Development of a bacterial challenge test for gnotobiotic sea bass (Dicentrarchus labrax) larvae

The use of probiotic microorganisms in aquaculture is gaining a lot of interest. Gnotobiotic model systems are required in order to fully understand the effects and modes-of-action of these microorganisms, as the native microbial communities present in non-sterile animals can lead to false conclusions. In this study, a gnotobiotic sea bass larvae ( Dicentrarchus labrax ) test system was developed. In order to obtain bacteria-free animals, the eggs were disinfected with glutaraldehyde and subsequently incubated in a solution of rifampicin and ampicillin. Axenity was confirmed using culture-dependent and -independent techniques. The gnotobiotic larvae were fed axenic Artemia sp. from 7 days after hatching onwards. In the challenge test, one of the three opportunistic pathogens, Aeromonas hydrophila , Listonella anguillarum serovar O1 and O2a, was added to the model system via the water and encapsulated in Artemia sp. Only serovar O2a led to increased mortality in the sea bass larvae. The presented gnotobiotic model can be used for research on, among others, reciprocal metabolic effects between microorganisms and the host (e.g. as measured by gene expression), immunostimulants, pharmacological research and the histological development of the gastrointestinal tract and growth of larvae.     

Evaluation of the probiotics Bacillus subtilis and Lactobacillus plantarum bioencapsulated in Artemia nauplii against vibriosis in European sea bass larvae (Dicentrarchus labrax, L.)

Two potential probiotics Bacillus subtilis and Lactobacillus plantarum were evaluated for use in aquaculture as preventive measures against vibriosis. In vitro evaluation of the probiotics using co-culture assays with the pathogen Vibrio anguillarum and testing for the production of antibacterial substances showed the presence of antagonism and confirmed the production of antibacterial substances. Both potential probiotics were administered to the live fish feed Artemia franciscana nauplii, offering protection against a subsequent challenge of the nauplii with the fish pathogen V. anguillarum, with best survival rates of the nauplii and the most efficient protection offered by B. subtilis. Nauplii enriched with B. subtilis were further used to evaluate the protection of sea bass larvae against vibriosis. The untreated group of fish challenged with V. anguillarum presented low survival of 36.7 %, while the fish treated with nauplii enriched with the probiotic B. subtilis showed significantly increased survival rates of 86.7 % after challenge with the pathogen. The survival of healthy unchallenged fish treated with the probiotic was not significantly different from control unchallenged fish (90-94 %). Our results indicate that B. subtilis is a probiotic suitable to be used for the prevention of vibriosis in fish larvae and can be safely administered through their live feed Artemia nauplii.     

Efficacy of a Listonella anguillarum (syn. Vibrio anguillarum) vaccine for juvenile sea bass Dicentrarchus labrax

The efficacy of a commercial bivalent Listonella anguillarum (serotype 01 and 02) vaccine (MICROViB, Microtek International) was tested on prime- and booster-immersion vaccinated sea bass Dicentrarchus labrax juveniles. We carried out 2 challenge tests on the prime-vaccinated fish, 50 and 90 d after initial vaccination. A second group of fish received a booster vaccination 60 d after the prime vaccination, and were tested with a single challenge 30 d later. Relative percent survival (RPS) was 92 and 84% (both p < 0.01) among the prime-vaccinated fish on the first and second challenges, respectively. The RPS of the booster-vaccinated sea bass was 100% (p < 0.01). Antibody titres were tested only among 10 prime-vaccinated and 10 unvaccinated (control) sea bass, 60 d post-immunisation, and were found to rise to 1/32 in the vaccinated fish. Our results demonstrate that MICROViB immersion vaccine can effectively protect juvenile sea bass from L. anguillarum infection.     

Survival and changes in growth of juvenile tench (Tinca tinca L.) fed defined diets commonly used to culture non-cyprinid species

The study is an attempt to evaluate the feasibility of intensive tench culture using non‐specific diets as a preliminary step to check the acclimatization of the species under intensive rearing systems. Five‐month‐old juvenile tench were reared in recirculating systems at mean water temperatures of 22°C for 75 days. Fish fed with four different commercial diets (trout starter, trout first feeding, sea‐bass and eel), showed significantly higher final weights than the fish fed either eel or sea‐bass diets. Initial weight for all treatments was 2.3 ± 0.53 g. Final weight for the commercial diet groups was 3.56 ± 0.4 g, compared with the remaining groups that reached 2.09 ± 0.47 g (P < 0.05). Significantly higher survival rates were observed in the eel and sea‐bass groups (84.7 and 51.5%, respectively) than in either of the trout diet groups (38%). Specific growth rates (1.26 vs –0.18) and condition factor (1.26 vs 0.93) were also higher than those fed with salmonid diets (P < 0.05). Results obtained in this study indicate that regardless of the species’ slow growth, when compared with other cyprinids, final growth rates and survival of tench fed exclusively on sea‐bass or eel diets can be considered satisfactory. It must be pointed out that these promising results were obtained at lower temperatures than previous studies of tench in culture systems. The use of belt feeders did not show improvement in growth compared with manually fed fish when trout diets were used.     

饲料中添加水解鱼蛋白对半滑舌鳎稚鱼生长及生理生化指标的影响

试验研究了饲料中添加不同水平(0%、20%、40%和60%)的水解鱼蛋白(Fish potein hdrolysate,FPH)对半滑舌鳎(Cynoglossus semilaevis Günther)稚鱼生长及生理生化指标的影响。28d饲养实验结束后,对试验鱼生长指标、鱼体消化酶活性、碱性磷酸酶(AKP)及肠道发育情况进行分析。结果显示:实验鱼的存活率为64.44%—78.88%,其中20%与40%添加组存活率显著高于其他两组(P0.05);实验鱼的特定生长率(SGR)随饲料中FPH添加水平升高呈下降趋势,20%添加组特定生长率最高,显著高于其他3组(P0.05),且60%添加组与对照组(FM)之间无显著差异(P0.05);各试验组鱼体消化酶(脂肪酶、淀粉酶、胃蛋白酶和胰蛋白酶)的比活力在14d与28d时的强弱关系不一致。鱼体碱性磷酸酶比活力大小依次为:20%组40%组对照组60%组,各组间差异均显著(P0.05);半滑舌鳎肠道微绒毛长度和黏膜厚度均以20%添加组为最优,显著优于其他3组(P0.05)。以上结果表明,在实验条件下,饲料中FPH的添加水平为20%时,能提高半滑舌鳎稚鱼的存活率及生长性能,并增强其对营养物质的吸收能力。     

不同开口饵料对西伯利亚鲟仔鱼生长、存活和体成分的影响

分别采用水丝蚓（Limnodrilus sp.）、卤虫无节幼体（Artemia nauplii）、枝角类（Moina sp.）和人工饲料饲养西伯利亚鲟仔鱼30 d,研究不同开口饵料对西伯利亚鲟仔鱼生长、存活率和体成分的影响.结果表明：卤虫无节幼体为西伯利亚鲟最适开口饵料,仔鱼的存活率最高（96.67%）; 投喂水丝蚓组生长速度最快;而人工饲料组生长速度明显低于其他组,且成活率最低.不同开口饵料组间仔鱼体成分差异显著,人工饲料组水分含量最高,且粗蛋白和粗灰分含量最低.采用卤虫无节幼体为西伯利亚鲟仔鱼开口饵料,然后采用水丝蚓进行强化培育,能获得较好的生长速度和存活率.     

Trypsin activity and physiological aspects in larval rearing of European sea bass (Dicentrarchus labrax) using live prey and compound diets

The aim of this study was to evaluate the potential of a compound diet as a live prey substitute for feeding European sea bass larvae ( Dicentrarchus labrax L.). The effect of a commercial diet (Nippai ML feed) and live prey ( Artemia nauplii) on tryptic enzyme activity, protein content, growth (standard length) and survival rates of sea bass larvae were tested during a 27-day rearing experiment. Sea bass larvae were divided into two groups. The live food group (control group) was fed exclusively on newly hatched Artemia nauplii (Inve AF grade), the test group was fed exclusively with the compound diet from day 15 onwards. As trypsin has been demonstrated to be a useful indicator for evaluating digestibility of food and the nutritional condition of fish larvae, individual tryptic enzyme activity was determined in both feeding groups. Larvae older than 14 days after hatching and fed on live food showed a significantly higher tryptic enzyme activity than larvae fed the compound diet. A similar relationship between tryptic activity and standard length in both test groups was detected only in small larvae (standard length < 7 mm). The usefulness of proteolytic enzyme activity measurements in larval fish research, as well as its use in aquaculture nutrition research, was confirmed. Protein content, increase in length and survival rates of the sea bass larvae were additionally determined in order to evaluate an influence on the diet. The protein content of larvae fed the Artemia nauplii was higher and the growth of larvae fed the compound diet was reduced. Larval mortality was not affected by the diet given.     

Comparison of the Growth and Survival of Larval Turbot in the Absence of Culturable Bacteria with Those in the Presence of Vibrio anguillarum, Vibrio alginolyticus, or a Marine Aeromonas sp

Larval turbot (Scophthalmus maximus) were reared on rotifers (Brachionus plicatilis) in the absence of culturable bacteria for up to 14 days and exhibited growth and high rates of survival (>55% in five experiments). Low numbers of known bacteria were introduced into similar cultures by exposure of the rotifers to a suspension of bacteria prior to addition of rotifers to the larval cultures; Vibrio anguillarum 91079 caused a highly significant decrease (P <0.01) in the proportion of survivors in two separate trials. With an Aeromonas sp. previously isolated from a healthy batch of copepod-fed larvae, there was no significant difference in survival compared with control larvae, even though the density of bacteria in the water of larval cultures reached 10(sup7) ml(sup-1). Bacteria colonized the gut of larvae exposed to Aeromonas-treated rotifers to levels similar to those in conventionally reared fish (>4 x 10(sup4) CFU per larva). Rearing of larvae in the presence of known bacteria provides a means of investigating the interaction of specific bacteria with turbot larvae and could provide a method for the selection of bacteria which may restrict the growth of opportunistic pathogens which would be harmful to turbot larvae.     

Diseases and Injuries Associated with Mortality of Hatchery Reared Baltic Cod (Gadus morhua L.) Larvae

A cod hatching plant was established in 1992 on the island of Bornholm in the Baltic Sea in order to elucidate the possibilites for restocking of cod fry in this brackishwater system. The disease prevalence in 3 batches of hatchery-reared yolksac larvae from the Baltic cod (Gadus morhua L.) was monitored during the posthatch period. High prevalences of bacteriosis/mycosis, lordosis/scoliosis, injuries and protozoan endoparasitism were recorded. Vibrio sp. and Vibrio anguillarum serovar 04, 06, 08 in addition to nontypable strains and saprolegniaceous fungi were isolated from the larvae. The dinoflagellate-like endoparasites were located in the yolksac of the cod larvae.     

Effects of feeding low fishmeal diets with increasing soybean meal levels on growth,gut histology and plasma biochemistry of sea bass

The aquaculture industry depends upon the development of sustainable protein sources to replace fishmeal (FM) in aquafeeds and the products derived from soybeans are some of the most studied plant feedstuffs. A key area of investigation for continuing to improve modern aquafeeds includes the evaluation of varying proportions and combinations of plant ingredients to identify mixtures that are more efficiently utilized by the fish. This study investigated the effects of increasing soybean meal (SBM) by replacing a mix of plant ingredients in low FM (20%) diets on growth, blood biochemistry profile and gut histology on European sea bass. Five isonitrogenous and isolipidic experimental diets were formulated: four diets containing increasing SBM levels (0, 10, 20 and 30%; 0SBM, 10SBM, 20SBM and 30SBM, respectively) with a low content of FM (20%) and one control diet (0% SBM; 35% FM). Diets containing SBM brought to comparable performance and protein utilization, while 0SBM had negative impact on feed conversion rate and protein utilization. Blood parameters suggested an optimal nutritional status under all feeding treatments, even though slightly decreased values were reported at increasing dietary SBM. Histology examination did not show any changes indicative of soy-induced enteritis. We can conclude that for European sea bass: (i) different blends of plant protein did not affect feed intake despite the 20% FM dietary level; (ii) the inclusion of SBM maintains optimal growth and feed utilization in low FM diets; (iii) blood biochemistry profile showed a good nutritional status under all feeding regimes; (iv) no evidence of soy-induced enteritis was reported in any group fed low FM diets. For formulation of practical diets in on-growing of European sea bass, SBM up to 30% can be successfully incorporated into feeds containing low FM inclusion.     

Expression of the B subunit of Escherichia coli heat-labile enterotoxin in a marine Vibrio and in a mutant that is pleiotropically defective in the secretion of extracellular proteins.

A marine Vibrio (designated Vibrio sp. 60) that is related to Vibrio anguillarum was used as a host for a plasmid that encodes the non-toxic B subunit (EtxB) of Escherichia coli heat-labile enterotoxin. Expression of EtxB in Vibrio sp. 60 resulted in the efficient and selective secretion of the B subunit into the extracellular growth medium. This indicated that Vibrio sp. 60, which does not normally produce cholera-like enterotoxins, nonetheless possesses a secretory machinery that permits these toxins to be translocated across its cytoplasmic and outer membranes. Expression of EtxB in a sec mutant of Vibrio sp. 60 (MVT1192), which had previously been shown to be defective in the secretion of several extracellular proteins, resulted in approximately 95% of the B subunit remaining entrapped within the periplasm of the bacterial cell envelope. This implies that the mutation in MVT1192 defines a locus that determines a common step in the secretion of extracellular proteins, including oligomeric toxins.     

饲料中不同水平鱼蛋白水解物对军曹鱼稚鱼生长及体组成的影响

设计4种等能等氮试验饲料,对照组以鱼粉为单一蛋白源,处理组分别以鱼蛋白水解物替代鱼粉蛋白的17%、34%和51%,军曹鱼(Rachycentron canadum)稚鱼的初始重为(3.79±0.22)g,实验在室内流水养殖系统中进行6周的饲养试验,每种饲料随机3个重复。试验结果显示不同水平鱼蛋白水解物对试验鱼的存活率没有显著影响(P>0.05),除了以鱼蛋白水解物替代鱼粉蛋白51%组外,其他各组的增重率(WG)和饲料摄入量(FI)都是随着饲料中鱼蛋白水解物替代鱼粉蛋白量的增加而增加。试验结果表明,在军曹鱼稚鱼饲料中用鱼蛋白水解物替代34%的鱼粉蛋白对于军曹鱼稚鱼的生长和饲料摄入量具有一定的促进作用。军曹鱼稚鱼血浆中胆固醇含量随着饲料中鱼蛋白水解物添加水平的升高而升高,而血浆中的总蛋白含量则无此规律,各处理组血浆中的甘油三酯无显著差异(P0.05)。       

Sulphur amino acid requirement of juvenile Asian sea bass Lates calcarifer

The dietary requirement of juvenile Asian sea bass Lates calcarifer Bloch for total sulphur amino acids was studied. Fish (average initial weight of 2.59 ± 0.08 g) were reared in twelve 500 L fibreglass tanks provided with flow-through seawater at 26°C and salinity of 31 p.p.t. for 12 weeks. They were fed semi-purified test diets containing 6.2, 7.2, 8.1, 9.0, 10.8, or 12.6 g methionine kg⁻¹ dry diet and a basal level of 3.1 g cystine kg⁻¹ dry diet. The mean crude protein of the diets (containing defatted Peruvian fishmeal, squid meal, soybean meal, and free amino acid mixture to simulate the pattern of hydrolysed sea bass protein) was 46.02%. The crude fat content of the diets was 10.51% from a 1 : 1 mixture of cod liver oil and soybean oil. Survival was 100% in all treatments. On the basis of the growth response, the total sulphur amino acid requirement of juvenile Asian sea bass was estimated to be 13.4 g kg⁻¹ dry diet (2.9% of protein). Fish fed low levels of l -methionine had significantly lower weight gains and feed efficiency ratios as well as slightly higher hepatosomatic indices. No nutritional deficiency signs were observed other than growth depression in fish fed on diets that were low in methionine. This information is valuable in further refinement of formulations of practical diets for the Asian sea bass.     

Fatty acids in liver and muscle of farmed and wild sea bass (Dicentrarchus labrax L.)

Growth rate and assimilation of essential fatty acids were analysed in sea bass (Dicentrarchus labrax L.) fed different diets. Gas Chromatographie analyses of total lipids in commercial diets indicated excessive presence of saturated, mono and dienoic fatty acids, while the content of to 3 HUFA was very low. Fatty acid patterns of liver and white muscle farmed sea bass reflected the contents of the dietary lipids. The concentration of polyunsaturated fatty acids, particularly ω3 HUFA was significantly lower when compared with samples from the wild population. The atherogenic and fish lipid quality indices confirmed the higher nutritional quality of wild sea bass when compared with farmed fishes.     

Selection and identification of autochthonous potential probiotic bacteria from turbot larvae (Scophthalmus maximus) rearing units

The purpose of this study was to select, identify and characterise bacteria as a disease control measure in the rearing of marine fish larvae (turbot, Scophthalmus maximus). Thirty-four out of 400 marine bacterial strains exhibited in vitro anti-bacterial activity against three fish larval pathogens. Two strains originated from culture collections and thirty two strains were isolated directly from turbot larvae rearing units using a pre-selection procedure to facilitate detection of antagonists. Approximately 8,500 colonies from colony-count plates were replica-plated on agar seeded with Vibrio anguillarum, and 196 of them caused zones of clearing in the V. anguillarum agar layer. Of these, 32 strains exhibited reproducible antibacterial properties in vitro when tested against the fish pathogens V. anguillarum 90-11-287, V. splendidus DMC-1 and a Pseudoalteromonas HQ. Seventeen antagonists were identified as Vibrio spp. and four of twelve tested were lethal to yolk-sac larvae. The 15 remaining strains were identified as Roseobacter spp. based on phenotypic criteria and 16S rDNA gene sequence analysis of two strains representing the two major RAPD groups. Most of the remaining 164 strains selected in the initial replica plating were identified as Vibrionaceae or Pseudoalteromonas. Roseobacter spp. were not lethal to egg yolk sac turbot larvae and in two of three trials, the mortality of larvae decreased (p > 0.001) in treatments where 10(7) cfu/ml Roseobacter sp. strain 27-4 was added, indicating a probiotic potential.     

Sea bass Dicentrarchus labrax nervous necrosis virus isolates with distinct pathogenicity to sea bass larvae.

Reproduction of nodavirus disease was performed by experimental infection of sea bass eggs during fertilization or at larval stage 4 with 2 genetically distinguishable nodavirus strains (Sb1 and Sb2) isolated from sea bass collected along the Atlantic and Mediterranean French coast. The pathogenicity of the virus strains was assigned after detection of the virus by ELISA and immunohistochemistry (IHC). The Atlantic (Sb1) strain was more pathogenic than the Mediterranean (Sb2) strain during the fertilization step whilst both strains were pathogenic following experimental exposure of 4 d old larvae. Virus lesions developed in the brain 4 to 6 d following experimental exposure. Experimental ELISA proved very sensitive for detecting the nodavirus in Sb1 or Sb2 experimentally infected larvae, as well as in naturally infected sea bass larvae collected in French hatcheries or in barramundi larvae reared in the Pacific area. The development of an ELISA specific for the 2 nodavirus strains isolated from the sea bass should be useful for the detection of the virus, in addition to other techniques recommended by the Office International des Epizooties (OIE).     

Phaeobacter gallaeciensis Reduces Vibrio anguillarum in Cultures of Microalgae and Rotifers, and Prevents Vibriosis in Cod Larvae

Phaeobacter gallaeciensis can antagonize fish-pathogenic bacteria in vitro, and the purpose of this study was to evaluate the organism as a probiont for marine fish larvae and their feed cultures. An in vivo mechanism of action of the antagonistic probiotic bacterium is suggested using a non-antagonistic mutant. P. gallaeciensis was readily established in axenic cultures of the two microalgae Tetraselmis suecica and Nannochloropsis oculata, and of the rotifer Brachionus plicatilis. P. gallaeciensis reached densities of 10(7) cfu/ml and did not adversely affect growth of algae or rotifers. Vibrio anguillarum was significantly reduced by wild-type P. gallaeciensis, when introduced into these cultures. A P. gallaeciensis mutant that did not produce the antibacterial compound tropodithietic acid (TDA) did not reduce V. anguillarum numbers, suggesting that production of the antibacterial compound is important for the antagonistic properties of P. gallaeciensis. The ability of P. gallaeciensis to protect fish larvae from vibriosis was determined in a bath challenge experiment using a multidish system with 1 larva per well. Unchallenged larvae reached 40% accumulated mortality which increased to 100% when infected with V. anguillarum. P. gallaeciensis reduced the mortality of challenged cod larvae (Gadus morhua) to 10%, significantly below the levels of both the challenged and the unchallenged larvae. The TDA mutant reduced mortality of the cod larvae in some of the replicates, although to a much lesser extent than the wild type. It is concluded that P. gallaeciensis is a promising probiont in marine larviculture and that TDA production likely contributes to its probiotic effect.     

Antimicrobial peptides protect coho salmon from Vibrio anguillarum infections

Fish losses from infectious diseases are a significant problem in aquaculture worldwide. Therefore, we investigated the ability of cationic antimicrobial peptides to protect against infection caused by the fish pathogen Vibrio anguillarum. To identify effective peptides for fish, the MICs of certain antimicrobial peptides against fish pathogens were determined in vitro. Two of the most effective antimicrobial peptides, CEME, a cecropin-melittin hybrid peptide, and pleurocidin amide, a C-terminally amidated form of the natural flounder peptide, were selected for in vivo studies. A single intraperitoneal injection of CEME did not affect mortality rates in juvenile coho salmon infected with V. anguillarum, the causative agent of vibriosis. Therefore, the peptides were delivered continuously using miniosmotic pumps placed in the peritoneal cavity. Twelve days after pump implantation, the fish received intraperitoneal injections of V. anguillarum at a dose that would kill 50 to 90% of the population. Fish receiving 200 microg of CEME per day survived longer and had significantly lower accumulated mortalities (13%) than the control groups (50 to 58%). Fish receiving pleurocidin amide at 250 microg per day also survived longer and had significantly lower accumulated mortalities (5%) than the control groups (67 to 75%). This clearly shows the potential for antimicrobial peptides to protect fish against infections and indicates that the strategy of overexpressing the peptides in transgenic fish may provide a method of decreasing bacterial disease problems.