Selected nondigestible carbohydrates and prebiotics support the growth of probiotic fish bacteria mono-cultures in vitro.

AIMS: To search for nondigestible but fermentable (NDF) carbohydrates and prebiotics with a potency to promote the growth of selected bacteria in vitro. METHODS AND RESULTS: The growth of three reference bacteria strains Bacillus subtilis LMG 7135(T), Carnobacterium piscicola LMG 9839, Lactobacillus plantarum LMG 9211 and one candidate probiotic bacteria Lactobacillus delbrueckii subsp. lactis was investigated over a minimum period of 48 h in the presence of beta-glucan, xylo-oligosaccharide, arabinoxylo-oligosaccharide, inulin, oligofructose and glucose. Besides the capability to grow on inulin and oligofructose containing media, a distinct high growth in beta-glucan based substrates and a low growth in (arabino)xylooligosaccharide containing media were evident for most bacteria tested. With the exception of B. subtilis and L. plantarum, other bacteria grew equally well or even better on different substrates than on glucose. The fermentation of studied carbohydrates by these micro-organisms was dominated by the production of acetic acid as the main short chain fatty acid. CONCLUSIONS: Selected bacteria are able to ferment and grow on NDF and prebiotic carbohydrates but in a substrate dependent manner. SIGNIFICANCE AND IMPACT OF THE STUDY: This study delivers a first screening of which NDF or prebiotic carbohydrates are the most promising for aquaculture feed supplementations.

Bioenergetics of fish spermatozoa during semen storage.

This mini-review focuses on changes in ATP and creatine phosphate concentrations in fish sperm under storage conditions. The storage of catfish sperm at 4 degrees C leads to ATP depletion and decreased sperm motility. The rate of intracellular ATP depletion can be diminished through the addition of energetic substrates to the sperm storage medium, with lactate + pyruvate being the most efficient substrates for maintaining ATP concentrations in catfish sperm. The decrease in ATP concentration is closely associated with increases in AMP and hypoxanthine content. In contrast to catfish sperm, carp sperm is able to maintain intracellular ATP concentration close to the physiological level during storage. Collectively, these results suggest that fish species differ in terms of the energy metabolism of their spermatozoa and that the semen storage medium must be carefully selected for a particular fish species so as to maintain the ATP concentration and adenylate energy charge close to physiological values as long as possible.

Morphological and morphometric study of crustacean parasites within the genus Lernaeocera.

Two species of Lernaeocera are present in the southeastern North Sea. Lernaeocera lusci infects bib Trisopterus luscus, dragonet Callionymus lyra and sand goby Pomatoschistus minutas. L. minuta is a junior synonym of L. lusci. The second valid species, L. branchialis, infects whiting Merlangius merlangus. The two species can be morphologically separated by the antennary processes, which are present in L. lusci and absent in L. branchialis. Discriminant functions allow complete separation between L. lusci and L. branchialis. There is high intraspecific, host-dependent variability within L. lusci. Length of L. lusci is significantly influenced by host size, and body form is influenced by the site of attachment of L. lusci on at least one host (bib). It is suggested that L. lusci consists of 3 forms: f. lusci, f. minuta and f. lyra.

Effect of route and frequency of administration of apomorphine on growth hormone release in African catfish (Clarias gariepinus).

Apomorphine is known to stimulate growth hormone release in African catfish following an intraperitoneal (IP) injection. In the present study the effect of apomorphine (5 or 20 mg/kg body weight) on plasma GH levels was evaluated after gastro-intestinal or parenteral delivery. Apomorphine increased the plasma GH concentration regardless of the route of administration, indicating that apomorphine can be absorbed from the intestinal tract. The effect of repeated administration of apomorphine differed clearly between the tested doses. Although a single IP injection with 20 mg apomorphine/kg body weight resulted in a clear increase in plasma GH levels, a second injection given 12 hours later was ineffective. In contrast the last of 4 consecutive injections with 5 mg apomorphine/kg body weight given at intervals of 12 hours stimulated the plasma GH levels in a similar way to a single IP injection with the same dose.

A presumptive role for GABA in the stimulatory effects of Des-Gly10, [D-Ala6]-LHRH-ethylamide and pimozide on the gonadotropin release in carp.

To investigate the effect of endogenous gamma-aminobutyric acid (GABA) on the blood maturating gonadotropin (GtH) levels, or to study its interaction with pimozide (dopamine antagonist) and a luteinizing hormone-releasing hormone analog (LHRH-a), sexually mature male and female carps were treated with drugs that may either inhibit GABA biosynthesis or GABA degradation. In females the irreversible inhibitor of GABA-transaminase, gamma-vinyl GABA (GVG), which was to increase the endogenous GABA-ergic tone, had no influence on GtH release. On the other hand, the increased GtH response to the combination of pimozide (PIM) and LHRH-a was clearly enhanced by the administration of 3-mercaptopropionic acid (MPA), an inhibitor of the rate limiting enzyme of GABA-biosynthesis. In males the GABA-ergic compound, valproic acid (DPA) decreased LHRH-a stimulated GtH levels. In male carps that received PIM to diminish the dopaminergic inhibition of GtH release, the spermiating response to LHRH-a was increased by administration of MPA. These data suggest that GABA interacts with the action of dopamine and the gonadotropin releasing hormone (GnRH) on the release of GtH.

One-step immunoaffinity purification and partial characterization of hypophyseal growth hormone from the African catfish, Clarias gariepinus (Burchell).

Growth hormone (GH) was purified from African catfish (Clarias gariepinus) pituitary extracts in a single step by use of immunoaffinity chromatography. A monoclonal antibody to chicken GH, which labels the catfish hypophyseal somatotropes in immunocytochemistry, was coupled to CNBr-activated Sepharose, and crude alkaline pituitary extracts were run over the immunoadsorbent. Reversed-phase high-performance liquid chromatography analysis of the eluted material suggested heterogeneity, whereas silver staining upon SDS-polyacrylamide gel electrophoresis showed one single band with an estimated molecular weight between 22,000 and 23,000 Da. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the same preparation revealed the presence of several components with molecular weights ranging from 20,170 to 20,900 Da. The amino terminus of the protein was homogeneous, and the first 50 residues matched the proposed sequence of GH from two other siluran species (Ictalurus punctatus and Pangasius pangasius), except for one substitution at position 3. These data unequivocally confirm the identity of the purified molecule as suggested by immunochemical evidence. The bioactivity of the GH preparation was demonstrated by the short-term effect of GH on T3 plasma levels in juvenile catfish.

Exposure of Chironomus riparius larvae to 17alpha-ethynylestradiol: effects on survival and mouthpart deformities.

Evidence from field studies shows that mouthpart deformities in chironomid larvae are a sublethal response to pollution. Interest has been shown to use this end-point in programs for monitoring sediment quality. During laboratory studies, however, deformities were induced in only a few single pollutant exposures. These deformities develop at the endocrine regulated molting stage and disruption of this complex process is likely at the base of their ontogeny. Aiming to clarify the processes involved in the rise of such deformities, we tested the effects of ethynylestradiol (EE2) in an in vivo lab study. Chironomus riparius larvae were exposed to 1, 10 and 100 microg l(-1) EE2 (nominal concentrations). No adverse effects on the larvae, for the investigated end-points (survival and deformity induction), were found.

Induction of mouthpart deformities in Chironomus riparius larvae exposed to 4-n-nonylphenol.

Chironomid mouthpart deformities have often been associated with sediment contamination and are, therefore, currently used to assess sediment quality. Deformities were only occasionally induced in laboratory bioassays. Mouthpart deformities results from a physiological disturbance during larval molting. In the past few years it has been shown that some chemicals can exert negative effects on both vertebrates and invertebrates at the level of endocrine regulation. As insect molting is hormonally regulated, we wanted to test the hypothesis that deformities are induced due to a hormonal disruption in the developmental process. The aim of the present study was to test whether the endocrine disrupter, 4-n-nonylphenol (4NP), induces mouthpart deformities in chironomids. A laboratory bioassay was performed exposing Chironomus riparius larvae to 10, 50 and 100 micrograms l-1 4NP. Survival of the larvae was not affected by the tested concentrations, but the frequency of mentum deformities increased significantly (P < 0.01) after exposure to 4NP.

Exposure of Chironomus riparius larvae (diptera) to lead, mercury and beta-sitosterol: effects on mouthpart deformation and moulting.

Mouthpart deformation in chironomid larvae is induced by exposure to chemical contaminants and is becoming an established bio-indicator in sediment assessment programmes. However, concentration-response relationships with causal agents have only been established occasionally and with varying success. In this laboratory study, instar II and III larvae were exposed to sub-lethal concentrations of lead, mercury and beta-sitosterol. A significant deformation response was induced in the pecten with lead and mercury. Deformation frequencies of the mentum after metal exposures were not significantly different from the control. Moulting was retarded by both metals and was well correlated with mouthpart deformation. The beta-sitosterol is an endocrine disruptor, which was used to test the hypothetical cause-effect relation between disruption of ecdyson functioning and chironomid deformation. In the present study, exposure to sublethal concentrations of beta-sitosterol did not result in any effect on deformation or moulting. As such, the proposed hypothesis of endocrine disruptors as primary causal agents of chironomid deformation could not be substantiated. Acetone, which was used as a solvent to apply beta-sitosterol caused a significant increase of mentum deformation. The ground filtration paper used as substrate seemed to induce deformities as well. Substrate contamination, acetone and (especially) inbreeding were most probably responsible for the high deformation frequencies in the control conditions.

Quality control of refrigerated and cryopreserved semen using computer-assisted sperm analysis (CASA), viable staining and standardized fertilization in African catfish (Clarias gariepinus).

A new integrated approach including computer-assisted sperm analysis (CASA), viability staining and fertilization was used to study the quality of cryodiluents used in fish sperm cryopreservation. As an example the sperm quality of an African catfish, Clarias gariepinus (Burchell, 1822), was assessed by its fertilizing ability, motility and viability at day 0 (fresh), after 2 days' storage at 4 degreesC and after 2 days, 5 months and 10 months frozen at -196 degreesC using solutions containing dimethyl sulphoxide (DMSO) or glycerol as permeating cryoprotectants. Four of the best freezing solutions were used, namely, Steyn's extender (S1, S4) and Mounib's extender (M3, M4) associating 10% hen's egg yolk. Progressive sperm movement measured by CASA and expressed by the straight line velocity (VSL), the average path velocity (VAP) and the curvilinear velocity (VCL) was highly correlated with hatching rates obtained from fertilization using minimal sperm:egg ratios. After 2 days, the motility of spermatozoa frozen with DMSO and 10% egg yolk had deteriorated less than that of spermatozoa stored at 4 degreesC. Post-thaw hatching rates reflected the post-thaw sperm viability, which was cryodiluent dependent: 14.9+/-2.0% (S4), 17.0+/-4.2% (S1), 25.9+/-3.7% (M4) and 52.1+/-3.4% (M3) after 5 months of cryopreservation. The percent motility of 10-months-frozen spermatozoa was high in M3 (70.7+/-11.4%) and M4 (64.0+/-2.0%) cryoprotected sperm when measured between 5 and 20 sec after activation, but decreased rapidly to 24.3+/-8.3% (M3) and 23.0+/-9.0% (M4) between 21 and 35 sec after activation. Mounib's extender (M3, M4) provided the best cryoprotection to the spermatozoa for all post-thaw sperm quality measurements and at all freezing durations. Sperm motility was positively related to fertility. Our method will make it possible to develop even better extenders and cryoprotectants.

Experimental Induction of Morphological Deformities in Chironomus riparius Larvae by Chronic Exposure to Copper and Lead

Five consecutive generations of Chironomus riparius Meigen larvae were chronically exposed from egg to fourth instar to four sublethal concentrations of copper (0, 1, 10, 100 µg L-1) and lead (0, 5, 50, 500 µg L-1) in artificially spiked water (static with renewal), with diatomaceous earth as substrate and tetraphyl(R) as food, in order to test the induction of morphological deformities by these metals. The use of diatomaceous earth was suboptimal because it caused high mortalities (>60%), independent of metal stress. The higher copper concentrations had a positive effect on the survivals relative to the control. Split medial mentum teeth were recorded in more than 10% of the larvae, but could not be related to metal stress. Deformities of the mentum and the mandibles were recorded in second, third, and fourth instars exposed to both metals. Concentration and generation effects were noted for unusual number of mentum teeth (0-5.3%, lead), unusual number of mandible inner teeth (0-10.4%, copper and lead), and small open mentum gap (0-6.5%, copper). These experiments demonstrated the potential of both an essential and a non-essential metal to induce weak deformities in a small proportion of a C. riparius population as well as the induction of deformities which are independent of metal stress or fluctuating over the generations. The study shows the potential of midge deformities as a biomonitoring tool, but at the same time warns for a careful interpretation of deformity scores because of the influence of population dynamics on the final outcome of deformity frequencies and of the existence of deformities not related to pollution.

PCR as a test for the presence or absence of Legionella in (cooling) water.

Legionella pneumophila, a Gram-negative bacterium, is the causative agent of legionellosis. Traditionally, culture methods are normally used to detect Legionella species in different types of water (e.g. surface or tap water, circulating systems, air conditioners and their cooling devices). In this study the PCR conditions to detect Legionella were optimised based on the EnviroAmp Legionella kit (Perkin-Elmer) which is no longer commercially available. The PCR is very sensitive and specific in indicating the presence or absence (no quantification with classical PCR) of Legionella spp in general and more specifically L. pneumophila. To identify L. pneumophila. DNA sequences from the mip (macrophage infectivity potentiator) gene were amplified. The mip gene is conserved and specific for L. pneumophila although mip-like genes are also present in other Legionella spp. The PCR techniques were able to detect small amounts of Legionella in tap water samples. Cooling water, however, often contained PCR-inhibiting substances that could result in false negative PCR results for Legionella.

A detection method for Legionella spp in (cooling) water: fluorescent in situ hybridisation (FISH) on whole bacteria.

Although traditional culture methods are appropriate for detection of Legionella species, such culture takes several days. Rapid detection (< 24 h) of individual Legionella is possible using fluorescent in situ hybridisation (FISH) on whole bacteria. Water samples were filtered and the concentrated bacteria were immediately detected (without culture) with a fluorescence microscope following appropriate labelling. The detection level was very high and quantification was possible. For the detection of all Legionella spp. the probe LEG705 was used, complementary to a 16S rRNA sequence conserved in all Legionella spp. For specific detection of L. pneumophila the probe LEGPNE1 was used. This probe is designed against a variable domain of the 16S rRNA sequence from L. pneumophila. CY3 and FLUOS labels were tested and CY3 showed clearly detectable bacteria with minimum background staining. This FISH technique is very sensitive, fast, reliable and individual bacteria are easily detected.

Detection of Naegleria spp. and Naegleria fowleri: a comparison of flagellation tests, ELISA and PCR.

To detect Naegleria spp, in particular Naegleria fowleri, the causative agent of human primary amoebic meningoencephalitis, a flagellation test (FT) is routinely used followed by a specific ELISA. A positive FT indicates the presence of Naegleria spp although some false negatives are likely to occur since parameters for enflagellation vary greatly. As negative FTs are not routinely screened any further for the presence of N. fowleri, this could result in an underestimation of the presence of this pathogen. Therefore, amoebae were further analysed using ELISA and standard PCR not only after a positive but also after a negative FT. In this study 39 cultures containing amoebae were tested with FT, ELISA and the two PCR assays with 11 positive for FT. These were submitted to ELISA and four confirmed as N. fowleri. PCR with the common primer-set on these 11 positive FTs revealed all as Naegleria spp. The specific PCR used on these cultures detected four positive for N. fowleri, corresponding totally with the ELISA results. The 28 negative flagellation tests were also submitted to ELISA and PCR. Of these, 11 were identified as Naegleria spp with common PCR and six as N. fowleri as well as with ELISA and the specific PCR. When the detection of Naegleria spp is based on intermediary processes, such as flagellation tests, false negatives are likely to occur leading to severe underestimations. This study has shown that amoebae taken from negative FTs can be identified as Naegleria spp and N. fowleri when using PCR and ELISA. The application of at least one of the specific N. fowleri tests is recommended for routine screening. The heterogeneous distribution of the false negative results between the different power plants suggested the presence of different genotypes.

Effects of peracetic acid and monochloramine on the inactivation of Naegleria lovaniensis.

Biocidal activities of monochloramine and peracetic acid were studied on cysts of Naegleria lovaniensis. Until recently the most commonly used biocide to disinfect cooling water systems was hypochlorite. Owing to its negative impact on the aquatic environment, ecologically less harmful alternatives have been sought. As the biocidal activity of monochloramine and peracetic acid makes them good candidates for inactivation of pathogenic Naegleria species, these biocides were tested against Naegleria lovaniensis, a relative of the pathogen Naegleria fowleri, as an alternative treatment to hypochlorite. Under laboratory conditions the biocidal activity of hypochlorite was 8- 10x stronger than that of the two investigated substances. Hypochlorite, at a concentration of 0.5 mg/L, killed 100% Naegleria lovaniensis after 1 h exposure (25 degrees C, pH 7.3- 7.4). To achieve similar results with monochloramine and peracetic acid, 3.94 mg/L or 5.33 mg/L had to be used respectively (25 degrees C, pH 8). It was known that the in situ biota of the biofilm, along with any organic material in the water column, had a negative impact on the efficiency of the biocides. There are, however, indications that the relative efficacy of monochloramine and peracetic acid was quite good under such conditions when compared with hypochlorite.

Receptor-mediated uptake of Legionella pneumophila by Acanthamoeba castellanii and Naegleria lovaniensis.

AIMS: Investigation of the attachment and uptake of Legionella pneumophila by Acanthamoeba castellanii and Naegleria lovaniensis, as these are two critical steps in the subsequent bacterial survival in both amoeba hosts. METHODS AND RESULTS: Initially, the mode of Legionella uptake was examined using inhibitors of microfilament-dependent and receptor-mediated uptake phagocytosis. Secondly, the minimum saccharide structure to interfere with L. pneumophila uptake was determined by means of selected saccharides. Bacterial attachment and uptake by each of the amoeba species occurred through a receptor-mediated endocytosis, which required de novo synthesis of host proteins. Legionella pneumophila showed a high affinity to the alpha1-3D-mannobiose domain of the mannose-binding receptor located on A. castellanii. In contrast, L. pneumophila bacteria had a high affinity for the GalNAcbeta1-4Gal domain of the N-acetyl-D-galactosamine receptor of N. lovaniensis. CONCLUSIONS: Our data pointed to a remarkable adaptation of L. pneumophila to invade different amoeba hosts, as the uptake by both amoeba species is mediated by two different receptor families. SIGNIFICANCE AND IMPACT OF THE STUDY: The fact that L. pneumophila is taken up by two different amoeba species using different receptor families adds further complexity to the host-parasite interaction process, as 14 amoeba species are known to be appropriate Legionella hosts.

Gnotobiotically grown aquatic animals: opportunities to investigate host-microbe interactions.

The culture of aquatic organisms is still hampered by the occurrence of unpredictable diseases in their early life stages, which are responsible for massive mortalities and considerable economic losses. A better understanding of the host-microbe interactions is certainly essential to develop effective solutions of disease control for the aquaculture industry. As demonstrated in terrestrial animals, the use of gnotobiotic systems (animals cultured in axenic conditions or with a known microflora) can be an excellent tool to extent the understanding of the mechanisms involved in host-microbe interactions and to evaluate new treatments of disease control. Several aquatic animals were cultured so far in germ-free conditions, such as fish, molluscs, crustaceans, rotifers, echinoderms, cnidarians, turbellarians, ascidians and echiurans. The aim of the present review is to recapitulate the findings obtained with gnotobiotic aquatic animals over the last decades, with special emphasis to the host-microbe interactions, as well as the perspectives for future research in this field. In addition, the procedures utilized to culture axenic aquatic animals and to verify contaminations are summarized, and the standardization of these procedures is proposed.

Impact of non-Legionella bacteria on the uptake and intracellular replication of Legionella pneumophila in Acanthamoeba castellanii and Naegleria lovaniensis.

In aquatic environments, Legionella pneumophila survives, in association with other bacteria, within biofilms by multiplying in free-living amoebae. The precise mechanisms underlying several aspects of the uptake and intracellular replication of L. pneumophila in amoebae, especially in the presence of other bacteria, remain unknown. In the present study, we examined the competitive effect of selected non-Legionella bacteria (Escherichia coli, Aeromonas hydrophila, Flavobacterium breve, and Pseudomonas aeruginosa) on the uptake of L. pneumophila serogroup 1 by the amoebae Acanthamoeba castellanii and Naegleria lovaniensis. We also investigated their possible influence on the intracellular replication of L. pneumophila in both amoeba species. Our results showed that the non-Legionella bacteria did not compete with L. pneumophila for uptake, suggesting that the amoeba hosts took in L. pneumophila through a specific and presumably highly efficient uptake mechanism. Living and heat-inactivated P. aeruginosa best supported the replication of L. pneumophila in N. lovaniensis and A. castellanii, respectively, whereas for both amoeba species, E. coli yielded the lowest number of replicated L. pneumophila. Furthermore, microscopic examination showed that 100% of the A. castellanii and only 2% of the N. lovaniensis population were infected with L. pneumophila at the end of the experiment. This study clearly shows the influence of some non-Legionella bacteria on the intracellular replication of L. pneumophila in A. castellanii and N. lovaniensis. It also demonstrates the different abilities of the two tested amoeba species to serve as a proper host for the replication and distribution of the human pathogen in man-made aquatic environments such as cooling towers, shower heads, and air conditioning systems with potential serious consequences for human health.

Comparative Serology of the Marine Fish Pathogen Vibrio anguillarum.

The different serotyping systems, based on thermostable O antigens, reported for Vibrio anguillarum and V. ordalii were compared by quantitative agglutination, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subsequent silver staining or Western blotting (immunoblotting) of purified lipopolysaccharide (LPS), using polyclonal rabbit antisera. The results demonstrate that 16 different serotypes within V. anguillarum (designated O1 to O16) can be distinguished. Each of these serotypes is characterized by a distinct polysaccharide banding pattern, as revealed by silver-stained gels of purified LPS. The comparative analysis allowed a complete alignment of the different serotypes for the first three serovars: O1, O2, and O3. Moreover, immunoblotting showed that strains belonging to each of these serotypes had the same LPS banding pattern independent of the origin of the strain. Serotype O2 contains different subtypes, O2a and O2b. While no differences were apparent between these subgroups in silver-stained gels, they could be separated by quantitative agglutination (titer determination) or immunoblotting. V. ordalii, the former biotype II of V. anguillarum, strongly reacts with anti-V. anguillarum O2a antiserum. Strains of the two species can be separated on the basis of different LPS profiles in the high-molecular-weight region of silver-stained gels of purified LPS. The silver-stained LPS profiles of the different serotypes of V. anguillarum that have been established are provided for further comparison in the future.

Ectoparasites of the whitespotted rabbitfish, Siganus sutor (Valenciennes, 1835) off the Kenyan Coast: distribution within the host population and site selection on the gills.

Different populations of the whitespotted rabbitfish, Siganus sutor, were examined for ectoparasites: adults from the Mombasa area (sampled in December 1990) and different age classes (adult, subadult and juveniles) from Gazi Bay (sampled in December 1992 and August 1993). The most common gill parasites were: the monogeneans Pseudohaliotrema sp., Tetrancistrum sigani and Microcotyle mouwoi, the copepods Hatschekia sp., Pseudolepeophtheirus sp. and juvenile Caligidae, and prazina larvae of the isopod Gnathia sp. Adult siganids had a higher parasite load than subadults. Juvenile rabbitfish did not harbour any gill parasites. Temporal differences in the parasite load of subadult rabbitfish were observed for M. mouwoi (highest in the December samples) and for juvenile Caligidae (highest in August). The microhabitat of the 5 most common gill parasites was species specific. Most parasite species showed distinct site preferences with respect to both gill arches and gill sectors, within the gill arches. Niche breadth of the different gill parasite species was independent of the abundance of any of the other species present. However, niche breadths of M. mouwoi, Tetrancistrum sp. and Hatschekia sp. increased with their own abundance. This suggests that interspecific competition for space is low and that intraspecific factors could play an important role in the microhabitat choice of these gill parasites. The hypothesis that niche restriction leads to higher intraspecific contact and an enhancement of chances to mate was tested on 2 monogenean species, Pseudohaliotrema sp. and Tetrancistrum sigani. Their highly aggregated distribution over the gill filaments, leading to increased intraspecific contact, is consistent with the hypothesis.