Simon J. Davies

Hydroxyl radicals (•OH) are associated with titanium dioxide (TiO2) nanoparticle-induced cytotoxicity and oxidative DNA damage in fish cells

TiO(2) nanoparticles (< 100 nm diameter) have been reported to cause oxidative stress related effects, including inflammation, cytotoxicity and genomic instability, either alone or in the presence of UVA irradiation in mammalian studies. Despite the fact that the aquatic environment is often the ultimate recipient of all contaminants there is a paucity of data pertaining to the potential detrimental effects of nanoparticles on aquatic organisms. Therefore, these investigations aimed to evaluate the potential cytotoxic and genotoxic effects of TiO(2) nanoparticles on goldfish skin cells (GFSk-S1), either alone or in combination with UVA. Whilst neutral red retention (NRR) assay (a measure of lysosomal membrane integrity) was used to evaluate cell viability, a modified Comet assay using bacterial lesion-specific repair endonucleases (Endo-III, Fpg) was employed to specifically target oxidative DNA damage. Additionally, electron spin resonance (ESR) studies with different spin traps were carried out for qualitative analysis of free radical generation. For cell viability, TiO(2) alone (0.1-1000 microg ml(-1)) had little effect whereas co-exposure with UVA (0.5-2.0 kJm(-2)) caused a significant dose-dependent decrease which was dependent on both the concentration of TiO(2) and the dose of UVA administered. For the Comet assay, doses of 1, 10 and 100 microg ml(-1) in the absence of UVA caused elevated levels of Fpg-sensitive sites, indicating the oxidation of purine DNA bases (i.e. guanine) by TiO(2). UVA irradiation of TiO(2)-treated cells caused further increases in DNA damage. ESR studies revealed that the observed toxic effects of nanoparticulate TiO(2) were most likely due to hydroxyl radical (OH) formation.

Microbial manipulations to improve fish health and production--a Mediterranean perspective.

The interactions between the endogenous gut microbiota and the fish host are integral in mediating the development, maintenance and effective functionality of the intestinal mucosa and gut associated lymphoid tissues (GALTs). These microbial populations also provide a level of protection against pathogenic visitors to the gastrointestinal (GI) tract and aid host digestive function via the production of exogenous digestive enzymes and vitamins. Manipulation of these endogenous populations may provide an alternative method to antibiotics to control disease and promote health management. Applications of probiotics for Mediterranean teleosts can stimulate immune responses, enhance growth performance, feed utilisation, digestive enzyme activities, antioxidant enzyme activities, gene expression, disease resistance, larval survival, gut morphology, modulate GI microbiota and mediate stress responses. Although considerably less information is available regarding prebiotic applications for Mediterranean teleosts, prebiotics also offer benefits with regards to improving immune status and fish production. Despite the promising potential benefits demonstrated in current literature, obtaining consistent and reliable results is often difficult due to our incomplete understanding of indigenous fish GI microbiota and their subsequent host interactions which mediate and drive both localised and systemic host immunological responses. Additionally, the probiotic and prebiotic (biotics) mechanisms which mediate host benefits at the mucosal interface are poorly understood. Future studies focused on these interactions utilising gnotobiotic techniques should provide a better understanding of how to extract the full potential of biotic applications to promote immune function of Mediterranean teleosts.

Dietary mannan oligosaccharide supplementation modulates intestinal microbial ecology and improves gut morphology of rainbow trout, Oncorhynchus mykiss (Walbaum).

A study was conducted to investigate the effect of mannan oligosaccharide (MOS) on the gut microbiota and intestinal morphology of rainbow trout under commercial farming conditions. Juvenile (mean initial BW 38.2 +/- 1.7 g) and subadult (111.7 +/- 11.6 g) trout were fed 2 dietary treatments for 111 and 58 d, respectively. The control treatment consisted of a standard commercial diet, and the MOS treatment consisted of the control diet supplemented with 0.2% MOS. Morphology of the anterior and the posterior intestine was examined with light and electron microscopy. Light microscopy demonstrated increased gut absorptive surface area in the subadult MOS group. Additionally, electron microscopy revealed an increase in microvilli length and density in the subadult MOS group compared with the control (P < 0.05). However, no significant improvements were detected in the juvenile group. Culture-based evaluation of the intestinal microbiota showed that MOS significantly reduced (P < 0.05) the viable intestinal bacterial populations (by approximately 2 log scales in all cases). Levels of Aeromonas/Vibrio spp. were significantly decreased (P < 0.05) in the juvenile MOS group (9% of the total microbiota) compared with the juvenile control group (37%). Additionally, analysis of microbial communities was conducted using denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA. The denaturing gradient gel electrophoresis fingerprinting revealed an alteration of bacterial populations; analysis of similarity, similarity percentages, and nonmetric multidimensional scaling analysis showed that MOS reduced species richness and increased similarity of bacterial populations found within the subadult and juvenile groups. The current study shows that MOS modulates intestinal microbial communities, which subsequently improve gut morphology and epithelial brush border.

Soybean meal alters autochthonous microbial populations, microvilli morphology and compromises intestinal enterocyte integrity of rainbow trout, Oncorhynchus mykiss (Walbaum).

Abstract Rainbow trout were fed either a diet containing fishmeal (FM) as the crude protein source or a diet containing 50% replacement with soybean meal (SBM) for 16 weeks. An enteritis-like effect was observed in the SBM group; villi, enterocytes and microvilli were noticeably damaged compared with the FM group. The posterior intestine microvilli of SBM-fed fish were significantly shorter and the anterior intestine microvilli significantly less dense than the FM-fed fish. Electron microscopy confirmed the presence of autochthonous bacterial populations associated with microvilli of both fish groups. Reduced density of microvilli consequently led to increased exposure of enterocyte tight junctions, which combined with necrotic enterocytes is likely to diminish the protective barrier of the intestinal epithelium. No significant differences in total viable counts of culturable microbial populations were found between the groups in any of the intestinal regions. A total of 1500 isolates were tentatively placed into groups or genera, according to standard methods. Subsequent partial 16S rRNA sequencing revealed species that have not been identified from the rainbow trout intestine previously. Compared with the FM group levels of Psychrobacter spp. and yeast were considerably higher in the SBM group; a reduction of Aeromonas spp. was also observed.

The effect of Pediococcus acidilactici on the gut microbiota and immune status of on-growing red tilapia (Oreochromis niloticus)

Aim:  To assess Pediococcus acidilactici as a dietary supplement for on‐growing red tilapia (Oreochromis niloticus).

High inclusion levels of poultry meals and related byproducts in diets for gilthead seabream Sparus aurata L.

The feasibility of replacing fish meal protein at high levels of 75 and 100% with a high quality poultry meat meal was assessed in diets for gilthead seabream. A combined mixture of poultry meat meal and feather meal was also tested at high inclusion levels of 75 and 100%. Finally, two lower grade poultry by-product meals produced for the Greek industry were tested at various levels for comparison. The diets were isocaloric containing 18 MJ/kg of gross energy, isonitrogenous (CP:45%) and had a lipid content of 13% on an as fed basis. The experiment was carried out in a semiclosed rearing system and its duration was 84 days. The groups of fish fed 75 and 100% poultry meat meal showed a slight reduction in growth parameters compared to fish fed the control diet containing white fish meal but was not statistically significant (P<0.05). Similar results were obtained for the fish fed the poultry and feather mixture. Feed efficiency, protein and energy utilisation followed the same trends. One of the locally produced meals at substitution levels of up to 50% produced no significant reduction in growth of seabream (P < 0.05). At the level of 75% of the protein, however, the material caused a severe decrease in growth performance, feed efficiency, protein efficiency ratio and apparent net protein utilisation. Inferior quality was also demonstrated by the other poultry meal available on the Greek market. Protein digestibility coefficients were measured for all diets and essential amino acid indices and chemical score values were calculated.

Influence of supplementation of practical diets with vitamin C on growth and response to hypoxic stress of seabream, Sparus aurata

Abstract Gilthead seabream were fed a fish meal based diet, supplemented with graded amounts of ascorbyl polyphosphate equivalent to 0, 25, 50, 100 and 200 mg of l -ascorbate (AA)/kg, for 12 weeks. Although there were no significant differences between growth rates of each group, the feed gain ratio and voluntary feed intake were significantly lower, and the protein efficiency ratio was significantly higher for the fish fed 200 mg AA/kg, when compared with the group fed 0 mg AA/kg. An increase of the ascorbate concentration within the liver and spleen occurred with the increasing vitamin supplementation. After 12 weeks, the fish were subjected to hypoxia for 24 h to determine the influence of AA supplementation on the physiological response to this stressor. A significant hyperglycaemia occurred in fish fed all the diets 3 h after the onset of stress, although a significantly higher resting plasma glucose was observed in fish fed the AA free diet. No significant difference was found in plasma cortisol concentration with stress, with the exception of fish fed the 100 mg of AA/kg diet, where a significantly lower cortisol level was found after 9 h of hypoxia. Fish fed the non-supplemented diet showed wider variation and a tendency to increase this variable, having significantly higher levels at 9 h and 24 h of stress then all the other groups and than fish fed the 200 mg AA/kg diet, respectively. Stress had no detectable effect on liver AA concentration in all groups. However, spleen AA showed significantly increased levels between 3–6 h of hypoxic stress in fish fed 25 and 200 mg AA/kg diet and a further increase after 9 h in fish fed the 200 mg of AA/kg diet. These results suggest that the ascorbic acid requirements for seabream is less than 25 mg/kg diet based on a 12-week growth study and that it requires about a four-field increase in weight before signs of deficiency can be observed. Also, the fact that no variation in liver vitamin C concentration was be detected as a response to stress, suggests that this kind of stress does not significantly increase the utilization of this vitamin. However, a possible relation between dietary ascorbate and the physiological response to hypoxia was found, since the fish receiving the non-supplemented diet showed increased plasma glucose and a tendency to have wider plasma cortisol variations than the fish fed the supplemented diets.

Potential of rapeseed meal as an alternative protein source in complete diets for tilapia (Oreochromis mossambicus Peters).

Abstract A 56-day feed trial was conducted to investigate the potential of rapeseed meal in practical diets for juvenile tilapias. Six isonitrogenous (38% crude protein) and lipid and carbohydrate balanced diets were formulated. The control diet contained fish and soyabean meals as principal protein sources; these were progressively substituted with rapeseed meal. Fifteen per cent rapeseed meal effectively replaced soyabean meal whilst higher inclusion levels led to progressively poorer feed performance. Levels of glucosinolate in the rapeseed meal were measured and effects on thyroid histology observed. The central colloid regions in thyroid follicles of fish receiving high rapeseed diets were found to be eroded or completely absent from the lumen. This study indicated a practical inclusion limit of 15% rapeseed meal in tilapia feeds. Various approaches towards the possible improvement of the nutritional value of rapeseed meal products for use in fish diets are also discussed.

Inclusion of an extruded dehulled pea seed meal in diets for juvenile European sea bass (Dicentrarchus labrax).

A feeding trial and subsequent digestibility trial were performed on juvenile European sea bass. The diet formulation allowed for the incorporation of up to 30% of a highly processed commercial pea seed meal as a replacement for both fish meal and the non-protein energy component of the diet (mainly starch). After 11 weeks, it was observed that a positive but non-significant trend existed for both growth and feed utilization with increasing incorporation of pea seed meal in the diets. There were, however, other nutritional parameters such as protein efficiency ratio (PER) and nitrogen deposition that increased according to graded levels of pea seed meal indicating that a high inclusion of this ingredient was beneficial to protein assimilation. Digestibility coefficients for protein, lipid and carbohydrates were not appreciably affected by the inclusion of this ingredient which was also reflected with respect to the overall carcass composition of sea bass at the end of the study.

Probiotic Pediococcus acidilactici modulates both localised intestinal- and peripheral-immunity in tilapia (Oreochromis niloticus).

The application of probiotics in aquaculture has received concerted research efforts but the localised intestinal immunological response of fish to probiotic bacteria is poorly understood. Therefore, a study was conducted to evaluate the probiotic effect of Pediococcus acidilactici on Nile tilapia (Oreochromis niloticus) with specific emphasis on intestinal health and probiotic levels as well as system level responses such as growth performance, feed utilization and haemato-immunological parameters under non-challenged conditions. Fish (9.19 ± 0.04 g) were fed either a control diet or a P. acidilactici supplemented diet (at 2.81 × 10(6) CFU g(-)(1)) for six weeks. At the end of the study the probiotic was observed to populate the intestine, accounting for ca. 3% (1.59 × 10(5) CFU g(-)(1)) of the cultivable intestinal bacterial load. Real-time PCR indicated that the probiotic treatment may potentiate the immune-responsiveness of the intestine as up-regulation of the gene expression of the pro-inflammatory cytokine TNFα was observed in the probiotic fed fish (P < 0.05). Light microscopy observations revealed elevated intraepithelial leucocyte (IEL) levels in the intestine of P. acidilactici fed tilapia after six weeks (P < 0.05) of feeding and a trend towards elevated goblet cells was also observed after six weeks feeding (P = 0.08). Concomitantly at week six, along with elevated IELs and elevated TNFα mRNA levels in the intestine, an increased abundance of circulating neutrophils and monocytes were observed in fish fed the probiotic supplemented diet (P < 0.05). This haemopoietic expansion of innate immune cells could be reflective of an elevated state of immuno-readiness. Together these results suggest that the probiotic has a protective action on the intestinal mucosal cells, stimulating the innate immune response after feeding for a period of six weeks. These immunological modulations did not impair growth performance or the remaining haematological and zootechnical parameters compared to the control group (P > 0.05).