From what we have learned about gut MC composition and functionality, we shall attempt to steer it favourably, as a barrier against infection by enteropathogens, as a guarantor of gut integrity, and more generally, as a possible contributor to digestion, immunity and disease prevention for the host. Particular attention will be paid to production of short chain fatty acids (SCFAs), by MC during the digestion process, since they are known to antagonize pathogens, to stimulate intestinal growth, and other metabolic benefits to the host (Clements, 1997). SCFAs may also be delivered from Polyhydroxyalkanoates (PHAs) that are linear polyesters naturally produced by bacteria, as a form of energy storage. Three factors are important to consider in attempts to steer the composition of the microbial community in fish. 1) the tools used for steering; 2) the life stage when a particular steering method can be applied with success, since the appropriate tools may not be same for initial settlement and for on-growing; 3) the fact that bacteria may enter the fish via both active uptake from water and via ingested feed means that both these sources may have to be manipulated.

Steering of the microbial community can be done in two different ways: (1) by adding live microbes through water or feed (probiotics), and (2) by selecting microbes in the gut and/or in the rearing system with particular substrates (prebiotics or other selective compounds). Prebiotics are non-digestible compounds present in the feed in small quantities - normally oligosaccharides and polysaccharides - for selecting beneficial bacteria. Such substrates may be considered to steer MC inside the fish. They can be used as purified feed additives, or introduced with plant-based protein sources, in the context of the urgent need for replacing fish meal by alternative sources of protein in aquafeeds. Nitrogenous compounds are other important substrates for bacteria, and their dietary supply, especially in readily usable forms like protein hydrolysates, may act upon MC and the host. In a more holistic approach, the C:N ratio in feeds will affect microbiota inside the fish, and in the rearing system after excretion and defecation.

In face of the wide variety of possible tools for steering, we must limit ourselves to a reasonable number of tasks. We deliberately selected the most advanced issues developed among the partners, and dedicate it to one of the three model species. The state of knowledge is still limited in this field for cod, but Partners 4 and 6 have identified five probiotic candidates (Fjellheim, 2006, see WP2).

The time window for successful steering during the larval stage needs to be defined in vivo, while further screening of prebiotics and probiotics will be done in vitro with cod microbiota. Analyses of feed degradation products and fermentation products will be carried out by new GC-MS based methods (Villas-Boas et al., 2003; 2006), established at Partner 5. The methods will be used as a "foot-printing" to compare different conditions and to follow specific compounds of interest, e.g. the consumption of feed components and formation of SCFAs. The consortium has already started to experiment the dietary microbial control in sea bass, and this will be pursued in three directions:

1. the impact of dietary carbohydrates in juveniles, and the effects of
2. polyhydroxybutyrate (PHB, the major PHA produced by MC), and
3. protein hydrolysates, on microbial colonization and larval development.

Partner 3 has experience on the effects of diet on digesta and excreta in tilapia, and the omnivorous behaviour of this fish makes feasible to study the effects of the dietary balance between carbohydrates and proteins on system MC. The ways of setting up selection for beneficial bacteria from the system level will be considered in WP 4. The basic knowledge from WPs 1 and 2 will be integrated when required, possibly inflecting the following experimental tasks.

The specific objectives are to:

• Test the time window for successful steering of the microbiota by candidate probiotics.
• Screen in vitro the effect of prebiotics and feed ingredients on selective stimulation of beneficial bacteria for cod larvae
• Document the contribution of intestinal microbiota to the catabolic process of carbohydrate in the gut, and the subsequent effects on the intermediary metabolism of seabass juveniles.
• Assess the dose response to PHB addition in the larval diet for seabass, with a particular attention to MC settlement and the onset of host immunity and digestive function.
• Study the effects of selected protein hydrolysates on larval development of seabass, with a similar analytical approach as for PHB.
• Modulate the effects of diet on system-MC by comparing several C:N ratios in the dietary regime for juvenile tilapia.
• Document the effect of the various steering tools on the functionality of the MC by analyses of low-molecular compounds resulting from feed digestion and the microbial metabolism.

1. Selection of protein hydrolysates
2. Larval rearing of cod for time-window assessment for probiotic steering
3. In vitro screening of prebiotics and probiotics
4. Larval rearing experiment on seabass fed protein hydrolysates
5. Larval rearing experiment on seabass fed PHB
6. Feeding experiment on seabass juveniles
7. Rearing experiment on tilapia

1. Report on time-window for probiotic steering, table of prebiotic characteristics of feed ingredients and report on new probiotics for larval cod
2. Report on protein hydrolysate characteristics suitable for larval fish diets
3. Report on PHB dose for larval diets of seabass
4. Report on plant-based feedstuffs for seabass juveniles
5. Report on protein supply for tilapia sustainable rearing, depending on the rearing system

• IFREMER
  • Joel Gatesoupe

• UGent
• WUR
• NTNU
• SINTEF-MC
• SINTEF-FA