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.     

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.     

Bacterial Colonization of Cod (Gadus morhua L.) and Halibut (Hippoglossus hippoglossus) Eggs in Marine Aquaculture

Aquaculture has brought about increased interest in mass production of marine fish larvae. Problems such as poor egg quality and mass mortality of fish larvae have been prevalent. The intensive incubation techniques that often result in bacterial overgrowth on fish eggs could affect the commensal relationship between the indigenous microflora and opportunistic pathogens and subsequently hamper egg development, hatching, larval health, and ongrowth. Little information about the adherent microflora on fish eggs is available, and the present study was undertaken to describe the microbial ecology during egg development and hatching of two fish species of potential commercial importance in marine aquaculture. Attachment and development of the bacterial flora on cod (Gadus morhua L.) eggs from fertilization until hatching was studied by scanning electron microscopy. The adherent microflora on cod (G. morhua L.) and halibut (Hippoglossus hippoglossus) eggs during incubation was characterized and grouped by cluster analysis. Marked bacterial growth could be demonstrated 2 h after fertilization, and at hatching eggs were heavily overgrown. Members of the genera Pseudomonas, Alteromonas, Aeromonas, and Flavobacterium were found to dominate on the surface of both cod and halibut eggs. The filamentous bacterium Leucothrix mucor was found on eggs from both species. While growth of L. mucor on halibut eggs was sparse, cod eggs with a hairy appearance due to overgrowth by this bacterium close to hatching were frequently observed. Vibrio fischeri could be detected on cod eggs only, and pathogenic vibrios were not detected. Members of the genera Moraxella and Alcaligenes were found only on halibut eggs. Caulobacter and Seliberia spp. were observed attached to eggs dissected from cod ovaries under sterile conditions, indicating the presence of these bacteria in ovaries before spawning. Adherent strains did not demonstrate antibiotic resistance above a normal level. Attempts to regulate the egg microflora by incubation of gnotobiotic eggs with defined antibiotic-producing strains did not result in persistent protection against subsequent colonization by the microflora of the incubator.     

Induction of triploidy and gynogenesis in teleost fish with emphasis on marine species

The induction of triploidy and gynogenesis by chromosome set manipulation has traditionally been studied more intensively in freshwater than in marine fish. In the last years, however, several studies have applied these manipulations in about a dozen marine species, including mainly sparids, moronids and flatfishes. This paper focuses on the methodologies used to induce, verify, and assess performance of both triploids and gynogenetics of these marine species. Since many of them are batch spawners and have small and fragile eggs and larvae, peculiarities relating to broodstock management, gamete quality and mortality assessment during early larval stages are also taken into account. However, data show that if handling is correct and the treatments are optimized, triploid and gynogenetic rates of 100% can be easily achieved. Survival of triploids with respect to the controls is about 70–80%, whereas in gynogenetics it is generally low and more variable, depending on the species considered. In the marine fish investigated so far, triploidy has not resulted in significantly higher growth rates. On the other hand, the induction of gynogenesis has resulted in the production of both all-female and mix-sex stocks. Throughout the paper, special reference is made to the European sea bass (Dicentrarchus labrax L.), a species of both basic and applied interest, for which a comprehensive study has been carried out on the induction, verification and performance of triploids and gynogenetics.Author for correspondence     

Nodaviruses as pathogens in larval and juvenile marine finfish

Nodaviruses have emerged as major pathogens of a wide range of larval and juvenile marine finfish in aquaculture worldwide. The causative agents are non-enveloped, icosahedral, RNA viruses with diameters in the range of 25–34nm. They display considerable serological and molecular homology, although the present evidence suggests that there is more than one agent causing disease in a range of species. The diseases produced by these nodaviruses invariably involve the central nervous system and the retina where they usually produce vacuolation and cell necrosis. Virus particles are numerous within the cytoplasm of affected cells and extracellularly. As a result of the lesions, affected larvae/juveniles exhibit a range of neurological signs usually culminating in high mortality rates (not uncommonly 100%). One virus, that of the European sea bass, has recently been cultured in a fish cell line, but to date techniques such as the fluorescent antibody test, enzyme-linked immunosorbent assay and polymerase chain reaction have relied upon the harvest of purified viral antigen from infected tissues rather than obtaining these reagents from viruses grown in cell cultures. The epidemiology of these diseases is only partly understood. All appear to transmit readily by cohabitation of infected fish with naive larvae or juveniles, but vertical transmission has only been recognized with striped jack nervous necrosis and sea bass nervous necrosis viruses. Consequently, some aspects of disease control are based on first principles, rather than application of a full understanding of epidemiological factors.     

The effect of early and late hatching on the escape response of walleye pollock (Theragra chalcogramma) larvae

Hatching of fish eggs fertilized at the same time occurs overa period of several days. Differences in the escape responseof fish larvae during the hatching period have not hithertobeen studied. In this study, the escape response of walleyepollock (Theragra chalcogramma) larvae over the hatching periodwas examined. Escape speed, response to multiple touches witha fine probe, response to water currents generated by a predatorand predation by euphausiids (Thysanoessa inermis) and amphipods(Pleusirus secorrus) were measured in the laboratory. Otolithmeasurements of field-collected larvae support a broad hatchingperiod for walleye pollock eggs in the sea similar to that observedin the laboratory. The escape response of walleye pollock larvaewas affected by rank in the order of hatching, thus with respectto predation, hatching order may affect the survival of larvaein the sea. Early hatching larvae were smaller, less sensitiveto tactile stimulation, had a slower, weaker escape responseand higher laboratory rates of predation mortality than thosethat hatched later.     

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.     

In Situ Mortality Experiments with Juvenile Sea Bass (Dicentrarchus labrax) in Relation to Impulsive Sound Levels Caused by Pile Driving of Windmill Foundations

Impact assessments of offshore wind farm installations and operations on the marine fauna are performed in many countries. Yet, only limited quantitative data on the physiological impact of impulsive sounds on (juvenile) fishes during pile driving of offshore wind farm foundations are available. Our current knowledge on fish injury and mortality due to pile driving is mainly based on laboratory experiments, in which high-intensity pile driving sounds are generated inside acoustic chambers. To validate these lab results, an in situ field experiment was carried out on board of a pile driving vessel. Juvenile European sea bass (Dicentrarchus labrax) of 68 and 115 days post hatching were exposed to pile-driving sounds as close as 45 m from the actual pile driving activity. Fish were exposed to strikes with a sound exposure level between 181 and 188 dB re 1 µPa².s. The number of strikes ranged from 1739 to 3067, resulting in a cumulative sound exposure level between 215 and 222 dB re 1 µPa².s. Control treatments consisted of fish not exposed to pile driving sounds. No differences in immediate mortality were found between exposed and control fish groups. Also no differences were noted in the delayed mortality up to 14 days after exposure between both groups. Our in situ experiments largely confirm the mortality results of the lab experiments found in other studies.     

Detecting the degree of establishment of a non-indigenous species in coastal ecosystems: sea bass <Emphasis Type="Italic">Dicentrarchus labrax</Emphasis> escapes from sea cages in Canary Islands (Northeastern Central Atlantic)

This article gives an insight into the degree of establishment and potential impact of escaped sea bass in shallow coastal habitats of Tenerife. We studied spatial distribution, abundance, size frequency and use of habitat by visual census. Stomach contents and gonadal maturity of escaped sea bass were analysed. Results show that abundance of escaped sea bass is related to distance from to the nearest fish farm and to the habitat complexity. The most frequent size found in the wild corresponds to that of first marketing size. Stomach contents indicate that escaped sea bass were able to exploit natural resources, especially fish. The presence of a female with mature gonads indicates that conditions for successful maturation exist. However, further studies are needed to ensure whether or not escaped sea bass are able to establish self-reproducing populations. Thus, we can conclude that sea bass seem to be already established in shallow coastal habitats of Tenerife, but the population may depend mainly on escapees. As a result of being an opportunistic piscivore, sea bass could be predating on and competing with other native species that are economically important. These results are useful to develop a risk assessment based on quantitative data to predict negative effects. Moreover, management policies such as communication of massive escapes or mitigation measures are needed to prevent future impact on the marine environment. Handling editor: P. Viaroli     

The effects of temperature change on the hatching success and larval survival of largemouth bass Micropterus salmoides and smallmouth bass Micropterus dolomieu

In this study, the effects of abrupt temperature change on the hatching success and larval survival of eggs, yolk-sac larvae (YSL) and larvae above nest (LAN), for both largemouth bass Micropterus salmoides and smallmouth bass Micropterus dolomieu were quantified. Temperature had a significant effect on hatching success and time to 50% mortality, with large heat shocks causing accelerated mortality. The temperature changes shown to influence survival of all life stages, however, were beyond what is typically experienced in the wild. Micropterus salmoides had greater egg hatching success rates and increased survival rates at YSL and LAN stages, relative to M. dolomieu. Additionally, egg hatching success and survival of LAN varied across nests within the study. These findings suggest that temperature alone may not account for variations in year-class strength and may emphasize the need for protection of the nest-guarding male Micropterus spp. to ensure recruitment.     

Detection of SJNNV and RGNNV genotypes using a relative quantification RT‐PCR assay

Viral Encephalopathy and Retinopathy (VER), is caused by a nodavirus included within the Betanodavirus genus of the Nodaviridae family. This disease affects more than 30 marine fish species worldwide and has been a major obstacle in the aquaculture industry; control of the disease is based on virus detection, essentially in carrier specimens. This study describes a real time PCR procedure for viral nervous necrosis virus detection from several organs of sea bass, Senegalese sole, and gilt‐head sea bream, from fish displaying either clinical symptoms or asymptomatic cases. The sensitivity of this technique was about 10⁶‐fold higher than that of the conventional RT‐PCR. The newly designed primers detected nodavirus isolates belonging to the RGNNV and SJNNV genotypes.     

Parental influence on early development in the European sea bass

Fertilization, survival during incubation and hatching were highly correlated and strongly influenced by the female parent and the interaction between both parents of the sea bass Dicentrarchus labrax. A single significant male effect was detected on hatching and may reflect a genetic component determining survival from fertilization to hatching. Egg size and total length of 8-day-old larvae (age of first feeding) were also subjected to the influence of the female but the two parameters were not correlated. A significant interaction between both parents was also observed for length of larvae. The survival rates were significantly correlated in two consecutive years indicating that reproductive performances may be repeatable. Although the effect of the male parent on early sea bass development is shown for the first time, it seems that most of the parental effects on early performance in this species are due to females even when hormonal stimulations are performed at very close prematurational stages of oocyte development.     

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.     

Evaluation of candidate probiotic strains for gilthead sea bream larvae (Sparus aurata) using an in vivo approach

AIMS: The aim of this study was to evaluate the effect of six bacterial strains on gilthead sea bream larvae (Sparus aurata). METHODS AND RESULTS: Six bacterial strains isolated from well-performing live food cultures were identified by sequencing fragments of their 16s rDNA genome to the genus level as Cytophaga sp., Roseobacter sp., Ruergeria sp., Paracoccus sp., Aeromonas sp. and Shewanella sp. Survival rates of gilthead sea bream larvae transferred to seawater added these bacterial strains at concentrations of 6 +/- 0.3 x 10(5) bacteria ml(-1) were similar to those of larvae transferred to sterilized seawater and showed an average of 86% at 9 days after hatching, whereas, survival rates of larvae transferred to filtered seawater were lower (P < 0.05), and showed an average of 39%, 9 days after hatching. CONCLUSION: Several bacterial strains isolated from well-performing live food cultures showed a positive effect for sea bream larvae when compared with filtered seawater. SIGNIFICANCE AND IMPACT OF THE STUDY: The approach used in this study could be applied as an in vivo evaluation method of candidate probiotic strains used in the rearing of marine fish larvae.     

Influence of diet on pepsin and some pancreatic enzymes in sea bass (Dicentrarchus labrax) larvae

Sea bass (Dicentrarchus labrax) larvae were weaned on day 25 with a basal compound diet as microparticles, enriched with a mixture of amino acid (SLAA) or protein (SLP). The control group was fed live prey (LP). Larval growth was significantly lower in the weaned groups. Trypsin activity was enhanced by the amino acid mixture. Pepsin was detected from day 24. Pepsin specific activity was significantly higher in the weaned groups, suggesting that the presence of pepsin is not such a determinant factor in the digestive process of sea bass larvae, since a poor growth was observed in these groups.     

Spawning response of mature female sea bass,Lates calcarifer (Bloch), to a single injection of luteinizing hormone-releasing hormone analogue: effect of dose and initial oocyte size1

The effect of various doses of luteinizing hormone-releasing hormone analogue (LHRHa) ranging from 1 to 100 μg/kg body weight on the spawning response of mature female sea bass, Lates calcarifer (Bloch) was tested. A single intramuscular injection of LHRHa resulted in a dose-related increase in the spawning rate (number of spawnings of each fish over four consecutive days) of mature fish. An LHRHa dose of 5 μg/kg and less induced low spawning rates of 16.7% to 37.5% or at least one spawning every four days. However, mature sea bass spawned more than once (43.8–58.3%) in four days at dose levels of 10 μg/kg and above. Hormone treatment within the dose range tested did not influence the number, fertilization and hatching rates of spawned eggs. The influence of initial oocyte size on the LHRHa-induced spawning response of mature sea bass was also examined. Sea bass with an initial oocyte diameter of 0.30–0.39 mm did not respond to the single injection of 100 μg LHRHa/kg. In contrast, LHRHa induced spawning among sea bass with an initial egg size of 0.40–0.49 mm, although two of four sea bass of the same stage of ovarian maturity spawned spontaneously. Fish having an initial oocyte size of 0.50–0.55 mm spawned with and without LHRHa treatment. Spontaneous spawning among saline-injected sea bass occurred at a later time (24–58 h post-injection) compared to fish induced to spawn by a single injection of LHRHa (8–36 h post-injection). The initial spawning response time interval for fish with an initial egg size of 0.50 mm or greater was further reduced to 8–9 h by LHRHa. These results indicate that LHRHa can successfully induce spawning in mature female sea bass which have attained a critical oocyte diameter and that the spawning response interval is reduced with a further increase in egg size beyond the critical oocyte diameter limit.     

Culture performance of different strains and crosses of the European sea bass (Dicentrarchus labrax) reared under controlled conditions at Eilat, Israel

The European sea bass (Dicentrarchus labrax) is one of the most important commercial marine fish species in the Mediterranean basin. However, broodstock domestication and selective breeding on a production scale have yet to be exploited. We reared progeny of sea bass from three different strains and their crosses throughout the entire growth cycle to market size. Data sets were analyzed to assess whether culture performance differs among different strains and crosses reared under controlled laboratory conditions using both separate and communal rearing techniques. Strains and crosses of sea bass varied significantly for traits of economic interest such as growth, survival, body composition, sex ratio, sexual maturation patterns of males, and frequencies of body shape abnormalities. Lack of evidence for substantial heterosis for growth among reciprocal crosses was also detected. Significant sexual dimorphism for length and weight was evident in all strains and crosses. At market size, the resulting weight advantages of females varied between strains and crosses; overall average weight advantage for females was about 39%. Length and weight of individual fish were strongly correlated during different time intervals. This may be useful for the choice of selection criteria, raising the possibility that the response to selection for weight at market time may be achieved by conducting selection on young fish. Generally, the results showed a high culture potential of sea bass strains originating from the southeastern areas of the Mediterranean Sea, suggesting that these domesticated strains can be exploited in future selective breeding programs.     

Carriage of potentially fish-pathogenic bacteria in Sparus aurata cultured in Mediterranean fish farms

A bacteriological survey of gilthead sea bream Sparus aurata from different fish farms and culture systems on the Spanish Mediterranean coast was conducted. Three different studies were performed. Study A included hatchery-reared larvae; Study B, periodic examination of randomly sampled growing fish; and Study C, growing fish sampled only during mortality/morbidity events. In Studies B and C, sea cages, earth ponds and indoor tanks were surveyed, and in both cases diseased (showing clinical signs) and non-diseased fish were included. In Study A, a shift from Vibrio spp. (30 d after hatching) to oxidative species (60 d after hatching) was detected, and no mortality events were registered. The percentage of fish yielding bacterial growth were similar in Studies B and C, reaching 57.4 and 61.3%, respectively. A statistically significant relationship between the bacterial carriage and the type of facility was only found in Study B, showing that fish from sea cages had a higher bacterial occurrence than fish from other facilities. A statistically significant relationship between bacterial carriage and signs of disease was found, although the pattern differed in each study. Thus, in Study B only 36.2% of fish yielding abundant bacterial growth were diseased, versus 68.0% in Study C. In total, 25.0% of the fish examined were diseased. Bacterial species composition was similar in asymptomatic and diseased fish, except for a group of V. ichthyoenteri-like isolates that occurred almost exclusively in asymptomatic fish. Dominant bacterial species were V. harveyi and V. splendidus, followed by V. ichthyoenteri-like isolates, Photobacterium damselae ssp. damselae and V. fisheri. Non-fermenters were less frequent but, among them, unidentified halophilic Cytophaga-Flavobacterium isolates and Pseudoalteromonas haloplanktis were the most abundant. An association of individual species with disease was not clear, which suggests the involvement of mixed infections.