15 OCTOBER 1999
MICROBIAL COMMUNITY ANALYSIS AND CONTROL IN THE CULTURE OF ARTEMIA JUVENILES FOR AQUACULTURE
Thesis submitted in fulfillment of the requirements for the degree of Doctor (Ph.D.) in Applied Biological Sciences by L. Verschuere, University of Gent, Belgium, 1999.
Summary:
The development of microbial control strategies is one of the recent challenges in aquaculture. In this thesis it was assessed whether the application of selected bacteria enhanced the zootechnical and hygienic performances of Artemia culture. In Chapter I (Introduction) the importance of the brine shrimp Artemia for aquaculture and aquarium pet shop industry is demonstrated. Furthermore, the branchiopod crustacean Artemia is a well-known organism that has been studied for decades. These facts designate Artemia as a good model organism to develop novel microbial control strategies. The present study examined the application of bacterial strains in the stagnant culture of Artemia juveniles with a threefold goal, i.e. the improvement of the nutritional value of the food for Artemia, microbial control of the associated microbiota, and better and more stable culture performances.
In Chapter II (Probiotic bacteria as biological control agents in aquaculture - a review) available literature reports about probiotic bacteria as biological control agents in aquaculture are listed and a rationale for the research and development concerning probiotics in aquaculture is proposed.
Chapter III (The contribution of individual populations to the Biolog pattern of model microbial communities) and Chapter IV (Modeling the color development in Biolog microtiter plates by the Gompertz function) deal with the Biolog system, which is a novel tool for phenotypical microbial community analysis. The possibilities and limitations of the characterization of microbial communities with the Biolog system were explored, as this tool has been applied in the subsequent chapters.
In Chapter III the use of model microbial communities with an a priori known taxonomical structure and carbon substrate utilization pattern of the separate community members allowed the detection and analysis of the contribution of the individual populations to the whole-community Biolog patterns. It was concluded that the Biolog system is a sensitive and reproducible tool to differentiate even closelyrelated microbial communities, but that interpretation of whole-community Biolog
patterns in terms of taxonomic composition must be dealt with carefully, as strains present in a low proportion can have the highest contribution to the whole-community pattern and as the activity of slower growing strains can be at least partially masked by faster growing strains.
To overcome the problem of choosing an appropriate incubation time when using Biolog, color development curves obtained from multiple readings of Biolog microtiter plates over a long incubation time were fitted to the Gompertz function (Chapter IV). This yielded - for each sole carbon source - the incubation time independent and biologically relevant Gompertz parameters A (maximal extent of color development), micro m (specific color development rate) and lambda (lagtime). The model was thoroughly statistically evaluated and showed to comply with the criteria in most cases. It was shown that this approach is practicable and may yield consistent results for environmental microbial community analysis.
In Chapter V (Monitoring Biolog patterns and r/K-strategists in the intensive culture of Artemia juveniles) the microbial communities occurring in the stagnant culture tanks of intensively reared Artemia were monitored over several culture periods. A parallelism between Artemia rearing success, Biolog fingerprint of the bacterial community and the proportion of r-strategists present, was observed. It was hypothesized that both deterministic factors and stochastic factors determine the microbial community colonizing the culture tanks. It was argued that the variability of the microbial community associated with the culture of Artemia juveniles could be restricted through the early application of a probiotic strain or a mixture of bacteria, provided they are well adapted to the prevailing conditions.
In Chapter VI (Microbial control of the culture of Artemia juveniles through preemptive colonization by selected bacterial strains) putative probiotic strains were selected based on monoxenic Artemia cultures. The in vivo positive effect of 9 selected bacterial strains was demonstrated in xenic culture of Artemia performed in pre-emptively colonized culture medium. The improved culture performances were accompanied by a clear shift of the microbial communities associated with the Artemia culture. It was demonstrated that the bacterial mixture provided as only food source sustained survival and growth of Artemia for at least 6 days and that incubation of the dry food with the bacterial mixture changed strongly its fatty acid profile. This indicated that the bacteria contributed to the nutritional value of the dry food for Artemia.
Chapter VII (Protection of Artemia against the pathogenic effects of Vibrio proteolyticus CW8T2 by selected bacterial strains) deals with the interaction between the strains selected in the previous chapter and the Artemia pathogen V. proteolyticus CW8T2. The infection route of V. proteolyticus CW8T2 was visualized with electron microscopy, showing its devastating activity of Artemia cells and tissues. The protective action of the selected bacterial strains against experimental infections with V. proteolyticus CWST2 was demonstrated in in vivo antagonism tests. It was shown that no extracellular products were involved in this protective action. It was suggested that the selected bacterial strains suppress the proliferation of the pathogen through competition for chemicals and available energy.
When bacterial strains are added to live food cultures, it is important to investigate whether the predator organisms are not negatively influenced. Therefore, in Chapter VIII (Effect of selected bacteria delivered via the diet or through injection on Litopenaeus vannamei postlarvae) the selected strains for Artemia were fed via Artemia nauplii to postlarvae of the shrimp L. vannamei, a typical predator of Artemia. Furthermore, injection of the bacterial strains in the postlarvae was performed in order to assess the safety of their potential application in practice. It was demonstrated that the bacterial strains selected for Artemia did not affect the L. vannamei postlarvae. At the contrary, the appetite of the postlarvae increased and the numbers of associated vibrionaceae decreased when they were fed Artemia loaded with the mixture of the 9 strains.
It has been shown in this research that the probiotic mixture both can contribute to the nutritional value of the dry food and, if necessary, prevent the development of a pathogen or opportunistic pathogen in the culture. Application of the bacterial mixture by pre-emptive colonization caused better and more stable Artemia culture performances, without affecting a typical predator organism.