Bacterial coloniza tion of winter flounder (pseudopleuronectes americanus)

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There is an increasing demand for fry from farmed fish, and most are already produced in intensive rearing installations. However, intensive rearing of marine fish larvae may lead to microbial problems, resulting in poor growth and mass mortality (Muroga, 1987; Nicolas, 1989; Munro et al. , 1994). The artificial aquaculture environment is characterized by a decrease in water quality and an accumulation of organic matter that promote proliferation of opportunistic bacteria (Skjermo & Vadstein, 1999; Olafsen, 2001). Pathogens often arise in the early stages of larval rearing because the culture of small and sensitive marine larvae often involves a period with no or low water exchange (e.g. , feeding period) (Skjermo & Vadstein, 1999).

The bacterial population in the water significantly affects the bacterial colonizing live prey (Nicolas, 1989) and fish digestive tracts (Munro et al. , 1994; Ringo et al. , 1996; Ringo & Birkbeck, 1999). This has been observed in flatfish larvae such as turbot Scophthalmus maximus (Nicolas, 1989) and summer flounder Paralichthys dentatus (Eddy & Jones, 2002). Marine larvae must take in water to maintain their osmotic balance, even before mouth opening (Reitan et al., 1998), so small amounts of bacteria could enter the digestive tract when water is ingested for osmoregulation (Tytler, 1988; Hansen & Olafsen, 1999; Ringo & Birkbeck, 1999); this facilitates the inoculation of opportunistic bacteria in larvae. Thus gills and intestinal surfaces are important sites of bacterial colonization. Nicolas (1989) showed that prior to exogenous feeding, turbot larvae had internaI microflora similar  to that of the water. Bacterial invasion through the skin is also possible (Vadstein, 1997). A way to decrease the impact of pathogenic bacteria is to stimulate immune function by nutritional supplements. Dietary lipids and their constituent fatty acids are fundamental for very small and rapidly developing larvae as the y supply energy and cell components for structural membranes (Sargent et al., 2002).

Several studies have characterized the bacterial flora associated with cold-water marine fish larvae without taking into account larval diet (review by Hansen & Olafsen, 1999). In addition, little information is available on the impact of commonly used enrichment media on the intestinal microbiota of fish larvae (Korsnes et al. , 2006) although a link has been revealed between dietary essential fatty acids and immune function in fish: essential fatty acids and their derivative products, eicosanoids, are highly biologically active and are involved in immunity (Tocher, 2003).

Experiments conducted on channel catfish Jeta/urus punctatus fed a diet enriched with n-3 fatty acids showed a positive correlation with macrophage activity while excess levels of highly unsaturated n-3 fatty acids may not be as effective (Sheldon & Blazer, 1991). Similar positive effects were observed in rainbow trout Onchorhynchus mykiss (Kiron et al., 1995). These authors showed that dietary levels of n-3 polyunsaturated fatty acids (PUF A) affect bacterial infections and leucocyte mobility. Experiments on essential fatty acids (EF A) deficiencies conducted on gilthead sea bream (Sparus aurata) juveniles showed a decrease in both neutrophil activity and lymphocyte abundance (Montero et al., 2004). Although the role of dietary lipids in fish immunity is not fully understood, the y may modulate the immune response by changing the physical properties of immune ceU membranes and ceU membrane interactions (e.g., phagocytosis, antigen- antibody cOlmections) through eicosanoid production from arachidonic acid (20:4n-6, AA) and eicosapentaenoic acid (20:5n-3 , EPA).

The main objectives of this work were two-fold: to study bacterial colonization of winter flounder Pseudopleuronectes americanus larvae fed rotifers enriched with three different diets and to examine the fatty acid response in larval membranes and lipid reserves. Winter flounder larvae were fed rotifers that had been enriched with three different commercial formulations for a period of 22 days to obtain rotifers with different contents of AA and 22:6n-3, docosahexaenoic acid (DRA) to EPA ratio.

As early as the time of mouth opening, winter flounder larvae have been shown to be equipped with enzymes involved in lipid digestion (Murray et al. , 2003), highlighting the importance of dietary lipids for larval growth. Arachidonic acid seems to be an important fatty acid for winter flounder larvae, as the y appear to have the ability to selectively incorporate dietary AA into their cellular membranes. Larvae fed rotifers enriched with the DPS and SEL diets may have encountered AA deficiencies since they retained this fatty acid in their PL, with values similar to larvae with a higher AA content in their NL (ALG treatment). AA has been shown to be highly conserved under conditions of dietary deficiency (Bell & Sargent, 2003) in the larvae of several flatfish species such as turbot (Rainuzzo et al., 1994), yellowtail flounder Limanda ferruginea (Copeman & Parrish, 2002), and summer flounder Paralichthys dentatus (Willey et al., 2003).

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Although we observed a higher relative percentage of AA in the NL of larvae fed rotifers containing a higher relative level of AA, no parallel increase was detected in PL. However, studies have shown that an increased AA content in the PL of gilthead sea bream Sparus auratus larvae was related to an increase in the dietary AA level (Bell et al. , 1995 ; Bessonart et al. , 1999). The same phenomenon was observed for EPAin turbot larvae (Reitan et al., 1994). The fatty acid composition of NL generally reflects that of the diet while the fatty acid composition of PL is more strongly regulated and reflects membrane requirements (Sargent et al. , 2002). We suggest that the relative AA level (6.6% of TF A) reached in the cellular membranes of 26 dph winter flounder larvae could be sufficient to sustain larval development.

The lower bacterial density in the intestinal lumen was related to the dietary treatment that resulted in a higher AA level and higher DHA to EPA ratios in rotifers (ALG treatment). High standard deviations observed for bacterial density data in intestinal lumen are indications of great differences among larvae from each treatment. This variability was also observed in intestinal flora of tilapia (Oreocrhomis mossambicus), carp (Cyprinus carpio) and goldfish (Carassius auratus) (Asfie et al. , 2003). Using four commercial enrichments for rotifers (Algamac 2000, AquaGrow Advantage [Advanced Bionutrition Columbia, USA], Marol E [SINTEF, Trondheim, Norway] and Protein Selco [INVE, Baasrode, Belgium D, Korsnes et al. (2006) observed an effect on bacterial colonization in the gastro-intestinal tracts of cod Gadus morhua larvae as weIl as on the bacterial concentration in enrichment cultures.

Direct bacterial counts in larval tank water by flow cytometry were low compared to values obtained in other studies using the plate method, which only takes into account culturable bacteria (corresponding to 0.1 – 1 % of the total bacteria) (Vadstein et al., 2004). Several studies have reported low levels of Vibrio in rotifer rearing (Verdonck et al., 1997; Skjermo & Vadstein, 1999). The low levels of Vibrio in larval tanks during the rotiferfeeding period could be explained by the use of microalgae, both as food for rotifers and as green water in the larval rearing tank. The addition of algae to the water in fish tanks can considerably alter the composition of the bacterial flora associated with rearing water, larval skin and gut (Skjermo & Vadstein, 1993) and can prevent Vibrio proliferation (Salvesen et al., 1999). Pavlova lutheri and lsochrysis galbana, which were present in the pseudo-green water used in our experiment, have been shown to increase the diversification of bacterial communities (St0ttrup & McEvoy, 2003). A larger fraction of slow growers and fewer opportunistic (potentially pathogenic) fast growing bacteria were observed compared to larval rearing in clear water. The Vibrio indexes of tank water at 46 dph (0.07- 0.15) were lower than the value of 0.24 observed by Eddy and Jones (2002) on summer flounder larvae of a similar stage.

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Table of contents :

INTRODUCTION GÉNÉRALE 
CHAPITRE 1. BACTERIAL COLONIZA TION OF WINTER FLOUNDER (PSEUDOPLEURONECTES AMERICANUS) LARVAE FED LIVE FEED ENRICHED WITH THREE DIFFERENT COMMERCIAL DIETS
1.0 Abstract
1.1 Introduction
1.2 Materials and methods
1.3 Results
1.4 Discussion
CHAPITRE II. ESSENTIAL FA TTY ACID ENRICHMENT OF CUL TURED ROTIFERS (BRACHIONUS PLICATILIS, MÜLLER) USING FROZEN-CONCENTRA TED MICROALGAE
11.0 Abstract
11.1 Introduction
II.2 Materials and methods
II.3 Results
11.4 Discussion
CHAPITRE III. EFFECT OF DIETARY ARACHIDONIC AC ID ON WINTER FLOUNDER (PSEUDOPLEURONECTES A MERICA NUS) LARVAL DEVELOPMENT AND BACTERIAL COLONIZA TI ON OF LARVAL TISSUES AND CULTURE TANKS
III. 0 A bstract
IILI Introduction
IIL2 Materials and methods
III.3 Results
IIL4 Discussion
DISCUSSION GÉNÉRALE
CONCLUSION GÉNÉRALE 

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