Protocol for quantitative shape analysis of deformities in early larval European seabass Dicentrarchus labrax.

This study established an optimized protocol for quantifying the shape variation of newly hatched larvae of European seabass Dicentrarchus labrax, focusing on the effect of fixatives and mounting on body shape from hatching until 14 days post hatching, while also minimizing the error introduced by handling. This assessment was performed based on both biometric and geometric shape data, with the latter relying on outline based elliptic Fourier analysis. The fixation and mounting effect on the total length and shape of newly hatched larvae of D. labrax was tested for four fixation treatments: (1) 8% formalin, (2) 70% ethanol, (3) 8% formalin for 48 h and then to 70% ethanol and (4) 3% phosphate-buffered glutaraldehyde. The analyses showed that no significant size and shape effect was observed on anaesthetized specimens 5 months post-fixation in glutaraldehyde, and that the glycerol mounting process of specimens fixed in glutaraldehyde provided the best results for further ontogenetic qualitative and quantitative analysis. The protocol proved to be sufficiently sensitive to even quantify and visualize subtle pre-fixation shape differences originating from a different egg batch.

Luminal uptake of Vibrio (Listonella) anguillarum by shed enterocytes--a novel early defence strategy in larval fish.

As adhesion and translocation through fish gut enterocytes of the pathogen Vibrio (Listonella) anguillarum are not well investigated, the effective cause of disease and mortality outbreaks in larval sea bass, Dicentrarchus labrax, suffering from vibriosis is unknown. We detected V. anguillarum within the gut of experimentally infected gnotobiotic sea bass larvae using transmission electron microscopy and immunogold labelling. Intact bacteria were observed in close contact with the apical brush border in the gut lumen. Enterocytes contained lysosomes positive for protein A-gold particles suggesting intracellular elimination of bacterial fragments. Shed intestinal cells were regularly visualized in the gut lumen in late stages of exposure. Some of the luminal cells showed invagination and putative engulfment of bacterial structures by pseudopod-like formations. The engulfed structures were positive for protein A-colloidal gold indicating that these structures were V. anguillarum. Immunogold positive thread-like structures secreted by V. anguillarum suggested the presence of outer membrane vesicles (MVs) hypothesizing that MVs are potent transporters of active virulence factors to sea bass gut cells suggestive for a substantial role in biofilm formation and pathogenesis. We put forward the hypothesis that MVs are important in the pathogenesis of V. anguillarum in sea bass larvae.

Bacterial host interaction of GFP-labelled Vibrio anguillarum HI-610 with gnotobiotic sea bass, Dicentrarchus labrax (L.), larvae.

The location and cell damage caused by Vibrio anguillarum, the causative agent of classical vibriosis, within the developing gut of the newly hatched sea bass, Dicentrarchus labrax (L.), is unknown. A gnotobiotic sea bass model was used to investigate the early interactions of V. anguillarum with sea bass larvae. In the present study, germ-free sea bass larvae were orally exposed to a V. anguillarum HI-610 pathogen labelled with the green fluorescent protein (GFP-HI-610) and sampled at regular intervals. Pathogenic colonization of gut enterocytes was observed 2 h post-exposure (p.e.) and onwards, whereas bacteria within the swim bladder were visualized 48 h p.e and onwards. Ultrastructural findings demonstrated direct bacterial contact with the host cell in the oesophageal mucosa and putative attachment to microvilli of mid- and hindgut enterocytes. The present findings form a starting point for studies assessing the impact of potential candidates (probiotics, prebiotics, antimicrobial peptides) to mitigate bacterial virulence.

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.

Chronic toxicity of dietary copper to Daphnia magna.

There is a growing concern that dietborne metal toxicity might be important in aquatic ecosystems. However, the science behind this matter is insufficiently developed to explicitly and accurately account for this in metal regulation or risk assessment. We investigated the effects of a chronic exposure of Daphnia magna to an elevated level of Cu (3000 microg Cu/g dry wt) in their diet (the green alga Pseudokirchneriella subcapitata). Compared to daphnids fed with P. subcapitata containing a background of 10.6 microg Cu/g dry wt, daphnids fed for 21 days with this Cu-contaminated food accumulated a total copper body burden of 325 microg Cu/g dry wt, which is about 30-fold higher than the control body burden of 12.1 microg/g dry wt. The exposed daphnids experienced a 38% reduction of growth (measured as final dry body weight), a 50% reduction of reproduction (total number of juveniles produced per daphnid), and only produced three broods versus four broods by the control daphnids. Unlike most other studies, we were able to demonstrate that these effects were most likely not due to a reduced nutritional quality of the food, based on C:P ratios and fatty acid content and composition of the Cu-contaminated algae. Life-history analysis showed that time to first brood was not affected by dietary Cu, while the second and third broods were significantly delayed by 0.7 and 1.5 days, respectively. On the other hand, brood sizes of all three broods were significantly lower in Cu exposed daphnids, i.e. by 32-55%. The variety of effects observed suggest the possible, and perhaps simultaneous, involvement of several toxicity mechanisms such as increased metabolic cost, reduced energy acquisition (potentially via inhibition of digestive enzyme activity), targeted inhibition of reproduction (potentially via inhibition of vitellogenesis), and/or direct inhibition of molting. Further research is needed to differentiate between these postulated mechanisms of dietary Cu toxicity and to determine whether they act separately or in concert.

Toxicological evaluation of a bioadhesive nasal powder containing a starch and Carbopol 974 P on rabbit nasal mucosa and slug mucosa.

The purpose of this study is the investigation of possible adverse effects of a powder formulation containing drum-dried waxy maize (DDWM) starch and Carbopol 974 P (90/10) on the nasal mucosa of rabbits and the foot mucosa of slugs after multiple administrations. In the rabbit, the effect of the formulation was measured by the release of proteins and lactate dehydrogenase (LDH) from the nasal mucosa with a new non-invasive in vivo method and also by histopathology. The mucosal toxicity of the formulation was evaluated using slugs by measuring the effect on the body weight and the amount of mucus produced during a repeated contact period. Additionally, the release of proteins, lactate dehydrogenase and alkaline phosphatase from the body wall of the slugs after a repeated treatment was measured. Twenty four hours after the powder administration to the rabbits the release of the marker molecules was comparable with the negative controls. The histopathological study showed only a slight increase of granulocytes in the epithelium. The formulation induced a higher mucus production in the slugs but no additional effects were detected on the body weight and on the release of proteins. No enzymes were released from the body wall. The results indicate that the effect of the bioadhesive powder consisting of DDWM/Carbopol 974 P (90:10, w/w) on the mucosa was negligible.

Interference with the quorum sensing systems in a Vibrio harveyi strain alters the growth rate of gnotobiotically cultured rotifer Brachionus plicatilis.

AIMS: To evaluate the effect of Vibrio harveyi strains on the growth rate of the gnotobiotically cultured rotifer Brachionus plicatilis, and to establish whether quorum sensing is involved in the observed phenomena. METHODS AND RESULTS: Gnotobiotic B. plicatilis sensu strictu, obtained by hatching glutaraldehyde-treated amictic eggs, were used as test organisms. Challenge tests were performed with 11 V. harveyi strains and different quorum sensing mutants derived from the V. harveyi BB120 strain. Brominated furanone [(5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone] as a quorum sensing inhibitor was tested in Brachionus challenge tests. Some V. harveyi strains, such as strain BB120, had a significantly negative effect on the Brachionus growth rate. In the challenge test with MM77, an isogenic strain of BB120 in which the two autoinducers (HAI-1 and AI-2) are both inactivated, no negative effect was observed. The effect of single mutants was the same as that observed in the BB120 strain. This indicates that both systems are responsible for the growth-retarding (GR) effect of the BB120 strain towards Brachionus. Moreover, the addition of an exogenous source of HAI-1 or AI-2 could restore the GR effect in the HAI-1 and AI-2 nonproducing mutant MM77. The addition of brominated furanone at a concentration of 2.5 mg l(-1) could neutralize the GR effect of some strains such as BB120 and VH-014. CONCLUSIONS: Two quorum sensing systems in V. harveyi strain BB120 (namely HAI-1 and AI-2-mediated) are necessary for its GR effect on B. plicatilis. With some other V. harveyi strains, however, growth inhibition towards Brachionus does not seem to be related to quorum sensing. SIGNIFICANCE AND IMPACT OF THE STUDY: Interference with the quorum sensing system might help to counteract the GR effect of some V. harveyi strains on Brachionus. However, further studies are needed to demonstrate the positive effect of halogenated furanone in nongnotobiotic Brachionus cultures and eventually, in other segments of the aquaculture industry.

The mucosal toxicity of different benzalkonium chloride analogues evaluated with an alternative test using slugs.

PURPOSE: The objective of this study was to evaluate the mucosal toxicity of different benzalkonium chloride (BAC) analogues using slugs as the alternative test organism. METHODS: The effect of different BAC analogues on the mucosal tissue of slugs was determined from the protein, lactate dehydrogenase, and alkaline phosphatase released from the foot mucosa after treatment. Additionally, mucus production and reduction in body weight of the slugs were measured. The eye irritation potency of the molecules was evaluated with the Bovine Corneal Opacity and Permeability (BCOP) assay. The antimicrobial activity of the different BAC analogues was also assessed. RESULTS: All BAC analogues induced severe damage to the mucosal epithelium of the slugs, and the irritation increased with decreasing alkyl chain length: BAC-C16 < BAC-C14 < BAC-C12 approximately BAC-mix. A similar ranking was obtained with the BCOP assay for eye irritation. The relative order of activities among the three BAC analogues was the same, i.e., BAC-C14 > or = BAC-C16 > BAC-C12. The BAC-C14 exhibited higher activity than the BAC-mix. CONCLUSIONS: The toxicity and activity of BAC analogues depend on the alkyl chain length. The use of BAC-C14 as a conservative agent in pharmaceutical preparations instead of the BAC-mix should be considered.