temperature increase from 15 to 25°C caused an increase of ammonia‐N susceptibility by 21.80%, 9.55%, 31.92% and 30.87%, after 24, 48, 72 and 96 h exposure respectively. Furthermore, we found that exposure of fish to ammonia‐N caused an elevation in total haemoglobin and blood glucose with an increase of 2 mg L^?1 concentration. Ammonia levels tolerated, especially in different temperatures levels, have important implications for the management of aquaculture.     

Menthol as anaesthetic for lambari Astyanax altiparanae (Garutti & Britski 2000): attenuation of stress responses

The anaesthetic potential of menthol was evaluated in lambari Astyanax altiparanae by exposing fingerlings to concentrations 50, 100, 150, 200, 250 and 300 mg L^?1 and measuring the induction and recovery times to deep anaesthesia, the mortality rates during and 96 h after procedure and after 6 min of continuous exposure. The effect of menthol on stress responses were evaluated by comparing glucose and cortisol levels of juveniles subjected to anaesthesia (50 mg L^?1), stress (air exposure) or pre‐anaesthesia associated to stress. All concentrations induced deep anaesthesia within 0.5 to 1 min, with recovery between 1.83 and 4.16 min, without mortality during the induction or up to 96 h after exposure. Induction time decreased and recovery time increased linearly as the menthol concentration increased. Continuous exposure to 50, 100 and 150 mg L^?1 concentrations resulted in mortality rates of 0%, 20% and 80% respectively. Anaesthesia or air exposure increase blood glucose but prior anaesthesia with menthol suppressed the elevation of cortisol caused by stress. Menthol has an anaesthetic effect and attenuates the stress response in lambari and 50 mg L^?1 is the most effective concentration for inducing deep anaesthesia in 1.0 min, safe for up to 6 min exposure.     

Effect of dissolved oxygen levels on growth performance,energy budget and antioxidant responses of yellow catfish,Pelteobagrus fulvidraco (Richardson)

This study was conducted to determine the effect of dissolved oxygen (DO) levels on growth performance, energy budget and antioxidant responses of yellow catfish Pelteobagrus fulvidraco. Yellow catfish was exposed to four levels of DO, consisting of hypoxia (2.28 mg L^?1), moderate hypoxia (4.04 mg L^?1), saturation (6.51 mg L^?1) and super‐saturation groups (9.11 mg L^?1), respectively, for 8 weeks. Specific growth rate and feed efficiency in dry matter were lowest in hypoxia and highest in the saturation and super‐saturation groups. Apparent digestibility coefficients of dry matter and energy increased with increasing DO levels. Gross energy and growth energy were lowest for hypoxia, followed by moderate hypoxia and the highest for other two groups. Faecal energy was highest in hypoxia and lowest in saturation and super‐saturation group. DO levels also significantly influenced activities of antioxidant enzymes and malondialdehyde level in liver and serum. Based on the observation described previously, saturated DO level helps to improve growth performance, feed utilization and antioxidant responses in yellow catfish. Super‐saturation did not increase fish performance. To our knowledge, this is the first study involved in the effect of DO levels on energy budget of fish and provides new insight into aeration regime for yellow catfish culture.     

Acute toxicity of nitrite on white shrimp Litopenaeus vannamei (Boone) juveniles in low‐salinity water

The nitrite toxicity was estimated in juveniles of L. vannamei. The 24, 48, 72 and 96 h LC₅₀ of nitrite‐N on juveniles were 8.1, 7.9, 6.8 and 5.7 mg L^?1 at 0.6 g L^?1; 14.4, 9.6 8.3 and 7.0 mg L^?1 at 1.0 g L^?1; 19.4, 15.4, 13.4 and 12.4 mg L^?1 at 2.0 g L^?1 of salinity respectively. The tolerance of juveniles to nitrite decreased at 96 h of exposure by 18.6% and 54.0%, when salinity declined from 1.0 to 0.6 g L^?1 and from 2.0 to 0.6 g L^?1 respectively. The safe concentrations at salinities of 0.6, 1.0 and 2.0 g L^?1 were 0.28, 0.35 and 0.62 mg L^?1 nitrite‐N respectively. The relationship between LC₅₀ (mg L^?1), salinity (S) (g L^?1) and exposure time (T) (h) was LC₅₀ = 8.4688 + 5.6764S – 0.0762T for salinities from 0.6 to 2.0 g L^?1 and for exposure times from 24 to 96 h; the relationship between survival (%) and nitrite‐N concentration (C) for salinity of 0.6–2.0 g L^?1, nitrite‐N concentrations of 0–40 mg L^?1 and exposure times from 0 to 96 h was as follows: survival (%) = 0.8442 + 0.1909S – 0.0038T – 0.0277C + 0.0008ST + 0.0001CT–0.0029SC, and the tentative equation for predicting the 96‐h LC₅₀ to salinities from 0.6 to 35 g L^?1 in L. vannamei juveniles (3.9–4.4 g) was 96‐h LC₅₀ = 0.2127 S² + 1.558S + 5.9868. For nitrite toxicity, it is shown that a small change in salinity of waters from 2.0 to 0.6 g L^?1 is more critical for L. vannamei than when wider differences in salinity occur in brackish and marine waters (15–35 g L^?1).     

Bioassay‐guided isolation and identification of active compounds from Macleaya microcarpa (Maxim) Fedde against fish pathogenic bacteria

Four alkaloids (Sanguinarine, 6‐Methoxyl‐dihydro‐chelerythrine, Cryptopine and β‐Allocryptopine) were isolated from aerial parts of Macleaya microcarpa (Maxim) Fedde using bioassay‐guided isolation method, and the inhibitory activity of ethanolic extract, various fractions and these four alkaloids against four fish pathogenic bacteria (Aeromonas hydrophila, Aeromonas salmonicida, Vibrio anguillarum and Vibrio harveyi) was assessed in vitro using the agar dilution method and the microdilution assay method respectively. A. hydrophila was the most sensitive strain to all the tested compounds. Minimum inhibitory concentration (MIC) values were lower for sanguinarine against all tested Gram‐negative strains than other three alkaloids, with MIC values of 12.5 mg L^?1 for A. hydrophila and 50 mg L^?1 to other pathogenic bacteria. Followed by 6‐methoxyl‐dihydro‐chelerythrine, which showed considerable antibacterial activity with MIC values of 80 mg L^?1 for A. hydrophila, 100 mg L^?1 for V. harveyi, and 125 mg L^?1 for both V. anguillarum and A. salmonicida. Cryptopine and β‐allocryptopine revealed similar inhibitory activity with MIC values of 100 mg L^?1 for A. hydrophila and 200 mg L^?1 for other three bacterial species. These finding provided evidence that extract, as well as isolated compounds from M. microcarpa might be potential sources novel antibacterial agents for the treatment of fish infectious diseases.     

Effects of dietary folic acid on growth,antioxidant capacity,non‐specific immune response and disease resistance of juvenile Chinese mitten crab Eriocheir sinensis (Milne‐Edwards, 1853)

An 8‐week study was conducted to determine folic acid requirement and its effect on antioxidant capacity and immunity in juvenile Chinese mitten crab Eriocheir sinensis (Milne‐Edwards, 1853), followed by a challenge assay with the pathogen Aeromonas hydrophila for 2 weeks. Folic acid was added to a basal diet at seven levels (0, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0 mg folic acid kg^?1 diet), and a diet free of folic acid and vitamin B₁₂ was also included as a control. Crabs were fed twice daily in 32 tanks with 7.76–8.17 mg oxygen L^?1, 25.0–31.0 °C and 7.5–8.3 pH. Growth and feed efficiency were significantly greater in crabs fed ≥2.0 mg folic acid kg^?1, but not significantly different between crabs fed diets >2.0 mg folic acid. The superoxide dismutase activity and glutathione S‐transferase activity were highest in crabs fed ≥2.0 mg folic acid kg^?1, followed by those fed 0.5 and 1.0 mg folic acid kg^?1, and the control diet. The malondialdehyde content was highest in crabs fed the control diet, followed by those fed 0 mg folic acid kg^?1, and the lowest value occurred in those fed ≥0.5 mg folic acid kg^?1. Phenoloxidase activity and total haemocytes were significantly higher in crabs fed ≥2.0 mg folic acid kg^?1 than other diets. Crabs fed 2.0 mg folic acid kg^?1 had the highest lysozyme, acid phosphatase and alkaline phosphatase activities but the lowest cumulative mortality. The optimum dietary folic acid requirement by E. sinensis was estimated at 2.29–2.90 mg kg^?1 diet.     

Determination of histological and genotoxic effects of formalin on Nile tilapia (Oreochromis niloticus L.)

Formalin (37–40% formaldehyde) is one of the most effective, widely used chemical in pisciculture for its antiparasitic, antifungal and prophylactic activities. It is used in paints, cleaning products and textile industry, as well. Genotoxic and histological effects of sublethal formalin exposure on Nile tilapia (Oreochromis niloticus) are investigated in this study. The semi‐static acute test was employed. One‐tenth, one‐fifth and half of the 96‐h LC₅₀ value, 148 mg L^?1, were used as sublethal exposure concentrations: 15, 30 and 75 mg L^?1 for 24, 48, 96 and 168 h respectively. The micronucleus test was applied to investigate the genotoxic effects on fish erythrocytes at the end of predetermined exposure periods and all tissues of the fish were sampled for histological examination. The micronucleus frequencies increased significantly in all exposure groups when compared with their control groups in a dose and time dependent manner (P < 0.05). Results of histological examination showed no histopathological findings in the gonads, heart, skeletal muscle, spleen and intestine tissues after sublethal formalin exposure. Passive hyperaemia of liver tissue in varying proportions, hydropic degeneration, fatty degeneration in high concentrations, branchitis on gill tissues in varying proportions, epithelial lifting, telangiectasia, bleeding in kidney and hyperaemia were found in Nile tilapia exposed to formalin at different concentrations and durations. As a result, it is thought that formalin leads to tissue damage and shows genotoxic effects even at 15 mg L^?1 concentration in O. niloticus.     

In vitro and in vivo efficacy of drugs against the protozoan parasite Azumiobodo hoyamushi that causes soft tunic syndrome in the edible ascidian Halocynthia roretzi (Drasche)

It was discovered recently that infection by a protozoan parasite, Azumiobodo hoyamushi, is the most probable cause for soft tunic syndrome in an edible ascidian, Halocynthia roretzi (Drasche). In an attempt to develop measures to eradicate the causative parasite, various drugs were tested for efficacy in vitro and in vivo. Of the 20 antiprotozoal drugs having different action mechanisms, five were found potent (24‐h EC₅₀ < 10 mg L^?1) in their parasite‐killing effects: formalin, H₂O₂, bithionol, ClO₂ and bronopol. Moderately potent drugs (10 < 24‐h EC₅₀ < 100 mg L^?1) were quinine, fumagillin, amphotericin B, ketoconazole, povidone‐iodine, chloramine‐T and benzalkonium chloride. Seven compounds, metronidazole, albendazole, paromomycin, nalidixic acid, sulfamonomethoxine, KMnO₄, potassium monopersulphate and citric acid, exhibited EC₅₀ > 100 mg L^?1. When ascidians were artificially infected with A. hoyamushi, treated using 40 mg L^?1 formalin, bronopol, ClO₂, or H₂O₂ for 1 h and then monitored for 24 h, very low mortality was observed. However, the number of surviving parasite cells in the ascidian tunic tissues was significantly reduced by treating with 40 mg L^?1 formalin or ClO₂ for 1 h. The data suggest that we might be able to develop a disinfection measure using a treatment regimen involving commonly available drugs.