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.     

Preparation and Characterization of Alginate Microparticles Containing a Model Protein for Oral Administration in Gnotobiotic European Sea Bass (<Emphasis Type="Italic">Dicentrarchus labrax</Emphasis>) Larvae

Aquaculture is the fastest growing animal production sector. However, the production of marine fish is still hampered by the high mortality rate in the first few weeks after hatching. Mortality in larvae is often caused by microbial infections. Today, the incorporation of immunostimulants into microparticles provides us new tools to enhance disease resistance in marine larviculture. In this study, we prepared alginate microparticles loaded with the model antigen fluorescein isothiocyanate conjugated-bovine serum albumin. Optimum concentrations of alginate and CaCl₂, the correct alginate viscosity and the appropriate preparatory conditions led to the creation of desirable microparticles with the correct size for oral feeding in gnotobiotic European sea bass larvae. The prepared alginate microparticles were stable in sea water and were successfully ingested by gnotobiotic sea bass larvae at day after hatching 7 without causing any negative effects. Results suggest the suitability of this drug delivery system for targeting the innate immune system of fish larvae in order to enhance disease resistance and thus reduce mortality in larviculture.     

Effects of different dietary levels of fish protein hydrolysates on growth, digestive enzymes, gut microbiota, and resistance to Vibrio anguillarum in European sea bass (Dicentrarchus labrax) larvae

Two fish protein hydrolysates (FPH) were incorporated into four diets prepared for start-feeding sea bass larvae, at two different levels (10% and 19% of total ingredients): a commercial FPH, CPSP, in which the molecular mass of the main fraction of soluble peptides (51%) was between 500-2500 Da, and an experimental FPH obtained by acidic silage of sardine offal, SH, with a main portion of soluble peptides (54%) ranging from 200 to 500 Da. The diet with 10% of the commercial FPH gave the best results in terms of growth, survival and intestinal development, as evaluated by the early activity of digestive enzymes in the brush border membrane (alkaline phosphatase and aminopeptidase N). This was related to the low level of Vibrio spp. counted in the larvae of group C10. The high dose of FPH, especially in the experimental preparation rich in short peptides, seemed to favour the dominance of Vibrio sp. TYH3, which behaved opportunistically. The effect of the experimental FPH was ambiguous, since early larvae challenged with Vibrio anguillarum were more resistant to the pathogen, especially at high FPH dose (group S19). This might be due either to direct antagonism between V. anguillarum and Vibrio sp. TYH3, or to the stimulation of the immune response in the larvae. These results indicate that different molecular weight fractions and concentrations of feed-soluble peptides may affect the growth performance and immunological status of sea bass larvae. Consequently, a low dose of commercial FPH seems advisable, both for larval development and for the bacterial environment, although further research is required to determine and characterize peptide fractions that may have a beneficial effect on growth and immune response, and to determine their optimal inclusion levels in diets for sea bass larvae.     

Tissue essential fatty acid composition and competitive response to dietary manipulations in white bass (Morone chrysops), striped bass (M. saxatilis) and hybrid striped bass (M. chrysopsxM. saxatilis)

The effects of wide changes in dietary levels of docosahexaenoic (DHA) or arachidonic (ArA) acids on growth, survival and fatty acid composition in body tissues of Morone larvae were examined. White bass (WB, Morone chrysops), striped bass (SB, Morone saxatilis) and sunshine hybrid bass (HSB, M. chrysopsxM. saxatilis) larvae (day 24-46) were fed Artemia nauplii enriched with algal sources of varying proportions of DHA and ArA (from 0 to over 20% of total fatty acids). WB larvae fed DHA-deficient Artemia diet retarded over 50% of their potential growth, however, increasing dietary DHA/ArA ratios were associated with a significant growth improvement. The highest proportion of polyunsaturated fatty acids was found in WB neural tissue (approx. 50% of total fatty acids), while HSB neural tissue contained the highest proportion of saturated fatty acids (approx. 35% of total fatty acids). Within the neural tissues of all Morone larvae, both DHA and ArA were generally the most dominant as well as the most responding fatty acids to dietary manipulations (except in WB fed DHA or ArA deficient diets). HSB neural tissue was particularly efficient in retaining a significant amount of DHA in the face of dietary deficiency. However, WB neural tissue was the most responsive to dietary increase in DHA, accumulating a significantly higher amount of DHA (P<0.05) than SB or HSB. Results demonstrate significant differences in fatty acid composition and growth responsiveness to dietary manipulations between Morone larvae species and within specific tissues. WB weight gain and neural tissue composition was affected most by dietary changes in both DHA and ArA whereas SB and HSB tissue compositions were generally less affected by dietary manipulations.     

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.     

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).     

Deficit of didocosahexaenoyl phospholipid in the eyes of larval sea bass fed an essential fatty acid deficient diet

Larval sea bass Dicentrarchus labrax of 27 days old were reared on Artemia enriched with Super Selco©, Tuna Orbital Oil or Yeast. The first diet is commonly used in mariculture for larval rearing, the second diet was designed to deliver an optimal docosahexaenoic acid (22: 6n-3) to eicosapentaenoic acid (20: 5n-3) ratio, and the third diet was deficient in docosahexaenoic acid (22: 6n-3). The eyes of these larvae were analysed after 28 days and the molecular species of the three main phospholipid classes, phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) determined. Eyes from larvae fed Artemia enriched with yeast showed large decreases in molecular species containing 22: 6n-3 compared to those supplemented with tuna orbital oil, most notably in 16: 0/22: 6n-3 PC which fell from 10.6 to 0.4%, in 22: 6n-3/22: 6n-3 and 18: 1/22: 6n-3 PE which fell from 29.6 to 0.3% and from 10.8 to 1.1% respectively, and in 22: 6n-3/22: 6n-3 PS which fell from 34.3 to 1.7%. Molecular species containing all other fatty acids, and especially 20: 5n-3, were elevated in eyes from the yeast-supplemented fish. In larvae fed Artemia enriched with Super Selco, amounts of eye 22: 6n-3/22: 6n-3 phospholipid were slightly lower in all three phospholipid classes compared to eyes from the tuna orbital oil-supplemented larvae. There was also a trend of decreased saturated fatty acid/22: 6n-3 and monounsaturated fatty acid/22: 6n-3 molecular species in all classes from the Super Selco-supplemented fish, the deficits being made up with molecular species containing 20: 5n-3 and 22: 5n-3. These results are discussed in relation to larval viability with particular respect to visual function.     

Ichthyophonus infections in cultured marine fish from Spain

Ichthyophonus sp. is reported for the first time in Mugil capito (thinlip grey mullet) and Li a saliens (leaping grey mullet). The fungus was also found in L. aurata (golden grey mullet), Dicentrarchus labrax (sea bass), Sparus aurata (gilthead sea bream) and Scophthalmus maximus (turbot), whereas Mugil cephalus (grey mullet) was not parasitized. In fish sampled periodically, the highest prevalences were observed in sea bass and the lowest in turbot. Among the fish sampled occasionally, the fungus was found associated to an epizootic in thinlip grey mullet. Ichthyophonus was never found in fish weighing <0·5 g. An increase in the prevalence of infection with the age of turbot and gilthead sea bream was observed. Gilthead sea bream and sea bass showed higher prevalences in a closed system than in open and semi-intensive systems. Multinucleate spherical spores, hyphae and endospores of Ichthyophonus sp. parasitized different organs of thinlip and leaping grey mullets, though infection intensity was maximal in the spleen. In the remaining fish, the fungus was found mainly in the trunk kidney, where it appeared frequently in a necrotic form. Ichthyophonus sp. can be considered a potential threat for marine fish aquaculture, especially in culture conditions which may favour the introduction and transmission of the fungus.     

Vitamin E in early stages of sea bass (Dicentrarchus labrax) development

This study reports titration of vitamin E levels in the sea bass (Dicentrarchus labrax) using high-pressure liquid chromatography. The first part of the work is devoted to vitamin E detection in: (1) plasma of maturing females and males characterized by different body sizes; (2) seminal fluid and eggs; and (3) developing embryos of sea bass fed with vitamin E. In the second part of the study, variations of vitamin E levels during larval development are analyzed. The results show a direct correlation between plasma vitamin E content and body size for both adult male and female sea bass. High vitamin E levels were found in seminal fluid, in eggs before and after fertilization, and in embryos during development and at hatching, whereas vitamin E level was low in dead embryos and in embryos with limited survival. During larval development, the vitamin E content decreased slowly but steadily during the first four days of larval growth; subsequently, it progressively increased from day 9 to day 40. In teratogenic larvae, vitamin E content was significantly higher than in normal larvae. This study provides evidence on how vitamin E exerts an antioxidant defense in sea bass reproduction.     

Virulence of luminescent and non-luminescent isogenic vibrios towards gnotobiotic Artemia franciscana larvae and specific pathogen-free Litopenaeus vannamei shrimp

Aims:  This study was conducted to test the virulence of luminescent (L) and non-luminescent (NL) isogenic strains of Vibrio campbellii LMG21363, Vibrio harveyi BB120 (wild type) and quorum-sensing mutant strains derived from the wild type such as Vibrio harveyi BB152, BB170, MM30 and BB886.
Methods and Results:  The NL strains could be obtained by culturing rifampicin-resistant luminescent strains in the dark under static condition. The virulence of the L and NL strains was tested in gnotobiotic Artemia franciscana larvae challenged with 10⁴ CFU ml⁻¹ of bacteria. All luminescent isogenic tested strains showed higher virulence compared to the NL strains. The virulence of L and NL V. campbellii and V. harveyi BB120 was also tested in specific pathogen-free juvenile shrimp upon intramuscular injection with 10⁶ CFU of bacteria. In contrast with Artemia , there was no significant difference in mortality between the groups challenged with L and NL strains ( P  > 0·05). The non-luminescent strains were not able to revert back to the luminescent state and quorum sensing did not influence this phenotypic shift.
Conclusions:  Luminescent Vibrio strains can switch to a non-luminescent state by culturing them in static conditions. The NL strains become less virulent as verified in Artemia .
Significance and Impact of the Study:  The luminescent state of Vibrio cells in a culture needs to be verified in order to assure maintenance of virulence.     

Drug assimilation in the tissue of European sea bass (Dicentrarchus labrax) fry delivered orally through bioencapsulation

Assimilation levels of the antibacterials trimethoprim (TMP) and sulfamethoxazole (SMX) in sea bass ( Dicentrarchus labrax ) fry tissue administered orally were investigated. A 1:5 TMP and SMX combination incorporated in an oil emulsion (Selco) at 20 % and 40 % concentrations (w/w) were bioencapsulated in Artemia (Instar II) nauplii. Chemotherapeutics-loaded ('medicated') nauplii were fed to the sea bass fry and drug concentrations in the tissue were analysed by high-performance liquid chromatrography (HPLC). Fish fed 40 %'medicated' Artemia assimilated significantly higher levels of chemotherapeutics compared with those fed 20 %'medicated' Artemia. Chemotherapeutics given at 40 % reached peak levels (19.3 μg TMP/g DW and 23.31 μg SMX/g DW) within 2 h while those at 20 % peaked (8.74 μg TMP/g DW and 6.73 fig SMX/g DW) after 5 h. TMP persisted in the tissues longer (up to 72 h) than SMX (12–16 h), suggesting a more efficient uptake and retention of the former and/or faster metabolism and elimination of the latter.     

Proteocephalus ambloplitis and Contracaecum sp. from largemouth bass (Micropterus salmoides) stocked into Boundary Reservoir, Saskatchewan

Nonresident (introduced) largemouth bass (Micropterus salmoides) from Boundary Reservoir, Saskatchewan were examined for helminths. Four species of parasites were found (Diplostomum sp., Proteocephalus ambloplitis, Pomphorhynchus bulbocolli, and Contracaecum sp.). Contracaecum sp. larvae were absent in age-0 and age-1 bass, but prevalence and mean intensity increased with age for bass age-2 or older. Similarly, the prevalence and mean intensity of P. ambloplitis plerocercoids in bass were low until age-2; older bass harbored significantly more plerocercoids. Analysis of stomach contents indicates that this pattern of recruitment for Contracaecum sp. and P. ambloplitis is probably due to increased feeding by largemouth bass on aquatic insects and cannibalism after age-2, respectively. Although Contracaecum sp. may have been established in the reservoir prior to the introduction of bass, we are certain that P. ambloplitis was introduced via stocking with infected fingerlings.     

Uptake and processing of a Vibrio anguillarum bacterin in Artemia franciscana measured by ELISA and immunohistochemistry

Nauplii of Artemia franciscana were incubated in two different concentrations (undiluted and 1:9 in autoclaved sea water) of a divalent bacterin composed of two different serovars of Vibrio anguillarum. In order to investigate uptake and further processing of a bacterin in the live feed organism A. franciscana, immunohistochemistry was applied, visualising the presence of whole bacterial cells and antigens from the bacterin in individual nauplii. By using ELISA, it was shown that approximately 1.5-2-5 x 10(5) cells were incorporated into each Artemia under the conditions used. Maximum incorporation of cells was measured after 30 min, whereas after 60 min there was a decline to levels of 0.9-1.6 x 10(5) cells per Artemia. Immediately after incubation in the bacterin solution, the nauplii were transferred to a culture of the alga Isochrysis galbana, in order to simulate transfer of the nauplii to rearing tanks for fish larvae. From the ELISA, it could be concluded that the incorporated bacterial cells were excreted from the Artemia nauplii rapidly, however a large variation among different nauplii could be visualised by immunohistochemistry.     

松褐天牛肿腿蜂对寄主松褐天牛三龄幼虫的功能反应

为评估松褐天牛肿腿蜂Sclerodermus sp.（膜翅目： 肿腿蜂科）对其寄主松褐天牛Monochamus alternatus 3 龄幼虫的控制作用, 通过功能反应试验, 研究了松褐天牛肿腿蜂对松褐天牛3 龄幼虫补充营养时期的捕食功能反应和补充营养后的寄生功能反应以及肿腿蜂的密度效应。结果表明： 当松褐天牛肿腿蜂密度固定为1 头/缸时, 肿腿蜂因补充营养对松褐天牛3 龄幼虫的捕食功能反应符合Holling Ⅱ型方程, 1 头松褐天牛肿腿蜂雌蜂对松褐天牛3 龄幼虫的最大致死量为9.48 头, 平均为3.75 头; 寄生作用的功能反应符合Holling Ⅰ型方程, 可知肿腿蜂的寄主密度阈限为4 头就能满足补充营养和寄生的需要; 在捕食和寄生两个阶段其对松褐天牛幼虫的寻找效应随着寄主密度的增加呈线性减弱。当松褐天牛幼虫密度固定为30 头/缸时, 随着肿腿蜂的密度增加, 其补充营养阶段的捕食作用和之后的寄生作用均呈线性升高, 寻找效应则呈线性减弱; 由蜂虫比和致死总量的模型可得肿腿蜂与天牛幼虫比例为0.9805（近1∶1）时, 可使松褐天牛幼虫死亡总数最大。这些结果表明, 松褐天牛肿腿蜂是松褐天牛幼虫期的有效天敌。本研究为评价松褐天牛肿腿蜂对寄主的控制能力提供了基础数据和方法, 并为其在野外的释放量提供了依据。     

Does Virulence Assessment of Vibrio anguillarum Using Sea Bass (Dicentrarchus labrax) Larvae Correspond with Genotypic and Phenotypic Characterization?

Background

Vibriosis is one of the most ubiquitous fish diseases caused by bacteria belonging to the genus Vibrio such as Vibrio (Listonella) anguillarum. Despite a lot of research efforts, the virulence factors and mechanism of V. anguillarum are still insufficiently known, in part because of the lack of standardized virulence assays.

Methodology/Principal Findings

We investigated and compared the virulence of 15 V. anguillarum strains obtained from different hosts or non-host niches using a standardized gnotobiotic bioassay with European sea bass (Dicentrarchus labrax L.) larvae as model hosts. In addition, to assess potential relationships between virulence and genotypic and phenotypic characteristics, the strains were characterized by random amplified polymorphic DNA (RAPD) and repetitive extragenic palindromic PCR (rep-PCR) analyses, as well as by phenotypic analyses using Biolog’s Phenotype MicroArray™ technology and some virulence factor assays.

Conclusions/Significance

Virulence testing revealed ten virulent and five avirulent strains. While some relation could be established between serotype, genotype and phenotype, no relation was found between virulence and genotypic or phenotypic characteristics, illustrating the complexity of V. anguillarum virulence. Moreover, the standardized gnotobiotic system used in this study has proven its strength as a model to assess and compare the virulence of different V. anguillarum strains in vivo. In this way, the bioassay contributes to the study of mechanisms underlying virulence in V. anguillarum.     

Diagnosis and seroprevalence of leptospirosis in California sea lions from coastal California

The sensitivity and specificity of the microscopic agglutination test (MAT) as a method for detection of exposure to Leptospira spp. in California sea lions (Zalophus californianus) were determined. Sera came from individuals that demonstrated clinical signs of renal disease, had lesions suggestive of leptospirosis at necropsy, and had visible leptospires in silver stained kidney sections as positive controls. Sera from unexposed captive individuals were used as negative controls. The test was 100% sensitive at 1:3,200 for confirming renal infection and 100% specific at negative < 1:100 for detection of Leptospira interrogans scrovar pomona antibodies by MAT in California sea lions. Leptospira interrogans serovar pomona was used as a screening serovar because it has been isolated previously from the kidneys and placentas of California sea lions, and there appears to be cross-reactivity between serovar pomona and other serovars. Sera from 225 free-ranging California sea lions presented to one of three participating California (USA) coastal marine mammal rehabilitation centers in 1996 were then evaluated for antibodies to serovar pomona using the MAT. The overall seroprevalence was 38.2% (86/225), although the prevalence varied among locations from 100% (38/38) in animals at the Marine Mammal Care Center (Fort MacArthur, California, USA) to 0% (0/14) at SeaWorld California (San Diego, California). At The Marine Mammal Center (Sausalito, California) [prevalence 27.8% (48/173)], the majority of seropositive animals were subadults and adults, and males were 4.7 times more likely to be seropositive to serovar pomona than females. When combining results from all three centers, subadult and adult animals were more likely to be seropositive than pups and juvenile sea lions, and the highest proportion of seropositive animals presented during the autumn months. Serum elevations of blood urea nitrogen, creatinine, phosphorus, and/or calcium were associated with seropositivity to serovar pomona. We found no association between potassium or sodium levels and seropositivity.