TABLE OF CONTENTS. List of Abbreviations. List of Tables. List of Figures. Chapter 1 Introduction Importance of aquaculture 2

Transcription

1 TABLE OF CONTENTS List of Abbreviations List of Tables List of Figures iii vi viii Chapter 1 Introduction Importance of aquaculture Situation of fish larviculture Overview Larviculture in Europe Larviculture in Japan Larviculture in Taiwan Larviculture in Southeast Asia Role of live food in marine larviculture Turbot larviculture Status of European production Larviculture techniques Critical aspects of turbot larviculture Microbial management in larviculture Fish-bacteria interactions in larviculture Techniques for microbial control in larviculture Disruption of quorum sensing a new strategy for microbial control Quorum sensing a means of bacterial communication Disruption of quorum sensing Limitations to manipulation of quorum sensing 42 i

2 1.6 Thesis outline 42 Chapter 2 What do we know about the functionality of probiotics in larviculture food chain? 58 Chapter 3 Gnotobiotically grown rotifer Brachionus plicatilis as a tool for evaluation of microbial functions and nutritional value of different food types 85 Chapter 4 Interference with the quorum sensing systems in a Vibrio harveyi strain alters the growth rate of gnotobiotically cultured rotifer Brachionus plicatilis 113 Chapter 5 N-acyl homoserine lactone degrading microbial enrichment cultures isolated from Penaeus vannamei shrimp gut and their probiotic properties in Brachionus plicatilis cultures 136 Chapter 6 An N-acyl homoserine lactone-degrading microbial community improves the survival of first-feeding turbot larvae (Scophthalmus maximus L.) 166 Chapter 7 Discussion and conclusions 187 Summary / Samenvatting 203 Curriculum Vitae 212 ii

3 LIST OF ABBREVIATIONS AFDW AFLP AHL Ash-free dry weight Amplified fragment length polymorphism Acyl homoserine lactone AI-2 Autoinducer 2 ANOVA ARC BAB BLAST Analysis of variance Artemia Reference Center Benzyldimethyldodecyl-ammonium bromide Basic local alignment search tool CAI-1 Cholerae autoinducer 1 CCAP cdna CFU CIAD DGGE DHA EC ELISA EPA EUROSCARF FAO FASW FISH GenBank Culture Collection of Algae and Protozoa Complementary deoxyribonucleic acid Colony forming unit Centro de Investigación en Alimentación y Desarrollo Denaturing gradient gel electrophoresis Docosahexaenoic acid Enrichment culture Enzyme-linked immunosorbent assay Eicosapentaenoic acid European Saccharomyces cerevisiae Archive for Functional Analysis Food and Agriculture Organization Filtered and autoclaved seawater Fluorescent in situ hybridization Genetic sequence database of the National Institute of Health, USA iii

4 GFP GI GR Green fluorescent protein Gastrointestinal Growth-retarding HAI-1 Harveyi autoinducer 1 HHL HUFA ICB LB LMG LPS MA MB MC MCR MOA NCBI NOEC NSS OD PCR PG QAC QS RAC RAPD Hexanoyl homoserine lactone Highly unsaturated fatty acid Immunocolony blot Luria-Bertani Laboratory of Microbiology of the Ghent University Lipopolysaccharide Marine Agar Marine Broth Microbial community Microbial community regrown Mechanism of action National Center for Biotechnology Information Non-observable effect concentration Nine Salt Solution Optical density Polymerase chain reaction Peptidoglycan Quaternary ammonium compound Quorum sensing Residual AHL concentration Random amplification of polymorphic DNA iv

5 RLLI RLU rpm RR rrna RS SD TCBS Agar T-RFLP TRO TRP VH WT YEPD Relative log luminescence intensity Relative light unit Rotation per minute Rifampicin resistant Ribosomal ribonucleic acid Rifampicin sensitive Standard deviation Thiosulfate Citrate Bile Salt Sucrose Agar Terminal restriction fragment length polymorphism Total residual oxidants Technical Rubber Product Vibrio harveyi Wild-type Yeast Extract Peptone Dextrose v

6 LIST OF TABLES Table 1.1 Major bacterial groups isolated from the intestinal microbiota of marine fish species 22 Table 1.2 The three elements in the strategy to achieve microbial control in the rearing of marine fish larvae 27 Table 3.1 Concentrations and exposure times of two disinfectants used for rotifer amictic eggs disinfection 91 Table 3.2 Outline of the experiments on the effect of microbial communities 93 Table 3.3 Effectiveness of BAB as a disinfectant for rotifer amictic eggs 95 Table 3.4 Effectiveness of glutaraldehyde as a disinfectant for rotifer amictic eggs 96 Table 3.5 Growth rate over 5 days of Brachionus plicatilis hatched from disinfected amictic eggs and fed three types of food: effect of food type 97 Table 3.6 Interaction between food type and bacterial treatment on Brachionus plicatilis growth rate over 5 days 101 Table 4.1 Vibrio harveyi strains used in this study 119 Table 4.2 Growth rate of Brachionus plicatilis over 72 h: effect of challenge with Vibrio harveyi strains 121 Table 4.3 Growth rate of Brachionus plicatilis over 72 h: effect of challenge with Vibrio harveyi strains 121 Table 4.4 Growth rate of Brachionus plicatilis over 72 h: effect of challenge with Vibrio harveyi mutants 122 Table 4.5 Growth rate of Brachionus plicatilis over 72 h: effect of challenge with the MM77 strain, with and without the addition of the washwater of the MM30 or the BB152 strain 123 vi

7 Table 4.6 Growth rate of Brachionus plicatilis over 72 h: effect of the addition of furanone 123 Table 4.7 Growth rate of Brachionus plicatilis over 72 h: effect of challenge with the BB120 strain, follow by the addition of furanone 124 Table 4.8 Growth rate of Brachionus plicatilis over 72 h: effect of challenge with Vibrio harveyi strains followed by the addition of 2.5 mg l -1 of furanone 125 Table 5.1 AHL molecules used in this study 140 Table 5.2 Vibrio harveyi strains used in this study 141 Table 5.3 HHL degradation capability of different isolates 148 Table 5.4 Growth rate of Brachionus plicatilis over 72 h: effect of challenge with Vibrio harveyi wild-type strain and single mutants, in the absence and presence of the enrichment culture EC5 153 Table 5.5 Growth rate of Brachionus plicatilis over 72 h: effect of challenge with the MM77 strain, with and without the addition of the MM30 washwater, in the absence and presence of the enrichment culture EC5 153 Table 6.1 Outline of the experiments conducted in this study 172 Table 6.2 Survival of turbot larvae on day 5 post-hatch, experiment Table 6.3 Survival of turbot larvae on day 8 post-hatch, experiment Table 6.4 Survival of turbot larvae on day 7 post-hatch, experiment Table 7.1 Studies conducted on the isolation of AHL-degrading bacteria 195 vii

8 LIST OF FIGURES Figure 1.1 World aquaculture production by major species groups in Figure 1.2 Microbial growth on the chorion of cod egg 18 days post-fertilization 19 Figure 1.3 Endocytosis of bacteria in enterocytes in the posterior part of the hindgut in a 14-day-old herring larvae 24 Figure 1.4 The three important factors for the probability of viable larvae and conditions that influence these factors 27 Figure 1.5 Model of the Vibrio harveyi quorum sensing systems 38 Figure 3.1 Growth rate over 5 days of Brachionus plicatilis hatched from disinfected amictic eggs and fed three types of food: effect of the addition of live MCs 98 Figure 3.2 Growth rate over 5 days of Brachionus plicatilis hatched from disinfected amictic eggs and fed three types of food: effect of the addition of autoclave-killed MCs 100 Figure 4.1 Vibrio counts in the Brachionus culture water at 0 h and 72 h 124 Figure 5.1 Cross-streak assay on LB agar plates supplemented with 0.1 mg l -1 HHL 147 Figure 5.2 HHL degradation curves of control cultures and enrichment cultures, as indicated by the residual HHL concentration in the medium over 48 h 149 Figure 5.3 Induction of bioluminescence in the reporter strains by cell-free washwater from the Vibrio harveyi strain BB120, in the absence and presence of the enrichment cultures (Exp. 1) 150 Figure 5.4 Induction of bioluminescence in the reporter strains by cell-free washwater from the Vibrio harveyi strain BB120, in the presence of 5 mg l -1 of exogenous AHL mixture (Exp. 2) 151 viii

9 Figure 5.5 Relative log luminescence intensity of pure cultures / co-cultures of Vibrio harveyi strains and ECs after 18 h incubation at 28 C 152 Figure 5.6 DGGE pattern of the enrichment cultures 154 Figure 6.1 PCR-DGGE profile of the enrichment cultures used in this study 169 Figure 6.2 Change in the number of EC5 bioencapsulated in the rotifers and total rotifer-associated bacteria with time 174 Figure 6.3 Numbers of ECs and total culturable bacteria in the turbot culture water on day 7 post-hatch, experiment Figure 6.4 Numbers of ECs and total culturable bacteria in the turbot larval gut on day 7 post-hatch, experiment Figure 6.5 Numbers of EC3 and EC5 colonizing the turbot larval gut during the post-experiment period 178 Figure 6.6 Residual AHL concentration in the rearing water, experiment Figure 7.1 Frequency distribution of control samples by growth rate class 192 ix

10 CHAPTER 1 INTRODUCTION

11 Chapter 1 INTRODUCTION 1.1 Importance of aquaculture Global production from capture fisheries and aquaculture supplied about 101 million metric tonnes of aquatic products in 2002, providing a per capita supply of 16.2 kg (FAO, 2004). Preliminary estimates for the year 2003 indicate that total world fishery production decreased slightly (-1 percent) compared with However, the total amount of fish available for human consumption increased to 103 million metric tonnes and the per capita supply was maintained. The decrease in capture fisheries was partly compensated for by increases in other food fisheries and especially aquaculture. According to FAO statistics, the contribution of aquaculture to global supplies of fish, crustaceans and molluscs continues to grow, increasing from 3.9 % of total production by weight in 1970 to 29.9 % in Worldwide, the aquaculture sector has grown at an average rate of 8.9 % per year since 1970, compared with only 1.2 % for capture fisheries and 2.8 % for terrestrial farmed meat production over the same period. Production from aquaculture has greatly outpaced population growth, with the world average per capita supply from aquaculture increasing from 0.7 kg in 1970 to 6.4 kg in In 2002, total world aquaculture production (including aquatic plants) was reported to be 51.4 million metric tonnes by quantity and 60.0 billion USD by value. This represents an annual increase of 6.1 % in quantity and 2.9 % in value, respectively, over the figures for The rapid growth in production of the different major species groups continues. Although the growth in production of crustaceans increased in the period , growth rates for the other species groups began to slow down, and the overall growth rate was lower than those experienced over the past 20 years. Aquaculture production in terms of quantity and value for major species groups in 2002 is presented in Fig

12 Chapter 1 Figure 1.1 World aquaculture production by major species groups in 2002 (After FAO, 2004). In terms of individual species, the largest production was reported for the Pacific cupped oyster (Crassostrea gigas 4.2 million tonnes), followed by three species of carp the silver carp (Hypophthalmichthys molitrix 4.1 million tonnes), grass carp (Ctenopharyngodon idellus 3.6 million tonnes) and common carp (Cyprinus carpio 3.2 million tonnes). Most aquaculture production of fish, crustaceans and molluscs continues to come from the freshwater environment (57.7 % by quantity and 48.4 % by value, in 2002). Mariculture contributes 36.5 % of total production and 35.7 % of the total value. Although brackish-water production represented only 5.8 % by quantity, it contributed 15.9 % of the total value, reflecting the prominence of high-value crustaceans and finfish. The aquaculture sector continues to expand, diversify and advance technologically, and dominates all other animal-producing sectors in terms of growth. Aquaculture is now 3

13 Chapter 1 perceived not only as an activity for meeting producers needs, but also as part of the engine for economic growth and for achieving social and environmental goals. 1.2 Situation of fish larviculture Overview As the production from aquaculture increases, the total demand for seed and fingerlings will also increase. According to FAO statistics, the total aquaculture production of finfish in 2002 was reported at over 25 million metric tonnes and at about 14 million metric tonnes of molluscs and crustaceans (Fig. 1.1). It can be estimated that about 160 billion fry were required to achieve this level of production. Besides the quantity, the quality of fingerlings is also important. Good-quality fingerlings can be achieved using advanced hatchery zootechniques, including broodstock management, larval nutrition, and microbial management (Lee, 2003). Nowadays, the complete life cycle of some species (e.g. sea bream, sea bass, turbot) is under control due to improvements in zootechniques, nutritional quality of live food and hygienic conditions of the rearing system. Survival, growth rate and quality of the fingerlings have improved considerably during the last decade (Planas and Cunha, 1999). According to Verreth (1994), bottlenecks in the development of techniques for larval rearing are: 1) the lack of knowledge of the optimal environmental conditions and feeding behaviour during the early life stages; 2) the unknown nutritional requirements which are difficult to assess in the absence of a suitable rearing technology; 3) the changes in specific husbandry and feeding practices from one species to another (due to ontogenic changes and growth); 4

14 Chapter 1 4) the small size of the larvae and the need for small particle sizes which pose specific problems on the feed technology. Planas and Cunha (1999) stated that new efforts should be directed towards the following problems: the cost per juvenile produced; optimisation of the rearing procedure and the variability and predictability of the results; nutritional composition and enrichment of live prey; microbiological composition of the larval environment and pathology; development and quality of the larvae Larviculture in Europe The major species cultured in Europe are Atlantic salmon Salmo salar (674,000 tonnes in 2002), gilthead sea bream Sparus aurata and sea bass Dicentrarchus labrax (114,000 tonnes combined in 2002), turbot Scophthalmus maximus (5,000 tonnes in 2002), and Atlantic halibut Hippoglossus hippoglossus (307 tonnes in 2002) (FAO, 2004). Selection of species to be cultured tends to be market driven. Hatchery production in Europe is mainly for providing juveniles for grow-out farming. Significant progress has been made in culture techniques for the European species, possibly because all available resources have been concentrated on only a few species. Future diversification of cultured species is a strategy to avoid overproduction of particular species and maintain profitable prices for the producers. Another possible benefit of diversification is fewer outbreaks of disease (Shields, 2001; Lee, 2003). While production of the anadromous Atlantic salmon in northern Europe accounts for the greatest tonnage of sea-reared fish, farming of true marine species is concentrated around the Mediterranean Sea. Larval rearing systems for these species range from simple installations utilizing endogenous plankton blooms to large industrial-scale hatcheries. Since the mid-1980s, aquaculture in northern Europe has concentrated on the production of a new species, Atlantic halibut. Production of halibut juveniles is now stabilizing due to the success of several key hatcheries. There is a renewed interest in this region in the intensive cultivation 5

15 Chapter 1 of Atlantic cod Gadus morhua as a direct alternative to cage-farmed salmon. In southern Europe, a small industry for turbot is located mainly in Spain and France. Recent diversification initiatives have centered on larviculture of sparid species, including red porgy Pagrus pagrus, sheepshead sea bream Puntazzo puntazzo and common dentex Dentex dentex. The viability of land-based farming of soleid flatfish Solea solea and cage cultivation of oceanic species are also under investigation (review by Shields, 2001). Comparing the historical figures of fry production from 2000 to 2005, the main production indices have drastically changed, but without a substantial growth of investments or hatchery volumes and areas. For example, the number of fry produced per employee per production season has more than doubled from 200,000 up to over 500,000 fry per person. Another visible change is the production of fry per production volume (cubic metre), that has risen from 20,000 fry in 2000 up to 50,000 fry in 2005 (de Wolf et al., 2005). Despite the wide range of specific larviculture techniques throughout Europe, a number of factors can be identified that have influenced production technology in recent years. Each of the European marine fish farming sectors has been affected by significant disease problems in recent years, requiring the development of preventive methods and prophylactic treatments (Rodgers and Furones, 1998). A major European research effort has been devoted to meeting the nutritional requirements of marine fish larvae. Techniques for administering essential nutrients via enriched live prey have been investigated (Sargent et al., 1997; McEvoy and Sargent, 1998; Sargent et al., 1999) and progress has been made in formulating inert feeds for the larvae (Cahu and Infante, 2001). In view of the nutritional deficiencies of rotifers and Artemia, cultivated marine zooplankton has been identified as an alternative diet source for marine fish larvae (Sargent et al., 1997). However, zooplankton-based larviculture accounts for only a small proportion of total 6

16 Chapter 1 European fry production, owing to the difficulty of producing sufficient copepod quantities under controlled conditions Larviculture in Japan In recent years, the studies on larval nutrition (as one of the factors to improve larval quality) in marine fish and crustaceans in Japan have been progressed on determining nutrient requirement and developing efficient microdiets for larval stages. Artificial microdiets have been developed for determining the nutrient requirements of larval aquatic animals. More practically, these are used for replacing live food such as rotifers and Artemia in seed production operation. The formulation of the microdiet was based on a combination of a peptide from milk casein and fatty acid-calcium containing a large amount of docosahexaenoic acid (DHA) obtained from fish oil. This diet has been successfully tested for red sea bream and Japanese flounder larvae. Highly unsaturated fatty acid (HUFA) enrichment is common practice in seed production of marine species due to the lack of HUFA in rotifers and Artemia nauplii (Koshio and Teshima, 2005) Larviculture in Taiwan (review by Liao et al., 2001) Taiwan has over 90 finfish species in which larviculture is possible. Billions of finfish fry can be produced annually. The use of modern and advanced techniques in larviculture has won Taiwan a leading position in the world. This achievement can be attributed to: 1) successful broodstock management, including broodstock collection, maturation and spawning, egg collection and incubation; 2) complete larval rearing using outdoor and indoor systems; 3) establishment of techniques of live food preparation for larval feeding. Cannibalism, difficulties in water quality control and disease outbreak often cause mass mortality in the larval rearing. Through a systematic understanding of the complex behavioural patterns of finfish larvae, cannibalism can be controlled under larviculture 7

17 Chapter 1 conditions. Control strategies involve physical manipulations such as grading and feeding adjustments. Food preparation includes techniques of live food culture, food enrichment, and artificial feed development. In 1997, there were 31 species of microalgae, 3 species of rotifers, 1 cladoceran species (Diaphanosoma aspinosum) and 1 copepod species (Apocyclops royi) that were being held as starter cultures in Taiwan (Su et al., 1997). Live food that are optimal for larviculture include algae, oyster trochophores, rotifers, copepods, nauplii and adult Artemia, cladocerans, and red worms. Both oyster trochophores and copepods are very nutritious larval foods, containing more than 10 mg g -1 dry weight of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Furthermore, copepods have the highest DHA to EPA ratio. On the contrary, rotifers and Artemia are poor in HUFA and need to be enriched before feeding Larviculture in Southeast Asia As the yield from the capture fisheries stagnates and the human population growth rate in the Southeast Asia region continues to be among the highest in the world, the requirement for proteins is expected to come from the increased production of low trophic level species such as milkfish (Chanos chanos) and tilapia (Oreochromis spp.). There is also an increasing demand for high value fish species such as groupers and snappers, particularly for the live food fish markets of affluent and developing countries in Asia. In order to meet the demand for more food fish and to develop new products for the export market, the most important component of any culture system must be met, that is the adequate supply of fry and juveniles for culture. Fry availability is an essential component in the development of culture systems for new species and in further increasing production of established culture species. A number of research institutions in the region are actively addressing the question of fry availability through research in breeding and larviculture of several marine species. To date, 8

18 Chapter 1 commercially viable technologies for breeding and seed production of milkfish, Asian sea bass (Lates calcarifer) and the humpback grouper (Cromileptes altivelis) have been developed and continue to be refined (review by Marte, 2003). The basic technique for intensive larviculture, adopted by hatchery operators in Thailand, Malaysia and Singapore for sea bass, and in Indonesia and the Philippines for milkfish, was developed through two decades by various research institutions in the region. These included the development of propagation techniques for microalgae and rotifers, investigations on the larval biology of these species, zootechnical aspects such as appropriate tank size and design of hatchery facilities, proper methods of egg collection, transport and handling of eggs and larvae, appropriate water management, feed and feeding management (reviews by Quinitio and Duray, 1996; Pechmanee, 1997) Role of live food in marine larviculture Microalgae Marine microalgae are widely used in the first-feeding stage of marine fish larvae (Naas et al., 1992; Reitan et al., 1993). Rotifers, commonly used as live prey in the early larval phase, may be grown on microalgae through the cultivation process (Witt et al., 1984). Microalgae are also added directly to the larval tanks together with the live food (Vásquez-Yeomans et al., 1990; Naas et al., 1992). This method (green-water technique), which is used for most marine fish species, requires relatively small amounts of microalgae. The algae may act as a food source for the early stages of fish larvae and also for the rotifers, which will consequently become short-term enriched in the larval tanks (Reitan et al., 1997). The use of microalgae during the first-feeding process is believed to improve the nutritional conditions of the larvae, either directly or through improving the nutritional value of the rotifers. Considerable attention has been given to the content of n-3 HUFA essential for normal growth, pigmentation, and survival of marine fish larvae, including EPA and DHA. In 9

19 Chapter 1 addition, both the protein and lipid levels of the rotifers are affected through consumption of microalgae (review by Reitan et al., 1997). It is suggested that microalgae may affect the microbial community of the water and the live food (Nicolas et al., 1989), which affects the microbiota of the larval fish s intestine (Skjermo and Vadstein, 1993). Moreover, algal addition to larval tanks also tends to change the light conditions of the tanks and the feeding behaviour of the larvae (Naas et al., 1992) Rotifers The rotifer Brachionus plicatilis is cosmopolitan and found typically in salt lakes and coastal lagoons. It is the only rotifer with commercial and applied importance through its use as live food for marine fish fry (Lubzens, 1987; Lubzens et al., 2001). Despite its commercial and scientific value, the taxonomic status of B. plicatilis remains controversial for many years. The B. plicatilis complex was originally classified as a single taxonomic species from which two morphotypes, L (large) and S (small), were distinguished using morphometrical data and isozyme analysis. These morphotypes were later characterized as B. plicatilis O.F. Müller and Brachionus rotundiformis Tschgunoff, respectively (Rumengan et al., 1991; Segers, 1995). However, data on nucleotide sequence variation from both nuclear and mitochondrial genes have revealed nine genetically divergent lineages within B. plicatilis species complex (Gómez et al., 2002). In a recent study, a number of molecular markers have been developed and applied to identify genetically the rotifer strains used in European hatcheries (Papakostas et al., 2006). The results indicated an absence of the typical B. plicatilis and B. rotundiformis species and a predominance of the Brachionus sp. Cayman biotype (Brachionus S morphotype). All the other biotypes/species were identified as B. plicatilis sensu stricto, B. sp. Nevada and B. sp. Austria, all belonging to the Brachionus L group. The above results have important implications for rotifer cultures, with respect to the different temperature and 10

20 Chapter 1 salinity optima of each biotype/species. Caution is needed regarding the common practice of exchanging samples based solely on morphological criteria. Culture methods for rotifers have been extensively studied and reviewed (reviews by Dhert et al., 2001; Lee, 2003). Batch cultivation, due to its simplicity, is the most common type of rotifer production in marine fish hatchery. A total harvest of rotifers is applied with part of the rotifers used as food for fish larvae and part used as inoculum for the next culture. Using an artificial diet, the density at harvest time is about 600 rotifers ml -1 after 4 days culture starting from rotifers ml -1 (Dhert, 1996). However, batch cultures are subjected to highly variable conditions, which contribute to unstable/unpredictable culture conditions and a relatively low production yield at high cost. The semi-continuous culture is known as thinning culture since the rotifer density is kept constant by periodic harvesting. Contrary to the batch culture, this long-term culture is maintained at low densities for a period of 7-14 days without water renewal (Lubzens, 1987). In the past decade, Japanese scientists have developed intensive ultra-high density rotifer culture techniques. The ultra-high density rotifer mass culture (maximum density from 20,000 to 35,000 rotifers ml -1 ) has been developed based on concentrated freshwater Chlorella as food (Fu et al., 1997; Yoshimura et al., 1997). A rotifer density of 160,000 rotifers ml -1 has been reached recently (Yoshimura et al., 2003). Cost-effective biomass production of rotifers relies on the use of a cheap food source and this explains why baker s yeast is used as an important diet. When applied as a sole diet, it may support the mass production of rotifers in non-axenic culture conditions where microorganisms provide essential nutrients (Hirayama, 1987). However, it is known that yeast-fed rotifers lack the essential fatty acids required for the proper development and survival of several species of marine fish (Watanabe et al., 1983; Watanabe, 1993). To ensure the nutritional value of rotifers, various enrichment methods have been developed to increase the essential nutrients, such as HUFAs. Rotifers can be enriched with good quality algae, such as 11

21 Chapter 1 Tetraselmis sp. Nannochloropsis oculata, Isochrysis galbana and Rhodomonas sp.; oil emulsions containing n-3 or n-6 HUFA; vitamins; proteins; and formulated diets, such as Culture Selco (Dhert et al., 2001). Several approaches can be followed for the enrichment of rotifers: (1) the adjustment of the lipid and vitamin content of the rotifers just before feeding them to other organisms (referred to as short-term enrichment); (2) the feeding of rotifers on a complete diet or long-term enrichment. Each technique has its benefits and disadvantages. The short-term enrichment technique is fast and flexible, but very often produces lower quality rotifers with a too high lipid content and poor hygienic quality. The biggest problem resides in the fact that a lot of rotifers are lost when they are concentrated at high density. Also, transfer of oil to larval rearing tanks with consequent loss of water quality and associated problems of larval viability have been reported. On top of that, the retention time of the nutrients, which are mainly accumulated in the digestive tract of the rotifers, is very short and can create problems when the rotifers are not eaten immediately (Dhert et al., 2001). Bacteria are always associated with the mass production of rotifers and may cause unexpected mortality or suppressed growth to rotifers. Although most bacteria are not pathogenic for rotifers, their proliferation must be avoided since a risk of accumulation and transfer via the food chain can cause detrimental effects on the predator (Dhert, 1996). It is assumed that rotifers, the first food administered to fish larvae, are the major carriers of bacteria (Munro et al., 1993b, 1994). In most hatcheries, the billions of rotifers and their accompanying food inevitably create a high load of organic material which is rapidly colonized by bacteria. These rearing conditions can change the normal interaction between bacteria and rotifers to the one that is detrimental (Vadstein et al., 1993). Most of the hatcheries still rely on batch cultures to obtain the live food for their larvae. In terms of microbiology, these systems are very unpredictable since normally, no microbial control is performed after the disinfection of the rearing water at the start of the rotifer culture 12

22 Chapter 1 (Dhert et al., 2001). The development of the microbiota in such an open system is expected to occur in two steps, based on the r/k-concept (Salvesen et al., 1999). During the first step, the r-strategists, i.e. fast-growing opportunistic bacteria will develop, whereafter K-selection will occur as the community starts to reach a mature stage. As rotifer batch cultures often do not reach the mature stage, as it takes only 4-7 days to complete one cycle, together with the fact that the microbiota is continuously disturbed due to the addition of feed, the selection for K- strategists is supposed not to happen. Therefore, it is important to select bacteria that can easily survive under the same conditions as the rotifers do, i.e. r-strategists and those which have a positive effect on the rotifers. On the contrary, for the new culture techniques based on long-term cultures (one month) and often associated with a recirculation system, the development of a mature microbiota and the presence of K-strategists are important (Dhert et al., 2001) Artemia Since no artificial feed formulation is yet available to completely substitute for Artemia, feeding live Artemia nauplii to marine fish larvae (after the rotifer-feeding stage) still remains essential in commercial hatchery operations. Although using Artemia cysts appears to be simple, several factors are critical for hatching large quantities needed in larval fish production. These include cyst disinfection or decapsulation prior to incubation, and hatching under the optimal conditions. All these factors affect the hatching rate and maximum output, and hence, the production cost of the harvested Artemia nauplii (Sorgeloos et al., 2001). As the nutritional quality of commercially available Artemia strains is relatively poor in some essential highly unsaturated fatty acids, such as EPA and DHA, it is essential and common practice to enrich these live prey organisms prior to feeding to the fish larvae. British, Japanese, and Belgian researchers have developed various enrichment products and procedures using selected microalgae, micro-encapsulated products, yeasts, self-emulsifying 13

23 Chapter 1 concentrates, and micro-particulate products, either singly or in various combinations (Léger et al., 1986). In the 1980s, most attention was dedicated to the presence of EPA in Artemia as a guarantee for successful production of marine fish larvae (Watanabe et al., 1983; Léger et al., 1985). However, in the late 1980s and early 1990s, more attention was paid to the level of DHA, since good survival appears to be correlated with EPA, but DHA improves larval quality and growth. The importance of DHA, more particularly the requirement for high DHA/EPA ratio in promoting growth, stress resistance, and pigmentation, was revealed (Reitan et al., 1994; Lavens et al., 1995). Although continuous disinfection of Artemia during hatching and enrichment is becoming a routine operation in many hatcheries, the interference of bacteria in hatching and enrichment remains an important study object. As more attention is given to the use of on-grown Artemia as a cheaper alternative to the use of nauplii, simple cost-effective production techniques have been developed (Sorgeloos et al., 2001). 1.3 Turbot larviculture Status of European production Turbot (Scophthalmus maximus) is a pleuronectiform teleost belonging to the family Scophthalmidae (Linnaeus, 1758). It is naturally found from North Africa to the North Atlantic up to Norway, in the Baltic, in the Mediterranean, and in the Black Sea. However, it is not very abundant in these locations. Total European capture production of this highly esteemed food fish is relatively low (6,329 tonnes in 2002, FAO data). Turbot reaches a size of kg. It lives on stony, sandy or mixed bottom at m depth. It is an active hunter of smaller fish but also feeds on crustaceans, mussels, polychaetes, etc., particularly in the younger life stages (Jones, 1973). 14

24 Chapter 1 The species supply and quality have gained a large interest in commercial turbot cultivation since early rearing trials in the 1970s. Following the initial commercialization of turbot farming in the UK and France during the 1980s, the emerging industry became centered on northern Spain, owing to favourable water temperatures for on-growing. Problems of oversupply were encountered in the early 1990s, associated with a proliferation of grant-assisted Spanish farms. The industry has subsequently consolidated and output has risen gradually to approximately 3,100 tonnes in The farmed product has increasingly gained in standing within the discerning Spanish and French markets, due in part to feed improvements and to the greater availability of larger-sized fish. Consolidation of production has taken place both in the on-growing and hatchery sectors. The Norwegian-owned company, Stolt Sea Farm, dominates the on-growing sector, having acquired five Spanish farms during the 1990s and involvement in two farms in France and Portugal. The company also acquired two Spanish hatcheries during this process, with a combined output capacity of approximately 1 million turbot fry per year. Juvenile supply is dominated by the company France Turbot, which produced approximately 3 million intensively reared turbot in This company has become a key juvenile supplier for the entire European industry (Shields, 2001) Larviculture techniques A basic rearing protocol for turbot larvae using rotifer and Artemia diets was developed in the early 1970s (Planas, 1994). Research and development on intensive rearing techniques continued thereafter in the UK and France, with the first commercial hatcheries being established in the late 1970s and early 1980s. These were joined by Norwegian semi-intensive rearing facilities in the mid- to late-1980s, based on the extraction of marine zooplankton from seawater lagoons. A further commercial production technique was introduced in 15

25 Chapter 1 Denmark in 1990, involving the controlled cultivation of calanoid copepods in large outdoor tanks (Urup, 1994). Rationalization of the turbot hatchery sector over the past ten years has been driven by a combination of limited market demand for fry and operational difficulties with certain production techniques. Faced with a rapidly expanding market for sea bass and sea bream juveniles, some hatcheries chose to switch production away from turbot. Regarding operational difficulties, the Norwegian semi-intensive production method suffered from inconsistent prey availability and exposure of larvae to pathogens (Urup, 1994). Therefore, the elements of this approach were taken forward in the Danish copepod-based technique (Urup, 1994) and in a combined extensive/semi-intensive method developed in Spain (Riaza and Hall, 1993). Only a few key producers have succeeded in obtaining consistent results with the intensive turbot rearing method, despite a large research effort in support of this approach Critical aspects of turbot larviculture Mortality during early larval development There are four phases in intensive turbot rearing corresponding to endogenous nutrition, feed initiation, transfer from rotifers to Artemia and subsequent survival up to weaning. Of these, the most critical is the transition phase from rotifers to Artemia, between week one and two, when catastrophic mortality can occur (Person-Le Ruyet, 1989; Minkoff and Broadhurst, 1994). Research on this critical phase has focused on the nutritional requirements of larvae during early exogenous feeding, together with the microbial characteristics of the intensive rearing environment (Shields, 2001) Nutritional factors Based on the results of food ingestion and survival in relation to ration, Planas (1994) and Dhert et al. (1994) considered that the rotifer densities applied in commercial hatcheries are sufficient to meet the energetic requirements of turbot larvae. Any nutritional effects on early 16

26 Chapter 1 mortality may therefore be poor digestibility of the diet, rather than the quantity of prey ingested. Evidence has been obtained that the dietary transition from rotifers to Artemia causes a nutritional disturbance to turbot larvae and this may contribute to mortality at this critical stage. In view of the limited capacity of turbot larvae to digest lipid, the suitability of HUFAboosted lipid enrichments for early larval stages has been questioned. Dhert et al. (1994) found that only a short duration on high DHA rotifers was required in order to increase the stress resistance of turbot larvae. DHA requirement during the rotifer-feeding phase appeared to be related to initial egg quality, with end-of-season larvae benefiting more from DHA supplementation than larvae obtained earlier in the breeding season. Dhert et al. (1998) suggested that it may be more productive to optimize the nutrition of turbot broodstock, rather than to rely on artificial enrichments during the larval phase. The presence of microalgae has been shown to increase the survival and growth rates of turbot larvae during the rotifer phase, relative to larvae reared in clear water (Reitan et al., 1993). Later on, Reitan et al. (1998) indicated that microalgae are taken up actively by turbot larvae, although the nutritional significance of this phenomenon is unclear. However, it has been demonstrated that the lipid and protein content of rotifers remains more stable in turbot rearing tanks containing microalgae (Øie et al., 1997). Naas et al. (1995) provided evidence that the physical effect of microalgae on water turbidity is important, in terms of feeding incidence and the numbers of prey ingested by turbot larvae Microbial factors The role of bacteria in the rearing success of turbot larvae has been the subject of sustained research, since early investigations demonstrated beneficial survival effects of antibiotictreated diets (Perez-Benavente and Gatesoupe, 1988). Further evidence for bacterial involvement was provided by Munro et al. (1995), who attained very high turbot survival 17

27 Chapter 1 rates in the absence of culturable bacteria. Although the application of antibiotics has been a valuable component of the hatchery process, the risks of generating bacterial resistance or disrupting the normal development of the gut microbiota have been recognized (Minkoff and Broadhurst, 1994). Recent research on live food-mediated delivery of chemotherapeutics (Robles et al., 1998) may offer a more controlled means of prophylactic treatment in the future. Alternative methods have also been sought to reduce the microbial loading associated with live food, to alter the composition of existing microbial populations in the rearing system and to promote beneficial bacteria (Ringø and Birkbeck, 1999; Skjermo and Vadstein, 1999). Although there is strong evidence for the involvement of bacteria in turbot mortality, no correlation has been found with the presence of known pathogens, or total microbial loading within the rearing system (Munro et al., 1993a, 1994). Munro et al. (1993a) suggested that the rate of development of the intestinal microbiota may be more significant in determining rearing success than total numbers of bacteria present. 1.4 Microbial management in larviculture Fish-bacteria interactions in larviculture Microbiota of fish eggs Poor egg quality and resulting mass mortalities have been serious problems in larval production systems. High-density incubation techniques, often resulting in eggs overgrown by bacteria, may affect the commensal relationship between the indigenous microbiota and opportunistic bacteria, hampering egg development and subsequently affecting hatching, larval health and growth (Olafsen, 2001). The eggshell consists of a thick lamellar inner layer, zona radiata, and a thinner outer layer, chorion or zona pellucida. The thickness and lamellar structure vary between fish species. The chorion and zona radiata of marine fish eggs are composed of a highly hydrophobic protein aggregate and glycoproteins with 2-3% 18

28 Chapter 1 carbohydrate dominated by galactose, mannose, N-acetylglucosamine, and N- acetylneuraminic acid. The glycoproteinaceous nature of the eggshell is well suited for adhesion and colonization by bacteria. The fish embryo secretes inorganic and low molecular weight organic compounds which diffuse through the eggshell and establish a gradient outside the eggshell. This gradient may act as a chemoattractant for bacteria that are able to utilize the secreted compounds. Through the phases of adsorption, adhesion, and colonization, a primary microbiota will eventually become established on the egg surface (Hansen and Olafsen, 1999). A study was undertaken by Hansen and Olafsen (1989) to describe the microbial ecology during egg development and hatching of two marine fish species, cod (Gadus morhua) and halibut (Hippoglossus hippoglossus). By means of scanning electron microscopy, substantial bacterial growth was observed on cod eggs 2 h after fertilization, indicating rapid primary colonization. Further on-grown and establishment of the egg primary microbiota proceeded more slowly, with eggs heavily overgrown after 10 to 12 days. At hatching (day 18), eggs were completely overgrown (Fig. 1.2). The filamentous bacterium, identified as Leucothrix mucor, was known to cause mortality problems in cod hatcheries. Figure 1.2 Microbial growth on the chorion of cod egg 18 days post-fertilization (After Hansen and Olafsen, 1989). 19

29 Chapter 1 Bacterial colonization may have adverse effects on the egg and on the developing embryo. It is evident that colonization of the egg surface by pathogens or opportunists may be detrimental and cause disease of eggs (Hansen et al., 1992) or larvae (Bergh et al., 1997). Additionally, the bacterial epiflora may cause problems other than disease for the developing embryo. Massive overgrowth by obligate aerobic bacteria may result in hypoxia in the developing embryo. As the oxygen demand increases toward hatching, the effect of oxygen deficiency may result in accumulation of lactic acid, retarded development, and possible neural injuries. Oxygen deficiency is probably the major cause of death in cod eggs heavily colonized by the obligate aerobic L. mucor, which is not known to penetrate the eggshell or to produce exotoxins or toxic metabolites (Johnson et al., 1971). Since the oxygen concentration is critical for successful hatching, bacterial overgrowth on the egg surface may also affect hatching. Moreover, exoproteolytic enzymes released by the adherent epiflora may damage the eggshell, resulting in release of hatching enzymes or leakage of cell constituents (Hansen et al., 1992). Eventually, bacterial exotoxins or toxic metabolites, e.g. NH 3 and H 2 S, released by the epiflora may harm the developing embryo (Barker et al., 1989). In intensive larviculture, the techniques used to sterilize the eggs (by removing the egg epiflora) may disturb the balance of microbial communities and favour exponential growth of opportunistic bacteria (Salvesen et al., 2000). It is necessary to keep control on the microbial community composition, including pathogens and opportunists, in the incubators. Thus, a precolonized non-pathogenic and diverse egg epiflora may be a barrier against colony formation by opportunistic pathogens (Olafsen, 2001) Microbiota of fish larvae It is known that microorganisms cannot be avoided in commercial aquaculture. The skin, gills and gastrointestinal tract are inhabited by microorganisms which are adapted to a life in intimate contact with these surfaces. Microbiota associated with the gastrointestinal tract of 20

30 Chapter 1 the early life stages of fish larvae are very diverse, and originate from the microbiota of the eggs, live food and rearing water. Microbes which can inhabit the gastrointestinal tract are specialized to survive and multiply in this region. However, the composition of the microbiota associated with the gastrointestinal tract is highly variable depending on the species and the development stage of the fish (Ringø and Birkbeck, 1999). In intensive larviculture, fish larvae are kept in incubators with hatching eggs and debris. As a result, bacterial counts of the ambient water increase with a factor of 1000-fold after hatching (Hansen and Olafsen, 1989). In order to osmoregulate, marine fish larvae start drinking water before the yolk sac is consumed (Tytler and Blaxter, 1988), and bacteria thus enter the digestive tract before active feeding commences. Older larvae may also ingest bacteria by grazing on suspended particles and egg debris (Olafsen and Hansen, 1992). Thus, the microbiota of eggs and other organisms in the system affect the primary microbiota of fish larvae. There is, however, little information on bacterial colonisation of mucosal surfaces of healthy fish larvae, apart from a few reports, reviewed by Hansen and Olafsen (1999). In fish, the existence and the roles of an indigenous intestinal microbiota have been disputed until the 1970s. However, it is now accepted that a primary transient microbiota will become established at the larval stage and develop into a persistent microbiota at the juvenile stage or after metamorphosis. Because of the immature nature of the immune system in fish larvae, they have to rely on non-specific defense mechanisms, of which a protective intestinal microbiota intimately associated with the gut mucosa will constitute a primary barrier. An understanding of the characteristics or features and the role of the indigenous microbiota of fish larvae may help to improve diets and incubation conditions for the intensive mass rearing of healthy fish (Hansen and Olafsen, 1999). The intestinal microbiota of various freshwater and seawater fishes has been described in a number of investigations. Although the majority of studies has concentrated on adult stages, 21

31 Chapter 1 several reports exist on the microbiota of larvae. Major bacterial genera/species encountered in the intestinal microbiota of various marine fish larvae and juveniles are summarized in Table 1.1. Table 1.1 Major bacterial groups isolated from the intestinal microbiota of marine fish species (adapted and amended from Hansen and Olafsen, 1999) Fish species Fish developmenttal stage Bacteria Reference Cod (Gadus morhua) larval/juvenile Vibrio, Lactobacillus, Bacillus, Pseudoalteromonas, Listonella anguillarum Strøm and Olafsen (1990); Korsnes et al. (2006) Herring (Clupea harengus) larval Pseudomonas, Alteromonas, Flavobacterium Hansen et al. (1992) Atlantic halibut (Hippoglossus hippoglossus) yolk sac larval Cytophaga, Flexibacter, Flavobacterium, Pseudomonas Vibrio, Aeromonas, Pseudoalteromonas Vibrio splendidus, Vibrio alginolyticus, Pseudoalteromonas, Photobacterium phosphoreum Bergh et al. (1994); Verner-Jeffreys et al. (2003) Bergh et al. (1994); Verner-Jeffreys et al. (2003) Turbot (Scophthalmus maximus) larval Vibrio fluvialis, Vibrio alginolyticus, V. pelagius, V. scophthalmi, V. splendidus, V. parahaemolyticus, Aeromonas hydrophila, Chromobacterium violaceum, Achromobacter sp., Acinetobacter calcoaceticus, Pseudomonas fluorescens, P. putida Nicolas et al. (1989); Gatesoupe (1990); Keskin and Rosenthal (1994); Munro et al. (1994); Blanch et al. (1997) Dover sole (Solea solea) yolk sac / metamorphosed larvae Pseudomonas, Alcaligenes, Vibrio, Aeromonas, Moraxella Campbell and Buswell (1983) Red sea bream (Pagrus major) larval/juvenile Vibrio, Pseudomonas, Enterobacteriaceae, Cytophaga, Aeromonas Muroga et al. (1987) Black sea bream (Acanthopagrus schlegeli) larval/juvenile Vibrio, Pseudomonas, Enterobacteriaceae, Cytophaga, Aeromonas Muroga et al. (1987) Gilthead sea bream (Sparus aurata) larval/juvenile Vibrio, Pseudomonas Savas et al. (2005) 22

32 Chapter 1 In summary, it is evident that an intestinal microbiota will become established very soon after hatching in fish larvae. The need for marine larvae to drink in order to osmoregulate will accelerate this establishment compared to that of freshwater larvae. The transient, primary intestinal microbiota will develop into a more diverse and specialized community due to impacts from developments in the intestinal structure and function. The bacterial load and composition of the diet and ambient water, together with external environmental factors, will also undoubtedly influence the development of the gut microbiota. It is likely that the pioneer strains, having adapted to the ecological niche formed by the larval gut, will persist and develop into components of the adult microbiota (Hansen and Olafsen, 1999) Endocytosis of bacteria Nutritional aspects Bacteria as food for marine invertebrates and also for fish have been the subject of a number of investigations. Bacteria play an important role for marine animals by furnishing cell substances or micronutrients such as essential fatty acids (Ringø et al., 1992), vitamins (Sugita et al., 1991), minerals or enzymes (Lindsay and Gooday, 1985). It is generally conceded that in fish larvae having a straight gut (such as cod, herring), ingested particles pass rapidly to the posterior part of the gut and accumulate. In cod larvae, fixed sheep s red blood cells and fluorescent latex spheres have been demonstrated to accumulate in the hindgut (Olafsen and Hansen, 1992), whereas bacteria are readily endocytosed. Ciliated epithelial cells, rich in mitochondria, were found in the posterior part of the foregut in herring larvae and in the oesophagus/foregut area in cod larvae (Olafsen and Hansen, 1992). They have also been demonstrated in halibut larvae (Morrison et al., 1997). In general, ciliated cells are believed to propel water, food particles, and mucus along the gut (Sleigh, 1990). It is likely that the ciliated epithelial cells in marine fish larvae are also involved in the process of propelling particles and bacteria toward the posterior part of the gut (Hansen and Olafsen, 1999). By means of electron microscopy, endocytosis of bacteria by 23

33 Chapter 1 enterocytes in the epithelial border in the posterior part of the hindgut of herring larvae has been demonstrated (Fig. 1.3). Figure 1.3 Endocytosis of bacteria in enterocytes (arrows) in the posterior part of the hindgut in a 14-day-old herring larvae (After Hansen et al., 1992). Ingestion of bacteria by drinking, propulsion of bacteria by ciliated epithelial cells, low levels of extracellular enzymes in the digestive tract, and subsequent endocytosis in the hindgut could contribute to the nutritional value, sustaining the larvae with exogenous nutrients before active feeding commences. This way of securing exogenous nutrition differs from the later larval and juvenile stages, in which high levels of digestive enzymes are present and extracellular digestion will be the major route of nutritional uptake (Hansen and Olafsen, 1999) Immunological aspects The presence of bacteria in fish eggs has provoked an interest in the presence of endogenous defense factors. Extracts from fish eggs may exhibit bactericidal activity (Kudo and Inoue, 1986). Immune molecules, such as immunoglobulins, are present in fish eggs (Kanlis et al., 24

34 Chapter ). Various agglutinating or lectin-like substances have also been demonstrated in fish eggs (Voss et al., 1978). Non-specific mechanisms constitute the most significant part of egg and larval defense, because of the immature state of the immune system at these stages (Manning et al., 1982). Passive immunity, in the form of transmission of maternally developed immunoglobulins against various antigens, has been demonstrated in eggs of various species, as reviewed by Hansen and Olafsen (1999), and may serve as protection in early life stages of these fish species. Immune competence, i.e. the ability to produce specific antibodies to the introduced antigens, appears to be related more to weight than to age. Grace et al. (1981) have reported immune competence in 4-day-old yolk sac larvae of trout, in which they demonstrated an effective system for particle trapping by macrophages under the skin and on the gills before maturation of the lymphoid tissues. In other marine fishes, a specific immune system is probably not fully mature until several weeks after hatching (Ellis, 1988). The fact that fish eggs hatch and develop into healthy larvae and juveniles in an environment often sustained with high numbers of potential pathogenic microorganisms supports the assumption that larvae have to recognize antigens at a very early stage, resulting in immune tolerance, which is necessary for the establishment of a protective microbiota (Hansen and Olafsen, 1999). Ingestion of bacteria by fish larvae may be of immunological importance by presenting antigen determinants to the developing immune system (Rombout and van den Berg, 1985). This may result either in antigen priming or in development of immune tolerance to the sequestered bacterial strains, thereby aiding the establishment of an indigenous microbiota. In juvenile and adult fish, local mucosal and secretory immune responses are important in protection against bacterial pathogens (Hart et al., 1988), but at what developmental stage these kinds of defense mechanisms start to function is not yet clear. It has been suggested that 25

35 Chapter 1 absorptive enterocytes in the intestinal epithelium may function as an antigen-sampling device, thereby presenting antigenic determinants to intraperitoneal lymphoid cells (Rombout and van den Berg, 1989). It is thus possible that the observed endocytosis of bacterial antigens in intestinal enterocytes of cod and herring larvae (Hansen et al., 1992; Olafsen and Hansen, 1992) is involved in the stimulation of a developing non-specific immune system Techniques for microbial control in larviculture The intensive larval rearing process is characterized by the exposure of larvae to considerable stresses of chemical, physical and biological nature. High loads of organic matter and bacteria introduced with the feed and high densities of larvae and live food organisms will result in a heavy internal load of organic matter and bacteria in the tanks (Vadstein, 1997). It is well known from ecological principles that irregular supplies of organic matter tend to destabilize the bacterial community and select for opportunistic bacteria (Andrews and Harris, 1986). These conditions, in turn, produce conditions that may be detrimental to the larvae (Vadstein et al., 1993). Taking these facts into consideration, microbial management is important in the intensive larviculture. Vadstein (1997) considers three interacting factors that are required for the development of conditions that ensure good survival of fish larvae (Fig. 1.4). These factors, which include the larvae, the biological and the physio-chemical environments, are in turn influenced by several conditions. How these conditions can be manipulated, will be described in further sections. 26

36 Chapter 1 Figure 1.4 The three important factors for the probability of viable larvae (P v ) and conditions that influence these factors (After Vadstein, 1997). Vadstein et al. (1993) proposed a general strategy for microbial control that takes into account both the ecological conditions and the various aspects described in Fig This strategy is based on the concurrent use of three different elements (Table 1.2). Table 1.2 The three elements in the strategy to achieve microbial control in the rearing of marine fish larvae, adapted and amended from Vadstein et al. (1993). Possible methods are suggested for the different elements. Element Element 1: Non-selective reduction of bacteria Element 2: Selective enhancement of beneficial bacteria / suppression of pathogenic bacteria Element 3: Improvement of larval resistance Method - Use of antibiotics - Surface disinfection of eggs - Disinfection of live food - Microbially matured water - Addition of selected beneficial bacteria - Phage therapy - Quorum sensing disruption - Stimulation of non-specific defense mechanism Use of antibiotics The accelerated growth of aquaculture has resulted in a series of developments detrimental to the environment and human health. These are illustrated by the widespread and unrestricted prophylactic use of antibiotics in this industry, to forestall bacterial infections resulting from 27

37 Chapter 1 sanitary shortcomings in fish rearing. The use of a wide variety of antibiotics in large amounts, including non-biodegradable antibiotics, ensures that they remain in the aquatic environment, exerting their selective pressure for long periods of time. This process has resulted in the emergence of antibiotic-resistant bacteria in aquaculture environments, in the increase of antibiotic resistance in fish pathogens, in the transfer of these resistance determinants to bacteria of land animals and to human pathogens, and in alterations of the bacterial community both in sediments and in the water column. The use of large amounts of antibiotics that have to be mixed with fish food also creates problems for industrial health and increases the opportunities for the presence of residual antibiotics in fish meat and fish products (Cabello, 2006) Surface disinfection of fish eggs The introduction of techniques for surface disinfection of marine fish eggs has been of significant importance in many hatcheries. At SINTEF (Norway), the disinfecting properties of several bactericidal chemicals were tested, resulting in a procedure for disinfecting marine fish eggs with 400 ppm glutaraldehyde for 10 min (Salvesen and Vadstein, 1995). Several studies have documented improved hatching, development and survival of larvae from different marine fish species after egg disinfection by this procedure (Harboe et al., 1994; Salvesen et al., 1997). The use of glutaraldehyde as a disinfectant has become a standard procedure in several hatcheries in Norway Disinfection of live food Since live food organisms appear to be the main source of gut colonization in larvae, methods to reduce the bacterial numbers associated with rotifers and Artemia play an important role in microbial control strategies (Ringø and Birkbeck, 1999). Different disinfection procedures were successfully applied in Artemia hatching and enrichment (Dehasque et al., 1993; Gomez-gil et al., 1994). However, these procedures are lethal for rotifers. In this respect, 28

38 Chapter 1 several attempts have been made to reduce bacterial numbers in rotifers. Munro et al. (1999) reported that exposure of rotifers to UV radiation (38 mw cm -2 ) was effective in reducing total bacterial load by approximately 90%. Total numbers of gut-associated bacteria in turbot larvae receiving these irradiated rotifers were approximately 85% lower compared to larvae fed seawater-rinsed rotifers on day 4 post-hatch. At the low stocking density (1.4 larvae l -1 ), survival on day 20 was the highest among larvae receiving irradiated rotifers. The authors attributed this higher survival rate to a slower rate of gut colonization. Rotifers can also be disinfected by ozone treatment (Davis and Arnold, 1997). Several reports concerned the use of disinfectants (Miyakawa and Muroga, 1988; Munro, 1993b). However, most of these treatments are either not effective since they only reduce the bacterial number without avoiding the risk for transfer to the predator or can even create additional problems (e.g. formation of by-products or toxic compounds) (Dhert et al., 2001). In some cases, the treatment of rotifers with antibacterial agents has resulted in a suppressed growth of Vibrionaceae which are suspected to cause heavy mortalities to turbot larvae and early juveniles (Tanasomwang and Muroga, 1992; Minkoff and Broadhurst, 1994). Other specific treatments such as rinsing (Verdonck et al., 1991), freshwater bath treatment (Rodriguez et al., 1991), and antibiotic mixtures (Perez-Benavente and Gatesoupe, 1988; Gatesoupe, 1989; Tanasomwang and Muroga, 1989) have been reported, but rotifers were not disinfected properly. In the past decade, several studies have been directed toward obtaining axenic rotifer cultures either from disinfected resting eggs (Douillet, 1998; Rombaut et al., 1999) or from disinfected parthenogenetic eggs (Martinez-Diaz et al., 2003), by using antibiotic mixtures or different kinds of disinfectants (e.g. glutaraldehyde). Axenic rotifers obtained from these studies were used as a tool for studying the role of specific bacterial strains or microbial communities, in both nutritional and probiotic aspects. 29

39 Chapter Microbially matured water Vadstein et al. (1993) postulated that selection of a microbial community dominated by nonopportunists may be promoted by a selective recolonization of filtered water before use (a process termed microbial maturation of water ). The theoretical background for this hypothesis was based on the r/k-concept, which differentiates between opportunists (rstrategists) and non-opportunists (K-strategists) in ecosystems. Since K-strategists have a high substrate affinity and thus good competitive ability at low substrate supply compared to r- strategists, low substrate availability per bacterium promotes K-selection (Andrews and Harris, 1986). In the system designed by Skjermo et al. (1997), seawater was successively filtered through a sand filter and a tangential flow filtration system with a membrane of pore size 0.2 µm. The system reduced the number of bacteria in the water by >95%. The recolonization and selection of bacteria (mainly K-strategists) was performed in a biofilter with a surface of 230 m 2 m -3 biofilter material. The biofilter secures a large surface which permits the development of a large bacterial biomass in the system, without reaching high bacterial density in the processed water. Heavy aeration was used to secure efficient oxygenation and circulation of the water, and to prevent anoxic zones in the filter. The water temperature was controlled with a heater in the maturation tank or by regulation of the room temperature. The maturation was done at a temperature similar to the temperature used in the larval experiments. Microbially matured water has been tested in several experiments with marine fish larvae. When used in incubation of Atlantic halibut (Hippoglossus hippoglossus) yolk sac larvae, 76% higher survival and improved feeding incidence have been obtained, as well as increased reproducibility between replicates. Similarly, turbot (Scophthalmus maximus) larvae maintained in matured water showed faster growth than those maintained in membrane filtered water, and reached 51% higher weight during the experimental period (14-16 days) 30

40 Chapter 1 (Skjermo et al., 1997). Microbiological studies of turbot larvae reared in membrane filtered water have shown that the onset of the first feeding may induce a 10,000-fold increase in the number of intestinal bacteria in the turbot larvae from day 1 to day 5 post hatching (Salvesen et al., 1999). For larvae maintained in tanks with microbially matured water with algae added, the increase in bacterial density was only 10-fold during the same period, and improved larval feeding rates and growth were obtained by using matured water. Munro et al. (1993a) also reported a correlation between a slower initial colonisation rate of turbot larvae and the increased larval survival (from 4.6% to 32.4%). In addition, Vanbelle et al. (1990), Vadstein et al. (1993) and Hansen and Olafsen (1999) stated that a protective barrier effect by the primary bacterial community may secure a better immunological status of the gut function, resulting in improved appetite and nutrition Use of probiotics Selection of probiotics The acquisition of a good pool of candidate probiotics is of major importance in the selection of probiotics. It is vital in this phase that the choice of strains is made as a function of the possible role of the probiotics to be developed, although there is no unequivocal indication that putative probiotics isolated from the host or from their ambient environment perform better than isolates completely alien to the cultured species or originating from a very different habitat. However, there is an elegant logic in isolating putative probiotics from the host or the environment in which the bacteria are supposed to exert their probiotic effect. It is assumed that strains showing a dominant colonization of the intestinal mucus of fish are good candidates to competitively exclude pathogens from the adhesion sites of the gut wall. Similarly, the presence of a dominant bacterial strain in high densities in culture water indicates its ability to grow successfully under the prevailing conditions, and one can expect 31

41 Chapter 1 that this strain will compete efficiently for nutrients with possibly deleterious strains (Verschuere et al., 2000). A scheme for the development of probiotics as biocontrol agents in aquaculture was proposed by Verschuere et al. (2000). Later on, Vine et al. (2006) has developed it into a detailed protocol for the selection of intestinal probiotics in marine larviculture. The protocol involves the following major steps. a. In vitro screening By systematically conducting in vitro tests on a large number of potential probiotics, less promising candidates can be excluded, thereby reducing the number of in vivo trials required to validate the effectiveness of the probionts. Ranking the four in vitro experiments as criteria for selection of probiotics, namely production of microbial compounds, attachment to intestinal mucus, growth characteristics in mucus, and production of beneficial compounds, must be performed with caution. For example, experiments investigating the production of antagonistic compounds cannot test the candidate probiont against all potentially competitive bacteria. b. Identification As not all initially isolated bacteria are to be used as probionts, identification of candidate probiotics would not need to be performed until after screening using in vitro tests. After the putative probiotic has been tested in vivo and found to be beneficial, it should be identified down to strain level. Identification of the candidate probiotic can provide useful information regarding its culture requirements, pathogenicity and hence suitability as a probiont. c. Pathogenicity/toxicity test A probiotic must not be pathogenic or toxic to its host. This can be determined by small-scale challenge tests of the host species using short-term baths in the bacterial suspension or direct 32

42 Chapter 1 addition to the culture water. If a microorganism is to be used as a probiotic for live food organisms, tests should also be performed on the larvae to which they will be fed. d. In vivo validation Based on the definition of probiotic, a researcher must be able to re-isolate the probiotic from the intestinal tract of the larvae. If not, it cannot be discounted that larval growth and/or survival may have been improved due to other factors, for example improved water quality, rather than the ability of the probiotic to exclude opportunistic or pathogenic bacteria. It has been suggested by Verschuere et al. (2000), that candidate probiotics should be tested by challenging them through the addition of a representative pathogen. However, larval mortality can usually be attributed to opportunistic rather than obligate pathogenic bacteria (Olafsen, 2001). Hence, a natural challenge (with all naturally occurring opportunistic bacteria) would be more relevant than a challenge test with specific pathogens. e. Cost-benefit analysis If the probiotic is to be commercialized, an economic analysis of the potential commercial production is required upon successful completion of the in vivo trials. Investigation into the economic viability of different product formulations, packaging options and dosing recommendations needs to be completed In vivo study on probiotic mechanisms of action During the past two decades, the use of probiotics as an alternative to the use of antibiotic has shown to be promising in aquaculture, particularly in fish and shellfish larviculture. However, the in vivo mechanisms of action of probiotics largely remain to be unravelled. Several methodologies are suggested for further in vivo research, including the study on the gut microbiota composition, the use of gnotobiotic animals as test model, and the application of molecular techniques to study the host-microbe and microbe-microbe interactions (see a review in Chapter 2). 33

43 Chapter Immunostimulation It is believed that fish larvae do not have the ability to develop specific immunity during the early stages of development. In this respect, the larvae are reliant on passive immunization from maternal antibodies. The non-specific immune system is probably the major defence against microorganisms in larvae (Vadstein, 1997). Many different groups of substances are known to act as immunostimulants. Some immunostimulants, such as lipopolysaccharide (LPS) from Gram-negative bacteria, peptidoglycan (PG) from Gram-positive bacteria or β-glucans, may act by triggering a number of mechanisms; others (e.g. mannuronic acid polymers) may have a more restricted stimulatory effect (Espevik et al., 1993). Considerable amount of data exists on stimulation of the non-specific defense systems of fish, with the first studies appearing two decades ago. Although most studies were on adult fish, a high diversity is evident with respect to immunostimulants tested, species studied, and different response parameters used. The immunostimulants studied include crude and purified compounds of bacterial origin, various polysaccharides and synthetic chemicals with a range of freshwater, seawater and anadromous species. Response parameters include both humoral and cellular mechanisms, as well as challenges to pathogens (review by Vadstein, 1997). In a study conducted by Yusoff et al. (2001), immunostimulants of bacterial origin (PG and LPS) and egg white were incorporated in black tiger shrimp diets as feed additives. Shrimp fed with these bacterial additives and egg white showed higher weight gain and specific growth rate, and higher survival when challenged with white spot syndrome virus, compared to those fed on commercial diet without bacterial additives. Some medicinal plants can be effective immunostimulant for fish, since plant materials as food additives caused an enhanced extracellular respiratory burst activity and phagocytosis in rainbow trout (Dugenci et al., 2003). 34

44 Chapter 1 There are several studies specifically dealing with larvae. In one study (Vadstein et al., 1993), yolk sac larvae of Atlantic halibut were stimulated by immersion in an alginate rich in mannuronic acid. The stimulation lasted for the whole yolk sac period (28 days) and a considerable improvement in survival was obtained. The second study was with turbot larvae using the immunostimulant FMI (Conceicao et al., 2001). Turbot larvae fed with rotifers enriched with FMI had significantly higher fractional rates of protein synthesis when compared to a control group. The third study included the use of Artemia as a carrier of an alginate immunostimulant (Skjermo and Bergh, 2004), which increased the resistance of halibut larvae against vibriosis Bacteriophage therapy In the 1980s, following early enthusiastic but uncontrolled studies on the application of phages for prevention and treatment of human bacterial infections, epoch-making studies were carried out by Smith et al. (1987). They indicated, using Escherichia coli models with mice and farm animals, that phages could be used for both treatment and prophylaxis against bacterial infections. Potential advantages of phage treatment over chemotherapy are: (1) the narrow host range of phages, indicating that the phages do not harm the normal intestinal microbiota; (2) the self-perpetuating nature of phages in the presence of susceptible bacteria (Barrow and Soothill, 1997). Bacteriophages, as specific pathogen killers, could be attractive agents for controlling fish bacterial infections. Phages of some fish pathogenic bacteria, such as Aeromonas salmonicida, A. hydrophila, Edwardsiella tarda and Yersinia ruckeri, have been reported (review by Nakai and Park, 2002). However, only a few studies on phages have been made toward preventing bacterial infections in fish (Nakai et al., 1999; Park et al., 2000). Recently, one study has been reported on the use of Vibrio harveyi bacteriophage to control luminous vibriosis in shrimp hatchery (Vinod et al., 2006). The phage used in this study belongs to the family Siphoviridae and was isolated from shrimp farm water from the 35

45 Chapter 1 west coast of India. Microcosm studies with Penaeus monodon larvae infected with V. harveyi showed that larval survival in the presence of bacteriophage was enhanced (80%) as compared with the control (25%). Field trials were conducted in a commercial hatchery where there was a natural outbreak of luminous bacterial disease. Treatment with bacteriophage improved larval survival and brought about a decline in luminescent V. harveyi counts in hatchery tanks. 1.5 Disruption of quorum sensing a new strategy for microbial control Quorum sensing a means of bacterial communication Bacteria communicate with one another using chemical signalling molecules. They release, detect, and respond to the accumulation of these molecules, which are called autoinducers. Detection of autoinducers allows bacteria to distinguish between low and high cell population density and to control gene expression in response to changes in cell numbers. This process, termed quorum sensing, allows a population of bacteria to co-ordinately control gene expression of the entire community. Many bacterial behaviours are regulated by quorum sensing, including symbiosis, virulence, antibiotic production, and biofilm formation (Schauder and Bassler, 2001). It is shown that highly specific as well as universal quorum sensing languages exist, which enable bacteria to communicate within and between species. Species-specific quorum sensing in Gram-negative bacteria is mediated by N-acyl homoserine lactones (AHLs), which share the core homoserine lactone moiety, but distinct acyl side chains are incorporated into the signal molecules by the various LuxI-type enzymes (Fuqua et al., 2001). In Gram-positive bacteria, species-specific quorum sensing is mostly facilitated through small oligopeptides which range from 5 to 17 amino acids in length (Merritt et al., 2003). 36

46 Chapter 1 The process of quorum sensing was first described in bioluminescent marine bacterium Vibrio fischeri (Nealson and Hastings, 1979). V. fischeri lives in symbiotic associations with a number of marine animal hosts. The host uses the light produced by V. fischeri for specific purposes such as attracting prey, avoiding predators, or finding a mate. In exchange for the light it provides, V. fischeri obtains a nutrient-rich environment in which to reside (Visick and McFall-Ngai, 2000). Bioluminescence only occurs when V. fischeri is at high cell number, and this process is controlled by quorum sensing. Communication via LuxI/LuxR signalling circuits appears to be the standard mechanism by which Gram-negative bacteria communicate to each other. This quorum sensing system has been shown to control gene expression in over 25 species of Gram-negative bacteria (Bassler, 1999; Miller and Bassler, 2001). In every case, an AHL is the signal molecule whose synthesis is dependent on a LuxI-like protein. A cognate LuxR-like protein is responsible for recognition of the AHL autoinducer and subsequent transcriptional activation of downstream target genes. AHLs were shown to be associated with the quorum sensing processes in various human and plant pathogens, such as Pseudomonas aeruginosa (Rumbaugh et al., 2000), Erwinia carotovora, Agrobacterium tumefaciens (Whitehead et al., 2001), as well as Vibrio harveyi (Manefield et al., 2000) and other fish pathogens (Bruhn et al., 2005). Vibrio harveyi is a pathogenic bacterium most notably associated with diseases in cultured shrimp worldwide (Liu and Lee, 1999). Quorum sensing in this bacterium utilizes three cellsignalling systems that function in parallel to regulate positively bioluminescence (Bassler et al., 1993, 1994; Henke and Bassler, 2004b), metalloprotease (Mok et al., 2003), siderophore, and exopolysaccharide production (Lilley and Bassler, 2000), and to regulate negatively type III secretion (Henke and Bassler, 2004a), in a cell population density-dependent manner. Fig. 1.5 depicts a model for the V. harveyi quorum sensing systems. 37

47 Chapter 1 Figure 1.5 Model of the V. harveyi quorum sensing systems (After Milton, 2006). The LuxM/N system relies on N-(3-hydroxybutanoyl)-L-homoserine lactone, which is synthesized by LuxM (Bassler et al., 1993). The LuxS/PQ system utilizes the signal molecule 3A-methyl-5,6-dihydro-furo(2,3-D)(1,3,2)diox-aborole-2,2,6,6A-tetraol (termed AI-2). The unborated AI-2 precursor is synthesized by LuxS (Schauder and Bassler, 2001; Chen et al., 2002). The CqsA/S system utilizes the signal molecule CAI-1, which is dependent on CqsA for its synthesis. The chemical structure of CAI-1 is unknown (Henke and Bassler, 2004b). The three signal molecules are distinct from each other and work synergistically in gene regulation. The population density regulated behaviour using quorum sensing makes good biological sense for pathogenic bacteria, enabling them to maintain a low profile of virulence gene expression to avoid triggering host defenses until a sufficient number of cells is present to mount an effective attack. Quorum sensing may also make sense in switching from the behaviours appropriate to isolated, free-living bacteria to the behaviours appropriate to cells in a colony or a biofilm (Bauer and Robinson, 2002). 38

48 Chapter Disruption of quorum sensing There are two major strategies for the control of bacterial infection, either to kill the organism or to attenuate its virulence such that it fails to adapt to the host environment and is readily cleared by the innate host defenses. The discovery that bacteria employ a low-molecularweight pheromone, namely quorum sensing molecules, to regulate the production of secondary metabolites and virulence determinants now offers a novel target for the second approach (Finch et al., 1998). Disruption of bacterial quorum sensing has been proposed as a new anti-infective strategy and several techniques that could be used to disrupt quorum sensing have been investigated. These techniques comprise: (1) the inhibition of signal molecule biosynthesis; (2) the application of quorum sensing antagonists; (3) the chemical inactivation of quorum sensing signals; (4) signal molecule biodegradation by bacterial enzymes; (5) the application of quorum sensing agonists. The few reports that deal with disruption of quorum sensing of aquatic pathogens, together with the results obtained with human and plant pathogens, indicate that this new approach has potential in fighting infections in aquaculture (Defoirdt et al., 2004) Inhibition of signal molecule biosynthesis This technique aims at blocking signal molecule biosynthesis, in other words, inhibiting the activity of LuxI-like protein. Parsek et al. (1999) found that analogues of S- adenosylmethionine (substrate for the homoserine lactone moiety synthesis) could inhibit activity of the Pseudomonas aeruginosa LuxI homologue RhlI by up to 97%. Therefore, it might be possible to use the S-adenosylmethionine analogues as specific quorum sensing 39

49 Chapter 1 inhibitors, without affecting other vital processes in prokaryotic and eukaryotic organisms (Defoirdt et al., 2004) Quorum sensing antagonists It was the applied research for compounds capable of preventing or disrupting bacterial biofilm formation that led to the discovery of the first quorum sensing signal-mimic compounds. The marine red alga Delisea pulchra was found to produce substances that were highly effective in preventing biofouling (de Nys et al., 1995). The active compounds comprise a range of halogenated furanones. Givskov et al. (1996) recognized that these halogenated furanones were similar in structure to N-acyl homoserine lactones. They subsequently demonstrated that these halogenated furanones inhibited AHL-regulated behaviours in a variety of Gram-negative bacteria. Thus, the furanones appear to mimic the AHL signals of these bacteria, and most likely do so by binding to the AHL receptor, LuxRlike protein (Manefield et al., 1999). Although all of the D. pulchra quorum sensing mimic compounds have inhibitory effects, higher plants secrete a variety of signal mimics that stimulate quorum sensing-regulated behaviours as well as mimic substances that inhibit quorum sensing-regulated behaviours (Teplitski et al., 2000). The compounds responsible for these signal-mimic activities in higher plants have not been identified yet, but their effects seem to be specific to quorum sensingregulated gene expression (Bauer and Robinson, 2002). A large number of other AHL-based molecules were reported to have quorum sensing inhibiting activity. The AHLs with longer acyl chains identified as autoinducers in one quorum sensing system antagonize receptors normally agonized by AHLs with shorter acyl chains, as reported in Aeromonas hydrophila, Chromobacterium violaceum and Vibrio fischeri. An analogous array of racemic sulfonamides, some of which were antagonists of LuxR in V. fischeri, was also reported recently (review by Persson et al., 2005). 40

50 Chapter Chemical inactivation of quorum sensing molecules The only chemical inactivation that has been studied so far is the reaction with oxidized halogen antimicrobials. These antimicrobials, at a concentration of approximately 0.14 mm, were found to decrease the concentration of 3-oxo-substituted AHLs to about 25% after 1 min incubation, but had no effect on unsubstituted ones (Borchardt et al., 2001) Enzymatic inactivation and biodegradation of quorum sensing molecules The ability to degrade AHLs is widely distributed in the bacterial kingdom. A strain of Variovorax paradoxus capable of utilizing AHL compounds as sole carbon, nitrogen and energy source was isolated from soil by enrichment culture, suggesting that there may be various bacterial species in natural environments that can metabolize AHLs and disrupt quorum sensing regulation in nearby bacteria (Leadbetter and Greenberg, 2000). Recent studies by Dong et al. (2002) and Dong and Zhang (2005) provide some mechanistic insight how one bacterial species might interfere with quorum sensing regulation in another species. They found that about 5% of the several hundred soil bacteria tested were able to inactivate AHLs. The AHL inactivation activity in a Bacillus cereus isolate was due to its synthesis and secretion of a lactonase capable of opening the homoserine lactone ring of AHLs, thereby reducing the effectiveness of the signal molecules by about 1000-fold Quorum sensing agonistic analogues Mäe et al. (2001) has tested an opposite strategy. They activated quorum sensing-regulated virulence factor expression by using quorum sensing agonists. The idea behind this strategy is that by adding the signal molecule of a pathogen, virulence factor expression would be activated at low population density. Subsequently, the virulence factors could trigger the activation of the host defense system allowing resistance to develop (Defoirdt et al., 2004). 41

51 Chapter Limitations to manipulation of quorum sensing Specificity is important with respect to targeting just those bacterial species and behaviours that are of interest (e.g. reducing the virulence of a particular pathogen). Various bacterial species use the same or very similar quorum sensing signals, indicating that they may be broadly rather than specifically affected by mimics or inactivators of those signals. Different strains within a given bacterial species may have diverged to use different pathways of quorum sensing regulation for a particular behaviour, making it difficult to consistently target virulence in a pathogen. In addition, substance/organism that disrupts quorum sensing may unintentionally affect unexpected species, which do not synthesize AHLs, but do have an AHL receptor which activates gene expression in response to AHLs from other bacterial species (Bauer and Robinson, 2002). 1.6 Thesis outline The general objective of the present thesis is to investigate the host-microbe interactions in a rotifer-turbot larvae food chain, and the isolation and application of N-acyl homoserine lactone (AHL)-degrading bacteria as a strategy for microbial control in turbot larviculture. In Chapter 1, a literature overview is presented summarizing the current status of aquaculture and larviculture in particular, the fish-microbe interactions, and the different aspects for microbial management in larviculture, with emphasis on the interference with quorum sensing as a new strategy. Chapter 2 gives a review of the way of verifying the mechanisms of action of probiotics in vivo, and suggests several approaches for future research. Chapter 3 describes the development of a technique to obtain axenic rotifers from disinfected amictic eggs. The gnotobiotically grown Brachionus plicatilis were used as a model to test the effect of different microbial communities (MCs) and different food types. 42

52 Chapter 1 In Chapter 4, the growth-retarding (GR) effect of various Vibrio harveyi strains towards gnotobiotically cultured Brachionus plicatilis is evaluated. Since quorum sensing has shown to be important for this GR effect, the effect of a brominated furanone as quorum sensing inhibitor was also tested. Chapter 5 aims at characterizing the in vitro properties of three AHL-degrading enrichment cultures (ECs) which were isolated from shrimp digestive tract. These ECs are also analyzed using DGGE and rrna sequencing techniques, and are tested for in vivo properties in Brachionus cultures. In Chapter 6, the technique of administering these ECs as biocontrol agent in turbot larviculture is investigated. In Chapter 7, the results obtained in the previous chapters are discussed, main conclusions are drawn, and the perspectives for further research are proposed. References Andrews, J.H., and Harris, R.F. (1986) r- and K-selection in microbial ecology. Adv Microb Ecol 9: Barker, G.A., Smith, S.N., and Bromage, N.R. (1989) The bacterial flora of rainbow trout, Salmo gairdneri Richardson, and brown trout, Salmo trutta L., eggs and its relationship to developmental success. J Fish Dis 12: Barrow, P.A., and Soothill, J.S. (1997) Bacteriophage therapy and prophylaxis: rediscovery and renewed assessment of potential. Trends Microbiol 5: Bassler, B.L. (1999) How bacteria talk to each other: regulation of gene expression by quorum sensing. Curr Opin Microbiol 2: Bassler, B.L., Wright, M., Showalter, R.E., and Silverman, M.R. (1993) Intercellular signalling in Vibrio harveyi: sequence and function of genes regulating expression of luminescence. Mol Microbiol 9: Bassler, B.L., Wright, M., and Silverman, M.R. (1994) Multiple signalling systems controlling expression of luminescence in Vibrio harveyi: sequence and function of genes encoding a second sensory pathway. Mol Microbiol 13:

53 Chapter 1 Bauer, W.D., and Robinson, J.B. (2002) Disruption of bacterial quorum sensing by other organisms. Curr Opin Biotechnol 13: Bergh, Ø., Hjeltnes, B., and Skiftesvik, A.B. (1997) Experimental infection of turbot Scophthalmus maximus and halibut Hippoglossus hippoglossus yolk sac larvae with Aeromonas salmonicida subsp. salmonicida. Dis Aquat Org 29: Bergh, Ø., Naas, K.E., and Harboe, T. (1994) Shift in the intestinal microflora of Atlantic halibut (Hippoglossus hippoglossus) larvae during first feeding. Can J Fish Aquat Sci 51: Blanch, A.R., Alsina, M., Simon, M., and Jofre, J. (1997) Determination of bacteria associated with reared turbot (Scophthalmus maximus) larvae. J Appl Microbiol 82: Borchardt, S.A., Allain, E.J., Michels, J.J., Stearns, G.W., Kelly, R.F., and McCoy, W.F. (2001) Reaction of acylated homoserine lactone bacterial signalling molecules with oxidized halogen antimicrobials. Appl Environ Microbiol 67: Bruhn, J.B., Dalsgaard, I., Nielsen, K.F., Buchholtz, C., Larsen, J.L., and Gram, L. (2005) Quorum sensing signal molecules (acylated homoserine lactones) in Gram-negative fish pathogenic bacteria. Dis Aquat Org 65: Cabello, F.C. (2006) Heavy use of prophylactic antibiotics in aquaculture: a growing problem for human and animal health and for the environment. Environ Microbiol 8: Cahu, C., and Infante, J.Z. (2001) Substitution of live food by formulated diets in marine fish larvae. Aquaculture 200: Campbell, A.C., and Buswell, J.A. (1983) The intestinal microflora of farmed dover sole (Solea solea) at different stages of fish development. J Appl Bacteriol 55: Chen, X., Schauder, S., Potier, N., Van Dorsselaer, A., Pelczer, I., Bassler, B.L., and Hughson, F.M. (2002) Structural identification of a bacterial quorum-sensing signal containing boron. Nature 415: Conceicao, L.E.C., Skjermo, J., Skjåk-Bræk, G., and Verreth, J.A.J. (2001) Effect of an immunostimulating alginate on protein turnover of turbot (Scophthalmus maximus L.) larvae. Fish Physiol Biochem 24: Davis, D.A., and Arnold, C.R. (1997) Tolerance of the rotifer Brachionus plicatilis to ozone and total oxidative residuals. Ozone-Sci Eng 19:

54 Chapter 1 De Nys, R., Steinberg, P., Willemsen, P., Dworjanyn, S.A., Gabelish, C.L., and King, R.J. (1995) Broad spectrum effects of secondary metabolites from the red alga Delisea pulchra in antifouling assays. Biofouling 8: De Wolf, T., Lenzi, F., and Moretti, A. (2005) Zootechnical improvements in the larviculture of European marine fish. In: Hendry C.I., Van Stappen, G., Wille, M., Sorgeloos, P. (Eds.), Larvi '05 - Fish & Shellfish Larviculture Symposium, Oostende, Belgium. European Aquaculture Society, Special Publication No. 36, pp Defoirdt, T., Boon, N., Bossier, P., and Verstraete, W. (2004) Disruption of bacterial quorum sensing: an unexplored strategy to fight infections in aquaculture. Aquaculture 240: Dehasque, M., Devresse, B., and Sorgeloos, P. (1993) Effective suppression of bacterial bloom during hatching and enrichment of Artemia and its applicability in fish/shrimp hatcheries. In: From Discovery to Commercialization - Book of Abstracts, Torremolinos, Spain, European Aquaculture Society, Special Publication 19, p Dhert, P. (1996) Rotifers. In: Lavens P., Sorgeloos, P. (Eds.), Manual on the production and use of live food for aquaculture. Fisheries technical paper no Food and Agriculture Organization of the United Nations, Rome, pp Dhert, P., Dehasque, M., and Sorgeloos, P. (1994) Improvements in the larviculture of turbot, Scophthalmus maximus: zootechnical and nutritional aspects, possibility for disease control. In: Lavens P., Remmerswaal, R.A.M. (Eds.), Turbot Culture: Problems and Prospects. Proceedings of the Satellite Workshop of World Aquaculture '93, May 1993, Torremolinos, Spain. European Aquaculture Society Special Publication No. 22, pp Dhert, P., Divanach, P., Kentouri, M., and Sorgeloos, P. (1998) Rearing techniques for difficult marine fish larvae. World Aquaculture March 1998, pp Dhert, P., Rombaut, G., Suantika, G., and Sorgeloos, P. (2001) Advancement of rotifer culture and manipulation techniques in Europe. Aquaculture 200: Dong, Y.-H., Gusti, A.R., Zhang, Q., Xu, J.-L., and Zhang, L.-H. (2002) Identification of quorum-quenching N-Acyl homoserine lactonases from Bacillus species. Appl Environ Microbiol 68: Dong, Y.H., and Zhang, L.H. (2005) Quorum sensing and quorum-quenching enzymes. J Microbiol 43: Douillet, P. (1998) Disinfection of rotifer cysts leading to bacteria-free populations. J Exp Mar Biol Ecol 224:

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56 Chapter 1 Grace, M.F., Botham, J.V., and Manning, M.J. (1981) Ontogeny of the lymphoid organ function in fish. In: Solomon J.B. (Ed.), Aspects of Developmental and Comparative Immunology. Pergamon Press, Oxford, pp Hansen, G.H., Bergh, Ø., Michaelsen, J., and Knappskog, D. (1992) Flexibacter ovolyticus sp. nov., a pathogen of eggs and larvae of Atlantic halibut, Hippoglossus hippoglossus L. Int J Syst Bacteriol 42: Hansen, G.H., and Olafsen, J.A. (1989) Bacterial colonization of cod (Gadus morhua L.) and halibut (Hippoglossus hippoglossus) eggs in marine aquaculture. Appl Environ Microbiol 55: Hansen, G.H., and Olafsen, J.A. (1999) Bacterial interactions in early life stages of marine cold water fish. Microb Ecol 38: Hansen, G.H., Strøm, E., and Olafsen, J.A. (1992) Effect of different holding regimens on the intestinal microflora of herring (Clupea harengus) larvae. Appl Environ Microbiol 58: Harboe, T., Huse, I., and Øie, G. (1994) Effects of egg disinfection on yolk sac and first feeding stages of halibut (Hippoglossus hippoglossus L.) larvae. Aquaculture 119: Hart, S., Wrathmell, A.B., Harris, J.E., and Grayson, T.H. (1988) Gut immunology in fish: a review. Dev Comp Immunol 12: Henke, J.M., and Bassler, B.L. (2004a) Quorum sensing regulates type III secretion in Vibrio harveyi and Vibrio parahaemolyticus. J Bacteriol 186: Henke, J.M., and Bassler, B.L. (2004b) Three parallel quorum-sensing systems regulate gene expression in Vibrio harveyi. J Bacteriol 186: Hirayama, K. (1987) A consideration of why mass culture of the rotifer Brachionus plicatilis with baker's yeast is unstable. Hydrobiologia 147: Johnson, P.W., Sieburth, J.M., Sastry, A., Arnold, C.R., and Doty, M.S. (1971) Leucothrix mucor infestation of benthic crustacea, fish eggs, and tropical algae. Limnol Oceanogr 16: Jones, A. (1973) The ecology of young turbot (Scophthalmus maximus L.) at Borth, Cardiganshire, Wales. J Fish Biol 5: Kanlis, G., Suzuki, Y., Tauchi, M., Numata, T., Shirojo, Y., and Takashima, F. (1995) Immunoglobulin in oocytes, fertilized eggs, and yolk sac larvae of red sea bream. Fish Sci 61:

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67 Chapter 1 CHAPTER 1 1 INTRODUCTIONINTRODUCTION 1 INTRODUCTION Importance of aquaculture Situation of fish larviculture Overview Larviculture in Europe Larviculture in Japan Larviculture in Taiwan (review by Liao et al., 2001) Larviculture in Southeast Asia Role of live food in marine larviculture Turbot larviculture Status of European production Larviculture techniques Critical aspects of turbot larviculture Microbial management in larviculture Fish-bacteria interactions in larviculture Techniques for microbial control in larviculture Disruption of quorum sensing a new strategy for microbial control Quorum sensing a means of bacterial communication Disruption of quorum sensing Limitations to manipulation of quorum sensing Thesis outline 42 References 43 58

68 CHAPTER 2 What do we know about the functionality of probiotics in larviculture food chain? Nguyen Thi Ngoc Tinh, Kristof Dierckens, Patrick Sorgeloos, Peter Bossier Submitted

69 MINI-REVIEW Title: What do we know about the functionality of probiotics in larviculture food chain? Authors: Nguyen Thi Ngoc Tinh 1*, Kristof Dierckens 1, Patrick Sorgeloos 1, Peter Bossier 1 1 Laboratory of Aquaculture & Artemia Reference Center, Ghent University, Rozier 44, 9000 Gent, Belgium *Corresponding author: Nguyen Thi Ngoc Tinh. Laboratory of Aquaculture & Artemia Reference Center. Rozier 44, 9000 Gent, Belgium. Phone: Fax: ntngoctinh@yahoo.co.uk Running title: Probiotics in larviculture food chain Keywords: Gnotobiotic; Gut microbiota; Host-microbe interaction; Larviculture; Mechanism of action; Molecular techniques; Probiotic

70 Chapter 2 Abstract During the past two decades, the use of probiotics as an alternative to the use of antibiotic has shown to be promising in aquaculture, particularly in fish and shellfish larviculture. This paper reviews the studies on probiotics in larviculture, focussing on the current knowledge of their in vivo mechanisms of action. The paper highlights that the in vivo mechanisms of action largely remain to be unravelled. Several methodologies are suggested for further in vivo research, including the study on the gut microbiota composition, the use of gnotobiotic animals as test model, and the application of molecular techniques to study the host-microbe and microbe-microbe interactions. Introduction The term probiotic was introduced in the 1970s to describe microbial feed supplements given to both humans and animals (Berg, 1998). Later, Fuller (1989) defined probiotic as a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance. The first studies on the screening of probiotic bacteria from the aquaculture environments were reported in the late 1980s (Dopazo et al., 1988; Kamei et al., 1988). In aquaculture systems, the interaction between the microbiota and the host is not limited to the intestinal tract. Probiotic bacteria can also be active on the gills or the skin of the host or in its ambient environment. In relation to this, Verschuere et al. (2000b) have proposed a new definition, which allows a broader application of the term probiotic in aquaculture. A probiotic is defined as a live microbial adjunct which has a beneficial effect on the host by modifying the host-associated or ambient microbial community, by ensuring improved use of the feed or enhancing its nutritional value, by enhancing the host response towards disease, or by improving the quality of its ambient environment. The use of probiotics is gaining great interest in the aquaculture industry in the past two decades as one 59

71 Chapter 2 of the alternatives to the use of antibiotics, since the latter has led to the development of resistance among bacterial populations. More attention in probiotic research has recently been given to fish and shellfish larvae and to the live food organisms (Marques et al., 2006c,d; Vine et al., 2006). The microbiota colonizing the larval stage of fish and shellfish appears to be transient and reflects the microbiota of the eggs, the rearing water, and the microbiota of live food organisms during the first-feeding stage (Olafsen, 2001). Therefore, manipulation of the larval microbiota using probiotics has been suggested as a strategy to pre-emptively colonize the larvae with beneficial microorganisms (Verschuere et al., 1999). On the other hand, the probiotic concept remains controversial, since there have been relatively few well-controlled studies on the mechanisms of action of probiotics in vivo (Atlas, 1999). Various mechanisms have been proposed to explain the beneficial effect of probiotics, such as antagonism towards pathogens, competition for adhesion sites, competition for nutrients, enzymatic contribution to digestion, improvement of water quality, and stimulation of host immune responses. However, in most of the studies, the explanation for the mechanisms of action of probiotics is largely based on in vitro observations, and the beneficial effect of the probiotics on the host has been wrongly attributed to these in vitro observations, neglecting that in vivo physiology might be different from the metabolic process in in vitro monocultures. Selective ingestion by the host (Riquelme et al., 2000), death in the digestive tract (Vine et al., 2006), or a failure of a probiotic to maintain its in vitro physiology under circumstances of more complex microbial interactions and/or nutritional environment, are some of the challenges that a probiotic might face inside a host. The interactions between the introduced probiotics and the indigenous gastrointestinal (GI) microbiota are still poorly understood. A probiotic can be allochthonous or indigenous to the host microbiota. However, the establishment and the role of a probiotic in the host gut are questionable. If the probiotic is allochthonous, how could it get established among a microbial community (MC) which is 60

72 Chapter 2 already in homeostasis? If the introduced strain gets established, how long can it last if the MC has not reached a homeostasis? Even if the introduced strain is indigenous (i.e. it was isolated from the same host species), would it cause any disturbance to the established microbiota? For newly-hatched fish larvae, the pre-emptive colonization by probiotics after mouth opening may be effective in the defense against invasion by pathogens. However, would the introduction of probiotics result in an alteration in the composition of the indigenous microbiota? In addition, as the host microbial community changes in the early life stages, is there a need for isolation of life stage-adapted probiotics? There have been several reviews recently on the use of probiotics in aquaculture (Irianto and Austin, 2002; Balcazar et al., 2006; Vine et al., 2006). These authors gave a comprehensive overview of the application of probiotics in aquaculture, with some discussions on the mechanisms of action (MOA). All the authors are aware that there is a lack of correlation between the results of in vitro and in vivo studies, and information regarding the MOA of probiotics in aquaculture is incomplete. Hence, the aims of this paper are (1) to present a critical overview of in vivo studies on the MOA of probiotics in larviculture food chain, and (2) to suggest the methodologies for future study on the MOA of probiotics in the context of host-microbe and microbe-microbe interactions. In vivo studies on the mechanisms of action of probiotics Colonization of the gut epithelium Mucosal adhesion has become one of the five criteria for selection of probiotics in fish (Nikoskelainen et al., 2001). A long residence time of probiotics in the intestinal tract is supposed to extent their potential beneficial effects on the site (Lee et al., 2004). The ability of a bacterial pathogen to attach to mucosal surface has been related to its virulence (Wilson and Horne, 1986; Bruno, 1988) and is considered the first step of bacterial infection 61

73 Chapter 2 (Bengmark, 1998). Therefore, competition for attachment sites may serve as the first barrier of defence against invading pathogenic bacteria (Vine et al., 2004a,b). The ability of a strain to colonize the gut and adhere to the mucus layer is considered a good criterium for preselection of probiotics in aquaculture (reviews by Verschuere et al., 2000b; Balcazar et al., 2006; Vine et al., 2006). However, there is little information on bacterial colonization of mucosal surfaces in fish larvae, apart from a few reports, reviewed by Hansen and Olafsen (1999). The introduction of a strain of lactic acid bacteria (Lactobacillus plantarum) into the rearing water of cod larvae (Gadus morhua) changed the bacterial composition in the fish gut, through reducing the colonization by opportunistic bacteria (Strøm and Ringø, 1993). The inoculation of a probiotic strain in a culture of Penaeus vannamei at an early stage (nauplii stage V) prevented colonization by a pathogenic strain, as the probiotic had succeeded in colonizing the gut of the larvae (Zherdmant et al., 1997, reviewed by Gomez-Gil et al., 2000). However, these observations do not unambiguously demonstrate that physical exclusion from adhesion sites by the probiotics was the MOA involved. Moreover, adhesion sites might change during the early larval stages, as the maturation of the GI tract might modify the chemical composition of the mucus, and among other physico-chemical characteristics, its hydrophobicity, which is an important factor in adhesion (Lee and Puong, 2002). Hence, one might wonder whether mucus isolated from adult fish (Fjellheim, 2006) can be the ideal compound to verify the adhesion property of a probiotic that is supposed to colonize the GI tract of developing larvae. In addition, surface characteristics (e.g. hydrophobicity) of the introduced probiotic will also be a factor. The latter can be influenced by the culture conditions (e.g. medium composition) and the growth stage (Strevett and Chen, 2003). Production of inhibitory substances 62

74 Chapter 2 Different microorganisms may release chemical substances that have a bactericidal or bacteriostatic effect on other microbial populations, which in turn influences the competition for chemicals or available energy (Fredrickson and Stephanopoulos, 1981). There are numerous studies demonstrating the in vitro inhibitory effect of selected bacterial strains towards the pathogens known to occur in larviculture. However, only in a few studies, mostly undertaken in shellfish larvae, it has been demonstrated that the production of inhibitory compounds is the cause of an in vivo probiotic effect of the strains. It should be emphasized that, the production of antagonistic or inhibitory compounds against other microbiota in vitro does not guarantee that the potential probiotics will be effective in vivo in that manner (Gram et al., 2001). Maeda and Liao (1992) conducted a trial to see the effect of bacterial strains isolated from the culture water of Penaeus monodon on the growth of the shrimp larvae. Among the seven strains tested, strain PM-4, added together with a diatom, gave higher survival and moulting rates of the larvae compared to those of the larvae receiving only this diatom. It was proven later by Maeda (1994) in a biocontrol assay, that the strain PM-4 repressed the growth of Vibrio spp., most probably by producing antibacterial substances. Riquelme et al. (1997) also selected naturally occurring beneficial bacteria with the aim to promote the growth and survival of Chilean scallop larvae Argopecten purpuratus. Eleven out of 506 bacterial isolates were found to produce substances inhibitory to a Vibrio anguillarum-related (VAR) larval pathogen. One of these strains (a Vibrio sp.) was able to protect the scallop larvae against the VAR pathogen in a subsequent challenge. Similarly, a Roseobacter sp. strain (BS107) secreted an antibacterial substance against a Vibrio anguillarum strain. The antibacterial activity was highest after 48 h of culturing V. anguillarum strain in the BS107 supernatant. In vivo, the cell-free supernatant of the BS107 strain significantly enhanced the survival of scallop larvae (Ruiz-Ponte et al., 1999). An Aeromonas media strain A199 protected the 63

75 Chapter 2 Pacific oyster larvae Crassostrea gigas when challenged with Vibrio tubiashii. In addition, A199 exhibited antagonistic activity against a wide range of fish/shellfish pathogens in vitro (Gibson et al., 1998). Later on, the in vitro production of an extracellular inhibitory substance was verified by Lategan et al. (2006) as the mode of action of strain A199. The inhibitory substance was identified as an indole (2,3-benzopyrrole), which had a wide antibacterial as well as antifungal activity. Lactobacillus brevis (10 8 bacteria ml -1 ) was used to control the load of Vibrio alginolyticus in the Artemia culture water. In vitro, the extracelular products from L. brevis were able to inhibit the growth of V. alginolyticus (Villamil et al., 2003). In all of the above studies, the probiotic effect, as measured by the improved larval growth and/or survival, has been attributed to an observed in vitro effect. However, in none of these studies, the concentration of the antibacterial substances was measured in vivo, attempting to establish a dose-response relationship. Control experiments with mutant probiotic strains lacking the capability to produce the inhibitory substances could be included in order to document the in vivo MOA. Competition for chemicals or available energy Competition for nutrients or available energy can play an important role in the composition of the microbiota of the gut or in the culture water of aquatic species. To date, there have been three in vivo studies on this subject, which were conducted in generally organic-poor live food cultures and in turbot larviculture. Rico-Mora et al. (1998) selected a bacterial strain (SK-05) which has the ability to grow on organic-poor substrates and inoculated it into a diatom (Skeletonema costatum) culture, where it prevented the establishment of an introduced Vibrio alginolyticus strain. By excluding the possibility that SK-05 had an inhibitory effect on V. alginolyticus, which was proven in vitro, the authors assumed that the strain was able to outcompete V. alginolyticus due to its ability to utilize the exudates of the diatom. Verschuere et al. (1999) selected nine bacterial strains which showed positive effects on survival and 64

76 Chapter 2 growth of Artemia juveniles. The pre-emptive colonization of the culture water with these strains protected Artemia against the pathogenic effects of a Vibrio proteolyticus strain CW8T2 (Verschuere et al., 2000a). Based on the results of the experiments using the supernatant of the selected bacterial strains, which showed that no extracellular bacterial compounds were involved, the authors postulated that the protective effect of the selected bacteria was due to the competition for chemicals and available energy and not due to the production of inhibitory compounds. However, the authors did not include the experiments where dead bacteria were used, to verify if a nutritional effect is involved in this protection. Siderophores are low-molecular-weight ferric iron chelating agents which can dissolve precipitated iron or extract it from iron complexes, thus making it available for microbial growth (Neilands, 1981). Harmless bacteria which can produce siderophores can be used as probiotics to compete with the pathogens whose pathogenicity is related to siderophore production and competition for ferric iron in an iron-stressed environment (Verschuere et al., 2000b). A vibrio E was able to improve the resistance of larval turbot (Scophthalmus maximus) challenged with a pathogenic strain of Vibrio splendidus, vibrio P (experiment 1). In vitro characterization showed that vibrio E was able to grow in an iron-depleted medium while producing siderophore, as revealed by chrome azurol S staining, while vibrio P did not. Moreover, turbot larvae fed with rotifers enriched with the vibrio E s siderophore deferoxamine had higher survival after the challenge with vibrio P, in comparison with the control group (experiment 2) (Gatesoupe, 1997). By relating the results of the in vitro assay and the in vivo experiments, the author made a cautious suggestion that the probiotic effect of vibrio E may be partly due to a competition for iron with the pathogen. Nutritional contribution It has been documented in a number of animals that GI microbiota plays an important role in the nutrition of the host organisms. Thus, various means of altering the intestinal microbiota 65

77 Chapter 2 to achieve favourable effects such as enhancing growth and digesting ability of the host organism have been investigated in livestock as well as in humans (Burr et al., 2005). Several studies have documented the nutritional effect of probiotic bacteria on the growth and survival of fish and shellfish larvae. Gnotobiotic oyster larvae (Crassostrea gigas), fed with axenic algae (Isochrysis galbana) supplemented with a bacterial strain CA2, had consistently enhanced survival (21-22%) and growth (16-21%) in comparison with control cultures (Douillet and Langdon, 1993). By using gnotobiotic oyster larvae fed with axenic food, the interference of the resident GI microbiota was excluded, and the observed effect could be attributed to the strain CA2. However, the authors did not include a control treatment where the larvae were fed with dead bacteria, to verify whether the improvement in growth and survival was contributed by a probiotic or a nutritional effect. Yeasts are well known in animal nutrition since they can act as a producer of polyamines, which enhance intestinal maturation (Peulen et al., 2000). Recently, various strains of yeast have been incorporated successfully in compound diets for weaning fish larvae. Tovar- Ramirez et al. (2002, 2004) investigated the secretion of digestive enzymes in sea bass larvae (Dicentrarchus labrax) fed a compound diet supplemented with different strains of Saccharomyces cerevisiae and Debaryomyces hansenii. The growth and survival of the larvae fed yeast-incorporated diet were higher than those of the larvae fed control diet. These authors suggested that the dose-dependent effect of yeast on larval performance could be attributed to the amount of polyamines secreted by yeast in the gut lumen of larvae. However, they could not prove whether the probiotic effect of yeast was due to a stimulation of the host digestive enzymes or due to an immunostimulatory effect. Similarly, Wache et al. (2006) tested the effect of two strains of S. cerevisiae and one strain of D. hansenii as feed supplement for rainbow trout fry (Onchorynchus mykiss). S. cerevisiae var. boulardii and D. hansenii seemed to stimulate the digestive maturation in rainbow trout through an unknown metabolism, which 66

78 Chapter 2 was indicated by the increased levels of intestinal enzymes. Artemia nauplii were cultured in the presence of ten bacterial strains (dead or live) combined with four different axenic live foods (two strains of the baker s yeast S. cerevisiae and two strains of the microalga Dunaliella tertiolecta), which differ in their nutritional value (Marques et al., 2005). In combination with poor or medium-quality live foods (i.e. yeast strains), dead bacteria, constituting only 5.9% of the total food biomass, exerted a strong effect on Artemia survival. These effects were reduced or even disappeared when good-quality food sources were used, possibly due to an improvement in the health status of Artemia. In addition, some probiotic bacteria (e.g. GR8, a Cytophaga sp. strain) improved Artemia performance beyond the effect of dead bacteria, independently of the food type. Green-water effect Microalgae have been suggested to have an effect on the bacterial composition of the culture water and on the growth and survival of fish larvae, both as an antibacterial agent and as a nutritional factor. Algal addition tends to modify and stabilize the nutritional quality of the rotifers added in the first-feeding larval tanks. Microalgae which are ingested by marine fish larvae at early life stages may trigger the digestion process or may contribute to the establishment of a beneficial gut microbiota (review by Reitan et al., 1997). The addition of a microalga Isochrysis galbana in sea bass rearing water increased the survival on day 32 by 18% and 26% for the larvae fed on live prey and on compound diet, respectively, compared to that of the larvae reared without algae (Cahu et al., 1998). The enhanced survival was attributed to the effect of algae on the digestive enzyme activities at both pancreatic and intestinal levels. The authors correlated the presence of algae in the rearing water with the increase in trypsin activity from day 8 to day 16 and the increase in alkaline phosphatase and maltase on day 26. Despite this correlation, the data do not provide evidence for a direct stimulation of the fish physiology. Alternatively, the observed changes in 67

79 Chapter 2 the enzymatic activity might be the result of an indirect effect on the gut MC composition or activity. The antimicrobial activity of microalgae was reported in shrimp larviculture and in Artemia culture. The inhibitory activity by Tetraselmis suecica towards Vibrio spp. was assessed in vivo in a commercial hatchery of Indian white shrimp Fenneropenaeus indicus (Regunathan and Wesley, 2004). Rearing experiments with axenic algae proved that the inhibitory effect was by Tetraselmis suecica and not by the algae-associated bacteria. However, a reduced Vibrio count in rearing water and in larval samples could not be attributed to a direct antagonism by the algae. Hence, an improved water quality in the presence of algae might partly explain the improved performance of the shrimp larvae. Marques et al. (2006c) observed that the daily supplementation of axenic Dunaliella tertiolecta conferred a straindependent partial or full protection on gnotobiotically grown Artemia towards two pathogenic Vibrio strains. This study demonstrated that the protective effect of algae can be strainspecific, without however elucidating the exact mechanism involved. Immunostimulatory function Reports on the effect of probiotics on the immune system of fish larvae are scarce. There are two recent studies on the use of baker s yeast (S. cerevisiae) and β-glucans to stimulate the non-specific defense mechanism in Artemia nauplii (Marques et al., 2006a,d). Two strains of axenic yeast, wild-type strain and its isogenic mutant mnn9, were used as feed for gnotobiotic Artemia. The mnn9 strain carries a null mutation resulting in a lower concentration of cell wall-bound mannoproteins and higher concentrations of chitin and glucans (Magnelli et al., 2002). This yeast strain was able to protect Artemia against an opportunistic (V. proteolyticus) and a virulent (Vibrio campbellii) bacterial strain. The authors suggested that the change in the cell wall composition (in favour of high level of β-glucans), rather than the better nutritional value of the mnn9 strain, is responsible for the protection against both Vibrios. 68

80 Chapter 2 Interference with quorum sensing During the past decade, the term quorum sensing has emerged as a process of bacterial cellto-cell communication. The quorum sensing signal molecules, AHL (N-acyl homoserine lactone) and/or AI-2 (autoinducer 2), were found to be involved in the regulation of virulence factors in many pathogenic bacteria, including fish pathogens (Federle and Bassler, 2003; Morohoshi et al., 2004; Bruhn et al., 2005). Vibrio harveyi is a pathogen of many aquatic organisms (Gomez-Gil et al., 2004). This bacterium possesses three quorum-sensing pathways, which regulate the expression of genes responsible for bioluminescence (Henke and Bassler, 2004). Recently, the AI-2-mediated system was discovered to be responsible for the virulence of V. harveyi towards gnotobiotic Artemia franciscana (Defoirdt et al., 2005), while both HAI-1 (Harveyi autoinducer 1) and AI-2-mediated systems are involved in the growth-retarding effect of this bacterium towards gnotobiotic Brachionus plicatilis (Tinh et al., 2007). Although these two models (gnotobiotic Artemia and gnotobiotic Brachionus) are hard to compare directly, the results demonstrated that the V. harveyi quorum sensing systems might operate in a host-dependent way. Disruption of quorum sensing (QS) has been suggested as a new anti-infective strategy in aquaculture (Defoirdt et al., 2004). Halogenated furanones, which are produced by the marine red alga Delisea pulchra (Manefield et al., 1999), have been investigated as a promising QS antagonist. These compounds, added at adequate concentrations, protected Brachionus, Artemia and rainbow trout from the negative effects of pathogenic Vibrios (Rasch et al., 2004; Defoirdt et al., 2006; Tinh et al., 2007). On the other hand, the exploitation of probiotic bacteria, which can act as QS-disrupting agents in aquaculture systems, remains a challenge for future research. In addition, the possibility of determining the concentration of QS molecules in vivo would on one hand provide better insight on the importance of QS in vivo, and on the other hand help to elucidate the mechanism of action of the QS-disrupting bacteria. 69

81 Chapter 2 Although the importance of QS in in situ gene expression regulation seems to become more evident, several issues need to be further addressed in order to provide better insight. At present, it is common practice to report the bacterial load in larvae as CFU per fish. However, if QS is important, the value would be better expressed as CFU per millilitre of GI tract, assuming that the GI tract is the main location for the host-microbial interference. Knowledge on the density of pathogens and/or the total MC in the GI tract might help to establish whether QS can be of importance in the expression of virulence. In addition, QS is more frequently studied in terms of negative host-microbial interactions. If the potential of probiotics is to be fully exploited, we need to know more about the importance of QS molecules in their interactions with the host. In this respect, it is interesting to mention that the QS signal molecule in Pseudomonas aeruginosa, N-3-(oxododecanoyl)-L-homoserine lactone, has been reported to have immunomodulatory activity in several mammalian systems (Hooi et al., 2004; Ritchie et al., 2005; Thomas et al., 2006). Proposed approaches for future in vivo study in fish larvae Based on the discussion above, it can be stated that although the beneficial effects of probiotic have been documented, the MOAs are generally unproven. In order to gain a better insight, we need to study the following issues: (1) the establishment of probiotic in the GI tract; (2) the effect of probiotic on the gut MC composition/activity; (3) the effect of probiotic on the host metabolism including immunomodulation; and (4) the effect of the developing host on the gut MC composition/activity. Dynamics of the composition of GI microbiota Marine fish larvae start drinking rearing water for osmoregulation purpose before the yolk sac is consumed and bacteria from the water enter the digestive tract before active feeding commences. Thus, a primary transient microbiota becomes established at this stage, which is 70

82 Chapter 2 influenced by the microbiota of the egg and the microbiota of the rearing water (Olafsen, 2001). Older larvae may also ingest bacteria by grazing on suspended particles and egg debris (Olafsen and Hansen, 1992). Whereas uptake of bacteria from the water is important during the first days after hatching, uptake of bacteria associated with the food dominates at later stages (Muroga et al., 1987; Eddy and Jones, 2002). Because of the immature nature of the immune system in fish larvae, they have to rely on the non-specific defense mechanisms. A protective intestinal microbiota might constitute a complementary barrier. An understanding of the characteristics and the role of the indigenous microbiota of fish larvae may help to improve diets and incubation conditions for the intensive larval rearing (Hansen and Olafsen, 1999). Therefore, in a first approach, detailed knowledge on the composition of the microbiota is of great importance for developing strategies for better controlling survival and growth of fish larvae in intensive larviculture. Traditionally, GI microbiota has been studied using cultivation-based techniques, which are labour intensive, require knowledge of individual nutritional and growth requirements, and characterize only the cultivable microbes. Recently, findings from culture-based methods have been supplemented with those molecular techniques, which enable characterization and quantification of microbiota, as well as studies of the gut colonization by probiotics. Possible techniques include (1) immunoassays: immunocolony blot (ICB) and enzyme-linked immunosorbent assay (ELISA); (2) molecular techniques: random amplification of polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), terminal restriction fragment length polymorphism (T-RFLP), immunohistochemical methods, and denaturing gradient gel electrophoresis (DGGE) of PCRamplified 16S rrna (Spanggaard et al., 2000; Cunningham, 2002; Temmerman et al., 2003). Recently, the bacterial composition in a Pacific white shrimp hatchery in Mexico was studied using fluorescent in situ hybridization (FISH) with rrna-specific oligonucleotide probes. 71

83 Chapter 2 Bacteria belonging to γ-proteobacteria were found to dominate the total picoplankton biomass in Penaeus vannamei larval culture, from nauplii to mysis stages (Garcia and Olmos, 2007). Fjellheim (2006) is the first person who characterized the dominant gut microbiota of fish larvae (i.e. cod larvae) on an individual basis within and between different rearing conditions, by using culture dependent and culture independent approaches (T-RFLP technique). He discovered a large inter-individual variation, both quantitatively and qualitatively, between cod larvae from the same rearing tank and between the larvae from three different rearing regimes (i.e. from three different hatcheries). The author attributed this inter-individual variation to stochastic factors in the colonization of the larvae and the genetic differences between the larvae. In our opinion, the health status of the larvae could be an additional factor in creating this type of variation. Since larval mortality is generally large, it could be that the MC measured in some individuals is the MC of weak or moribund larvae. In order to clearly distinguish between the MC of a healthy larva and that of a weak larva, we should look at the MC composition in combination with a health marker for the individual larva. Probably the inter-individual variation among healthy fish larvae (although hypothetical for the time being) is considerably smaller than that among a total pool of larvae. The inter-individual variations in the gut microbiota of fish larvae add complexity to microbial management strategies. Thus, the microbial management should focus on the early stages to obtain a controlled colonization of the larvae (Fjellheim, 2006). If a probiotic is to be introduced into the fish larvae, the factors related to the genetic difference and the difference in health status of the larvae need to be taken into consideration. There are several issues to be verified. How well can the same probiont perform in the larvae of different development stages? Since the larval MC is individual-based, do we need to measure the ecological success of a probiont at the individual level as well? Does the dynamics of a MC along the larval life and the individual differences cause problems to the 72

84 Chapter 2 introduced probionts? Can this be overcome by introducing appropriate probiotic mixtures? ( revolving microbial community concept, see Verschuere et al., 2000b). Gnotobiotic animals as model systems The use of gnotobiotic systems (animals cultured in axenic conditions or with a known microbiota) can be an excellent tool to extend the understanding of the mechanisms involved in host-microbe interactions and to evaluate the new methodologies for disease control (Marques et al., 2006b). For studying the mechanism of action of a probiotic, the use of gnotobiotic animal will be essential for understanding the impact of the probiont on the composition and functioning of the gut microbiota. This might help to establish whether the effect of a probiotic is mediated through the resident microbiota versus direct signalling to the host and the impact of host genotype on probiotic responses. A combination of different methodologies for studying the host functions, that are modulated by different factors (e.g. probiotics and prebiotics, immunostimulants, antimicrobial peptides), has been employed by several research groups using gnotobiotic (or germ-free) animals. Sonnenburg et al. (2006) employed a gnotobiotic mouse model to study the three-component relationship between a host, a prominent component of its microbiota (Bacteroides thetaiotaomicron), and an intentionally introduced microbial species (Bifidobacterium longum). The results of this study revealed that the presence of B. longum elicited an expansion in the diversity of polysaccharides targeted for degradation by B. thetaiotaomicron, independently of host genotype. One of the possible explanations for this phenomenon is that, the complementary enzyme activities during the co-colonization allow the two bacterial species to synergistically harvest some glycans, which cannot be degraded if each species is present alone in the cecum. In addition, 52 genes were found to be up-regulated in the host epithelial response to the presence of both species. The group of genes showing highest expression in the co-colonized state included the members of the interferon-responsive genes, 73

85 Chapter 2 which are involved in the host innate immunity. Apart from the above study, gnotobiotic mouse model was employed widely in a number of studies on the effect of different probiotic strains, as well as in the studies on host-microbe interactions (Aiba et al., 1998; Maia et al., 2001; Prioult et al., 2003; Bjursell et al., 2006; Samuel and Gordon, 2006). The studies of gnotobiotic mice have disclosed that the gut microbiota affects a wide range of biological processes in the host. Similarly, gnotobiotic zebrafish (Danio rerio) provided an opportunity to investigate the molecular mechanisms underlying these interactions, taking advantage of its transparency during larval and juvenile stages. Rawls et al. (2004) have developed the methods for producing and rearing germ-free zebrafish through late juvenile stages. By using this model, it was revealed that 212 genes in zebrafish were regulated by the gut microbiota, and 59 responses were conserved in the mouse intestine, including those involved in stimulation of epithelial proliferation, promotion of nutrient metabolism, and innate immune responses. The gut microbiotas of zebrafish and mice share six bacterial divisions, although the specific bacteria within these divisions differ. By performing reciprocal transplantations of these microbiotas into germ-free zebrafish and mouse recipients, Rawls et al. (2006) indicated that the differences in microbiota structure between zebrafish and mice arose from the distinct selective pressures imposed within the gut habitat of each host. Since the microbiotas of zebrafish and mice have many characteristics that are conserved during evolution, zebrafish, having several unique features (Rawls et al., 2004), can be considered an excellent model for studying the microbial-attributed functions in vertebrates and aquaculture species in particular. However, this model has a disadvantage that it does not accommodate for the use of live food, making it probably less suitable as a model for marine fish larvae. The gnotobiotic models for invertebrates have also been established. Gnotobiotic oyster larvae (Douillet and Langdon, 1993), gnotobiotic Artemia (Defoirdt et al., 2006; Marques et 74

86 Chapter 2 al., 2006c,d), and gnotobiotic Brachionus (Tinh et al., 2006, 2007) have been used in different studies on host-microbial interactions. These model systems can be further exploited to understand the gene expression in the host and in the probiont and how they mutually influence each other. Apart from those general gene expression responses due to the presence of probiotics, gnotobiotic models could also be used to study in more detail the mechanisms of probiotic action. For instance, by creating bacterial mutant strains, using gene deletion techniques, the effect of probiotic strain defective in some characteristics (e.g. siderophore production, production of inhibitory compound, QS production, QS degradation) can be compared with that of the wild-type strain. Subsequently, by performing experiments using a three-component system, i.e. gnotobiotic host model resident microbiota probiotic (intact and engineered), and by evaluating the host performance and host gene expression, more convincible conclusions can be drawn on the MOA of the probiont in question. Microarray technology is a powerful tool for host gene profiling in the studies of hostmicrobe interaction, since it allows simultaneous interrogation of the transcriptional status of thousands of genes. By using this technique, it was revealed that specific host gene expression patterns reflect the differences between virulence determinant functions of different pathogens, suggesting their adaptive survival strategies inside the host (Hossain et al., 2006). In a recent study, cdna microarray was employed in a transcriptome analysis of the aquatic cnidarian dinoflagellate intracellular symbioses (Rodriguez-Lanetty et al., 2006). It was demonstrated that this type of symbiosis is maintained by altering the expression of genes involved in the vital cellular processes, such as lipid metabolism, cell proliferation, apoptosis, and oxidative stress. Microarray technique was also employed in the study with gnotobiotic zebrafish (Rawls et al., 2004), a non-aquaculture species. It is likely that a lot of genes are homologous enough so that this model can be used for aquaculture species. However, the 75

87 Chapter 2 diversity of aquaculture species is constantly growing, increasing the need for species-specific microarrays, which might be un-economical to design. Hence, molecular techniques that do not require previous genomic knowledge, such as cdna-aflp, can be helpful (Hegarty et al., 2005; de Diego et al., 2006). Green fluorescent protein (GFP) from the bioluminescent jellyfish Aequorea victoria has become an important tool in molecular and cellular biology as a transcriptional reporter or biosensor. Mutagenesis studies have generated GFP variants with new colours, improved fluorescence and other biochemical properties (Miyawaki, 2002). The application of the GFPlike proteins as a bacterial marker in microbial ecology is increasingly being used, as it is a powerful method for in situ monitoring the presence and behaviour of microbes which are intentionally introduced into the host organisms. Differential fluorescent induction in Streptococcus pneumoniae was used as a method for the discovery of genes activated in specific growth environments, using murine infection models (Bartilson et al., 2001). In addition, GFP translational fusions with genes of interest in probiotics, when introduced into translucent larvae might provide additional data on gene functioning. Conclusions Since the probiotic concept is controversial and the studies on probiotic mechanisms of action in vivo are still disperse, more in-depth research is essential. Acquisition of knowledge of the composition and dynamics of the host GI microbiota, as well as the activity of microbiota and its interactions with the host, are indispensable steps in future research, allowing for an optimized selection and use of probiotics. In this respect, gnotobiotic fish, Artemia and Brachionus can be excellent test models for studying the mechanisms of action of probiotics in larviculture systems, and specifically, for studying the behaviour of probiotics 76

88 Chapter 2 along the food chain. In addition, molecular techniques can be employed to support these studies. Acknowledgements This study was supported by a doctoral grant for candidates from developing countries (Bijzonder Onderzoeksfonds, grant number B/ DS502) by Ghent University, Belgium. References Aiba, Y., Suzuki, N., Kabir, A.M.A., Takagi, A., and Koga, Y. (1998) Lactic acid-mediated suppression of Helicobacter pylori by the oral administration of Lactobacillus salivarius as a probiotic in a gnotobiotic murine model. Amer J Gastroenterol 93: Atlas, R.M. (1999) Probiotics - snake oil for the new millennium? Environ Microbiol 1: Balcazar, J.L., de Blas, I., Ruiz-Zarzuela, I., Cunningham, D., Vendrell, D., and Muzquiz, J.L. (2006) The role of probiotics in aquaculture. Vet Microbiol 114: Bartilson, M., Marra, A., Christine, J., Asundi, J.S., Schneider, W.P., and Hromockyj, A.E. (2001) Differential fluorescence induction reveals Streptococcus pneumoniae loci regulated by competence stimulatory peptide. Mol Microbiol 39: Bengmark, S. (1998) Ecological control of the gastrointestinal tract. The role of probiotic flora. Gut 42: 2-7. Berg, R.D. (1998) Probiotics, prebiotics or "conbiotics"? Trends Microbiol 6: Bjursell, M.K., Martens, E.C., and Gordon, J.I. (2006) Functional genomic and metabolic studies of the adaptations of a prominent adult human gut symbiont, Bacteroides thetaiotaomicron, to the suckling period. J Biol Chem 281: Bruhn, J.B., Dalsgaard, I., Nielsen, K.F., Buchholtz, C., Larsen, J.L., and Gram, L. (2005) Quorum sensing signal molecules (acylated homoserine lactones) in Gram-negative fish pathogenic bacteria. Dis Aquat Org 65:

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93 Chapter 2 Rawls, J.F., Mahowald, M.A., Ley, R.E., and Gordon, J.I. (2006) Reciprocal gut microbiota transplants from zebrafish and mice to germ-free recipients reveal host habitat selection. Cell 127: Rawls, J.F., Samuel, B.S., and Gordon, J.I. (2004) Gnotobiotic zebrafish reveal evolutionarily conserved responses to the gut microbiota. Proc Natl Acad Sci USA 101: Regunathan, C., and Wesley, S.G. (2004) Control of Vibrio spp. in shrimp hatcheries using the green algae Tetraselmis suecica. Asian Fish Sci 17: Reitan, K.I., Rainuzzo, J.R., Øie, G., and Olsen, Y. (1997) A review of the nutritional effects of algae in marine fish larvae. Aquaculture 155: Rico-Mora, R., Voltolina, D., and Villaescusa-Celaya, J.A. (1998) Biological control of Vibrio alginolyticus in Skeletonema costatum (Bacillariophyceae) cultures. Aquaculture Eng 19: 1-6. Riquelme, C., Araya, R., and Escribano, R. (2000) Selective incorporation of bacteria by Argopecten purpuratus larvae: implications for the use of probiotics in culturing systems of the Chilean scallop. Aquaculture 181: Riquelme, C., Araya, R., Vergara, N., Rojas, A., Guaita, M., and Candia, M. (1997) Potential probiotic strains in the culture of the Chilean scallop Argopecten purpuratus (Lamarck, 1819). Aquaculture 154: Ritchie, A.J., Jansson, A., Stallberg, J., Nilsson, P., Lysaght, P., and Cooley, M.A. (2005) The Pseudomonas aeruginosa quorum-sensing molecule N-3-(oxododecanoyl)-Lhomoserine lactone inhibits T-cell differentiation and cytokine production by a mechanism involving an early step in T-cell activation. Infect Immun 73: Rodriguez-Lanetty, M., Phillips, W.S., and Weis, V.M. (2006) Transcriptome analysis of a cnidarian-dinoflagellate mutualism reveals complex modulation of host gene expression. BMC Genomics 7: Art. No. 23. Ruiz-Ponte, C., Samain, J.F., Sanchez, J.L., and Nicolas, J.L. (1999) The benefit of a Roseobacter species on the survival of scallop larvae. Mar Biotechnol 1: Samuel, B.S., and Gordon, J.I. (2006) A humanized gnotobiotic mouse model of hostarchaeal-bacterial mutualism. Proc Natl Acad Sci USA 103: Sonnenburg, J.L., Chen, C.T.L., and Gordon, J.I. (2006) Genomic and metabolic studies of the impact of probiotics on a model gut symbiont and host. PLOS Biology 4: Spanggaard, B., Huber, I., Nielsen, J., Nielsen, T., Appel, K.F., and Gram, L. (2000) The microflora of rainbow trout intestine: a comparison of traditional and molecular identification. Aquaculture 182:

94 Chapter 2 Strevett, K.A., and Chen, G. (2003) Microbial surface thermodynamics and application. Res Microbiol 154: Strøm, E., and Ringø, E. (1993) Changes in the bacterial composition of early developing cod, Gadus morhua (L.) larvae following inoculation of Lactobacillus plantarum into the water. In: Walther B.T., Fyhn, H.J. (Eds.), Physiology and biochemical aspects of fish development, Bergen, Norway, University of Bergen, pp Temmerman, R., Scheirlinck, I., Huys, G., and Swings, J. (2003) Culture-independent analysis of probiotic products by denaturing gradient gel electrophoresis. Appl Environ Microbiol 69: Thomas, G.L., Bohner, C.M., Williams, H.E., Walsh, C.M., Ladlow, M., Welch, M., et al. (2006) Immunomodulatory effects of Pseudomonas aeruginosa quorum sensing small molecule probes on mammalian macrophages. Mol Biosystems 2: Tinh, N.T.N., Linh, N.D., Wood, T.K., Dierckens, K., Sorgeloos, P., and Bossier, P. (2007) Interference with the quorum sensing systems in a Vibrio harveyi strain alters the growth rate of gnotobiotically cultured rotifer Brachionus plicatilis. J Appl Microbiol 102. Tinh, N.T.N., Phuoc, N.N., Dierckens, K., Sorgeloos, P., and Bossier, P. (2006) Gnotobiotically grown rotifer Brachionus plicatilis sensu strictu as a tool for evaluation of microbial functions and nutritional values of different food types. Aquaculture 253: Tovar-Ramirez, D., Infante, J.Z., Cahu, C., Gatesoupe, F.J., and Vazquez-Juarez, R. (2004) Influence of dietary live yeast on European sea bass (Dicentrarchus labrax) larval development. Aquaculture 234: Tovar-Ramirez, D., Zambonino, J., Cahu, C., Gatesoupe, F.J., Vazquez-Juarez, R., and Lesel, R. (2002) Effect of live yeast incorporation in compound diet on digestive enzyme activity in sea bass (Dicentrarchus labrax) larvae. Aquaculture 204: Verschuere, L., Heang, H., Criel, G., Sorgeloos, P., and Verstraete, W. (2000a) Selected bacterial strains protect Artemia spp. from the pathogenic effects of Vibrio proteolyticus CW8T2. Appl Environ Microbiol 66: Verschuere, L., Rombaut, G., Huys, G., Dhont, J., Sorgeloos, P., and Verstraete, W. (1999) Microbial control of the culture of Artemia juveniles through preemptive colonization by selected bacterial strains. Appl Environ Microbiol 65: Verschuere, L., Rombaut, G., Sorgeloos, P., and Verstraete, W. (2000b) Probiotic bacteria as biological control agents in aquaculture. Microbiol Mol Biol Rev 64:

95 Chapter 2 Villamil, L., Figueras, A., Planas, M., and Novoa, B. (2003) Control of Vibrio alginolyticus in Artemia culture by treatment with bacterial probiotics. Aquaculture 219: Vine, N.G., Leukes, W.D., and Kaiser, H. (2004a) In vitro growth characteristics of five candidate aquaculture probiotics and two fish pathogens grown in fish intestinal mucus. FEMS Microbiol Lett 231: Vine, N.G., Leukes, W.D., and Kaiser, H. (2006) Probiotics in marine larviculture. FEMS Microbiol Rev 30: Vine, N.G., Leukes, W.D., Kaiser, H., Daya, S., Baxter, J., and Hecht, T. (2004b) Competition for attachment of aquaculture candidate probiotic and pathogenic bacteria on fish intestinal mucus. J Fish Dis 27: Wache, Y., Auffray, F., Gatesoupe, F.J., Zambonino, J., Gayet, V., Labbe, L., and Quentel, C. (2006) Cross effects of the strain of dietary Saccharomyces cerevisiae and rearing conditions on the onset of intestinal microbiota and digestive enzymes in rainbow trout, Onchorynchus mykiss, fry. Aquaculture 258: Wilson, A., and Horne, M.T. (1986) Detection of A-protein in Aeromonas salmonicida and some effects of temperature on A-layer assembly. Aquaculture 56: Zherdmant, M.T., San Miguel, L., Serrano, J., Donoso, E., and Miahle, E. (1997) Estudio y utilización de probióticos en el Ecuador. Panorama Acuícola 2:

96 CHAPTER 3 Gnotobiotically grown rotifer Brachionus plicatilis as a tool for evaluation of microbial functions and nutritional value of different food types Nguyen Thi Ngoc Tinh, Nguyen Ngoc Phuoc, Kristof Dierckens, Patrick Sorgeloos, Peter Bossier (2006) Aquaculture 253:

97 CHAPTER 3 Gnotobiotically grown rotifer Brachionus plicatilis as a tool for evaluation of microbial functions and nutritional value of different food types Nguyen Thi Ngoc Tinh, Nguyen Ngoc Phuoc, Kristof Dierckens, Patrick Sorgeloos, Peter Bossier Aquaculture 253 (2006):

98 Chapter 3 Abstract Axenic rotifers (Brachionus plicatilis sensu stricto, clone 10) were obtained by treating amictic eggs with glutaraldehyde. Depending of the batch of rotifers, total disinfection could be obtained by exposure to ppm for one to two hours at 28 C. The hatched axenic neonates were used to test the effect of microbial communities (MCs) which were isolated from either normal-performing or crashed rotifer cultures. These MCs were either used directly or were first regrown on Marine Agar. MCs were introduced to gnotobiotic Brachionus cultures in combination with three different food types, i.e. Chlorella, wild-type baker s yeast and the mnn9 yeast mutant, which is deficient in cell wall-bound mannoprotein. In the absence of MCs or when autoclave-killed MCs were added, Chlorella was always the best food, while lower growth rates were observed with wild-type yeast and the mnn9 mutant as food. In the presence of live MCs and when rotifers were fed with Chlorella, the added MCs had no effect on rotifer performance. When yeasts were used as major food, all the tested MCs were able to increase the rotifer growth rate. The experiments with autoclavekilled MCs yielded no increase in rotifer growth rate, suggesting that the observed enhancement in rotifer growth rate was truly a probiotic effect rather than a nutritional effect. The results of this study demonstrate that gnotobiotic rotifer cultures obtained from axenic amictic eggs can be used as a test system for studying microbial-attributed as well as nutritional functions in the aquatic food chain. In addition, since the MCs originating from the crashed rotifer cultures did not decrease the growth rate in the tested rotifer cultures, it is likely that the observed crashes were not due to the presence of a standing deleterious MC. Introduction Rotifers (Brachionus spp.) have been found to be valuable and indispensable food organisms in the industrial larviculture of fish and crustaceans throughout the world (Lubzens et al., 86

99 Chapter ; Lee and Ostrowski, 2001; Liao et al., 2001; Shields, 2001; Marte, 2003). Rotifers possess several characteristics that make them suitable live prey for the newly-hatched fish larvae, e.g. relative small size, slow swimming behaviour, rapid reproduction rate, possibility to be cultured at high densities (Lubzens, 1987; Lubzens et al., 1989, 2001), possibility of bioencapsulation with highly unsaturated fatty acids, vitamins or antibiotics, which are required for the growth and survival of fish larvae (Gatesoupe, 1982). Several studies have shown that rotifers can be used for transferring probiotic bacteria to fish larvae (Makridis et al., 2000; Martinez-Diaz et al., 2003; Rombaut et al., 1999). One of the bottlenecks in using rotifers as food, is the large diversity of microbiota associated with this filter-feeding organism. Although most of the associated bacteria are not pathogenic to rotifers, they can be easily transferred via the food chain to their larval predators and can cause detrimental effects (Dhert, 1996). The dominant bacterial groups in rotifer cultures were classified as Pseudomonas, Vibrio and Aeromonas (Nicolas et al., 1989). Verdonck et al. (1997) revealed that Vibrio species were dominantly present in the microbiota of rotifer cultures. Vibrio spp. account for 45 73% of culturable bacteria in the startcultures and 15 67% in mass production. Nicolas et al. (1989) observed 10 7 CFU ml -1 of total bacteria in the culture water by direct counts, and from 10 4 to 10 5 CFU rotifer -1 were observed in the rotifers. Skjermo and Vadstein (1993) reported large variations in the number of rotifer-associated ( x 10 3 CFU rotifer -1 ) and free-living bacteria ( x 10 7 CFU ml -1 ). Hayashi et al. (1975) and Miyakawa and Muroga (1988) came up with total aerobic rotifer-associated bacterial numbers ranging from 10 7 to CFU g -1 dry weight of rotifers, while the numbers in the culture water ranged from 10 4 to 10 7 CFU ml -1. Treatment of rotifers, as well as other live food organisms, with antibiotics or disinfectants prior to feeding to the fish larvae has become a routine practice in many hatcheries, as it can improve the larval survival rates. Various chemical and physical methods for reducing the 87

100 Chapter 3 rotifer external bacterial load have been developed by Munro et al. (1993). The bacteriolytic enzyme, lysozyme, was considered as a potential decontaminating agent. Tanasomwang and Muroga (1992) have found that the Vibrio numbers rapidly declined by four log units of CFU g -1 wet weight of rotifers after five-hour exposure to sodium nyfurstyrenate. Physical methods for surface disinfection of rotifers have been widely studied, although complete decontamination can not be achieved. Exposure of rotifers to ultraviolet radiation can reduce the bacterial load by 90% within 2 min (Munro et al., 1999). Utilization of ozone-treated seawater is also suitable for rotifer disinfection, as long as the total residual oxidants (TRO) value does not exceed the NOEC (non-observable effect concentration) value (Davis and Arnold, 1997). On the other hand, the approaches of disinfection of live food as well as fish eggs or culture water may disturb the balance of microbial communities in the larval rearing environment and result in unfavourable conditions (Olafsen, 2001). In recent years, research has focused on the bioencapsulation of rotifers and other live food organisms with selected bacteria, which can favour the growth and survival of the predating fish larvae. Axenic rotifers were used as a tool for studying the role of specific bacterial strains or microbial communities, in both nutritional and probiotic aspects. The first attempts to obtain axenic cultures of rotifers were reported a few decades ago (Dougherty et al., 1960; Plasota et al., 1980). Recently, several studies have been directed toward obtaining axenic rotifer cultures either from disinfected resting eggs (Douillet, 1998; Rombaut et al., 1999) or from disinfected parthenogenetic eggs (Martinez- Diaz et al., 2003), by using antibiotic mixtures or different kinds of disinfectants. The aim of this study is to develop a technique to obtain axenic rotifer cultures from amictic eggs using disinfectants of non-antibiotic nature. Gnotobiotic rotifer cultures obtained this way are used for evaluating the effect of different microbial communities as well as the effect of food types on the rotifer growth performance. 88

101 Chapter 3 Materials and methods Rotifer strains Clones L1 and L3 of Brachionus plicatilis were obtained from the University of Valencia (Instituto Cavanilles de Biodiversidad y Biología Evolutiva) in Spain. Clone 10 was obtained from CIAD (Centro de Investigación en Alimentación y Desarrollo, Mazatlan Unit for Aquaculture) in Mexico. All the clones were confirmed to belong to the Brachionus plicatilis sensu stricto species (Papakostas et al., 2006). The rotifer stocks were maintained at controlled culture conditions: 28 C, light intensity 2000 lx, 25 g l -1 seawater, and fed with Chlorella at 1.5 x 10 6 cells ml -1. Preparation of food An axenic inoculum of Chlorella sp., strain CCAP 211/76, was obtained from the Culture Collection of Algae and Protozoa (Dunstaffnage Marine Laboratory, Dunberg) in Scotland. Axenic Chlorella was grown in closed 500 ml bottles provided with 0.22 µm-filtered aeration. The culture was maintained at 19 C, light intensity lx, using a standard Walne medium supplemented with vitamins and 0.22 µm-filtered and autoclaved regular seawater (FASW), which was diluted with tap water to have a salinity of 25 g l -1. The wild-type strain of baker s yeast (Saccharomyces cerevisiae) and its isogenic mutant strain mnn9 were obtained from EUROSCARF (Institute of Microbiology, University of Frankfurt) in Germany. The mnn9 strain is deficient in mannose chain elongation of mannoproteins resulting in a low concentration of mannoproteins in the outer layer of the cell wall (Magnelli et al., 2002; Marques et al., 2004). Axenic yeast cultures were grown in sterile Erlenmeyer s on a shaker at 150 rpm and 30 C. The culture medium used was YEPD (Yeast Extract Peptone Dextrose) medium, containing yeast extract (Sigma, 1% w/v), peptone 89

102 Chapter 3 bacteriological grade (Sigma, 1% w/v) and D-glucose (Sigma, 2% w/v). This medium was prepared in 25 g l -1 FASW. Cultures of axenic Chlorella and two yeast strains were harvested by centrifugation (1600 g for 5 min) at exponential growth phase (after seven days for Chlorella, 24 h for yeast strains). Cells were resuspended twice in sterile falcon tubes (TRP, γ-irradiated) with 10 ml of 25 g l -1 FASW. All the manipulations were performed in a laminar flow hood to maintain axenicity. Chlorella and yeast densities were determined by measuring twice the cell concentration, using a Bürker haemocytometer. Sources of microbial communities Two types of MCs were used in the experiments, which were isolated either from normalperforming or from crashed rotifer cultures. The latter were isolated from 2-L cultures which were maintained under controlled conditions (25.5 C, aeration, light regime 12 h day -1, Chlorella was used as food). The rotifer densities in these cultures dropped drastically from rotifers ml -1 to 0 rotifers ml -1 within three days. In addition, the crash conditions were preceded by very low egg ratio (< 0.1 eggs rotifer -1 ) and reduced food consumption. Resting egg production was observed in L3 culture following the crash. Experimental protocol Disinfection of rotifer amictic eggs Glutaraldehyde (Fluka, Germany) and Benzyldimethyldodecyl-ammonium bromide (BAB) (Sigma, Germany) were used as disinfectants for the rotifer culture (clone 10). Different concentrations and exposure times were tested at a temperature of 28 C, each treatment was done in triplicate (Table 3.1). The rotifer culture was first filtered through a 250-µm mesh to remove all the algal flocci which may affect the disinfectant s activity, then washed three times with an equal volume of 25 g l -1 FASW using a 60 µm mesh, before being transferred to sterile falcon tubes which contained different concentrations of disinfectants in 40 ml of 25 g 90

103 Chapter 3 l -1 FASW. The initial rotifer number was animals per tube. The swimming behaviour of the rotifers was checked every 30 min under a binocular microscope. As soon as all the rotifers were dead (i.e. no more cilia movement) in any treatment, the content of each falcon tube was filtered through an autoclave-sterilized 30-µm mesh mounted to a Büchner filter (Nalgene ). The rotifer adults were not separated from the amictic eggs. The mesh with all the retained dead rotifers and amictic eggs was subsequently placed in another falcon tube containing 40 ml of fresh 25 g l -1 FASW. The falcon tube was placed on a rotor for hatching. The culture was checked after 30 min, to verify that all the rotifers were killed. After 3 h of incubation, the presence of newly-hatched neonates was observed under a microscope. Table 3.1 Concentrations and exposure times of two disinfectants (glutaraldehyde and benzyldimethyldodecyl-ammonium bromide) used for rotifer amictic eggs disinfection Experiment 1 Experiment 2 Experiment 3 Disinfectant Concentration (ppm) Exposure time Concentration (ppm) Exposure time Concentration (ppm) Exposure time BAB 5 2h 50 1h, 2h 10 2h, 3h, 4h, 5h min, 30 min 25 1h, 2h Glutaraldehyde 10 1h 10 6h, 9h, 12h, 24h 60 2h 25 1h 25 6h, 9h, 12h, 24h 80 2h 50 1h, 2h, 3h 100 2h 100 1h After disinfection, the amictic eggs were tested for axenicity. Ten ml of the culture from each replicate was filtered through an autoclave-sterilized 30-µm mesh mounted to a Büchner filter. The mesh was then placed aseptically in a sterile plastic bag containing 10 ml of autoclaved Nine Salt Solution (NSS), which was composed of 17.6 g l -1 NaCl, 1.47 g l -1 Na 2 SO 4, 0.08 g l -1 NaHCO 3, 0.25 g l -1 KCl, 0.04 g l -1 KBr, 1.87 g l -1 MgCl 2, 0.41 g l -1 CaCl 2, g l -1 SrCl 2, and g l -1 H 3 BO 3. The content of the plastic bag was homogenized for 6 min by means of a stomacher blender (Seward, UK). Three sub-samples of 50 µl from the suspension were spread plated on Marine Agar (MA) (Difco, Detroit, USA). After 24 h of incubation at 28 C, the presence of bacterial colonies was checked. Axenicity was also 91

104 Chapter 3 verified by staining vital bacterial cells. Each sample was treated with MTT (-3-(4,5- dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Sigma, 0.5% w/v) in a sterile eppendorf (1 part of MTT to 9 parts of sample) and incubated at 30 C for 30 min. Under a light microscope (1000x magnification), the samples were checked for the presence of blue-stained viable cells (Sladowski et al., 1993). Experiments on the effect of microbial communities and effect of food types Rotifers (clone 10), hatched from axenic amictic eggs, were used in all experiments. Disinfection of amictic eggs was done using ppm of glutaraldehyde with 1-2 h exposure time at 28 C, depending on rotifer batch. After 3 h of incubation in fresh FASW, the cultures were allowed to stand for 5 min during which most of the dead rotifers were settled down. The density of newly-hatched animals was counted afterwards, and the neonates were distributed to sterile falcon tubes containing 20 ml of 25 g l -1 FASW, to have a density of 8 rotifers ml -1 at the start of each experiment. Axenic cultures of Chlorella and two yeast strains were used as food for rotifers in all the experiments. The optimum feeding level for each food type was determined in previous experiments (unpublished data). The optimum feeding levels for Chlorella, wild-type yeast and mnn9 was 150,000 cells rotifer -1, 240,000 cells rotifer -1 and 106,000 cells rotifer -1, respectively. The different feeding levels in cell number were due to the difference in the cell ash-free dry weight (Marques et al., 2004). Feeding was done once a day under a laminar flow hood, using MultiGuard TM Barrier pipette tips (Sorenson BioScience, UT, USA). Two series of experiments were conducted (Table 3.2). Live MCs (freshly-isolated or preserved at -80 C and regrown on MA) were added in the first series (Exp ). For isolation of MCs, the culture water collected from respective rotifer culture was filtered through 250-µm and 60-µm meshes to remove big food particles and all the rotifers, respectively, and was subsequently centrifuged at 1600 g for 5 min to remove the algal cells, 92

105 Chapter 3 thus only the MCs were retained in the supernatant. The MCs from normal-performing cultures were collected the same day when experiment started. There was only one experiment where the MC from crashed culture was used fresh (Exp. 1.1). Table 3.2 Outline of the experiments on the effect of microbial communities Exp Bacterial treatment Control Treatment 1 Treatment No bacteria No bacteria No bacteria No bacteria MC from clone 10 normal culture MC from clone 10 normal culture MC from clone 10 normal culture MC from clone 10 normal culture MC from L1 crashed culture MCR from L1 crashed culture MCR from L3 crashed culture MCR from clone 10 crashed culture No bacteria No bacteria No bacteria Autoclaved MC from clone 10 normal culture Autoclaved MC from clone 10 normal culture Autoclaved MC from clone 10 normal culture Autoclaved MCR from L1 crashed culture Autoclaved MCR from L3 crashed culture Autoclaved MCR from clone 10 crashed culture MC: Live and freshly-collected microbial community. MCR: Live microbial community, which was preserved at -80 C and regrown on MA. Part of the supernatants collected from L1, L3 and clone 10 crashed cultures was centrifuged at 4450 g for 15 min, the yielded bacterial pellets were resuspended in autoclaved Nine Salt Solution. These MCs were preserved for further experiments in 1 ml eppendorfs containing 20% glycerol and 80% bacterial suspension and kept at -80 C. Before starting each experiment, the eppendorfs were defrosted, subsequently 50 µl of the corresponding MC suspension was spread plated on Marine Agar. After 24 h of incubation at 28 C, the bacteria were harvested by swabbing the MA plate and resuspending in autoclaved NSS. The optical density measurement (OD 550 ) was taken for each bacterial suspension, appropriate volume to be added to each treatment was calculated in order to have a density of 10 6 CFU ml -1 at the 93

106 Chapter 3 start of each experiment. It is acknowledged that the microbial composition of the culture obtained in this way cannot be the same as the one originally available in the rotifer cultures. The second experimental series (Exp ) was run in order to investigate whether the effects observed in the first series of experiments are nutritional or probiotic/pathogenic in nature. The MCs used in these experiments, either freshly-isolated or preserved and regrown on MA, were killed by autoclaving at 121 C for 20 min before addition to the rotifer cultures. All the treatments took place in 50-ml sterile falcon tubes (TRP, γ-irradiated) containing 20 ml of 25 g l -1 FASW, with four replicates per treatment. The falcon tubes were put on a rotor (4 rpm) which was placed inside a temperature-controlled room (28 C, light intensity 2000 lx). Axenicity was tested on the starting day (day 1) and on the last day (day 5) for the experiments where no bacteria were added. Fifty µl of the culture water was spread plated on MA plate, bacterial growth was checked after incubation at 28 C for 24 h. Axenicity was also checked by bacterial staining using MTT. All the results were discarded if contamination was found in any replicate. Data analysis Two sub-samples of 500 µl were withdrawn daily from each replicate for estimation of rotifer densities. Population growth rate was calculated as: µ = (lnn t lnn o ) / t where N o is the initial rotifer density, N t is the rotifer density on day t of culture, t is the duration in days. Parametric assumptions were evaluated using Levene s test for homogeneity of variances and Shapiro-Wilk s test for normality. As all the data were normal-distributed and homoscedastic, the growth rates on day 5 were compared by food type between treatments using one-way ANOVA, followed by Tukey test. The interaction between food type and bacterial treatment 94

107 Chapter 3 was evaluated using two-way ANOVA. All the tests were performed using the computer program SPSS release Results Disinfection of rotifer amictic eggs Different concentrations and different exposure times were tested for two kinds of disinfectants (glutaraldehyde and BAB). The effectiveness of each treatment was evaluated based on the observation of the following parameters: the mobility of adult rotifers after exposure time, the hatchability after incubation in new seawater, the bacterial count of amictic eggs after disinfection. The results of disinfection treatments with BAB are presented in Table 3.3. The rotifers were found dead in all the treatments after a certain exposure time. However, hatchability of amictic eggs was zero in the treatments with high concentrations of disinfectant or with prolonged exposure times. In addition, axenicity could not be obtained in any treatment. Hence, BAB is not suitable for disinfection of rotifer amictic eggs. Table 3.3 Effectiveness of BAB as a disinfectant for rotifer amictic eggs Concentration Exposure time Mobility after exposure to disinfectant Hatchability of treated amictic eggs Axenicity of treated amictic eggs MA plate count (log CFU ml -1 ) (Mean ± SD) 5 ppm 2h ± ppm 2h ± h ± h ± h ± ppm 1h ± h ± ppm 1h ± h ± ppm 15 min ± min ±

108 Chapter 3 Table 3.4 shows the results of glutaraldehyde treatment with different combinations of concentration and exposure time. The concentration of 10 ppm at 1 h, 6 h and 9 h exposure time and the concentration of 25 ppm at 1 h exposure time were not sufficient to kill all the rotifer adults. In contrast to BAB, all of the treatments did not affect the hatchability of amictic eggs. However, only one treatment (100 ppm at 2 h exposure time) showed a complete elimination of the microbiota associated with amictic eggs. The four treatments 50 ppm, 60 ppm, 80 ppm and 100 ppm were later repeated four times for different rotifer cultures (results not shown). In all the cases, the hatched rotifers showed normal swimming behaviour and ability to produce eggs afterwards. However, the disinfecting efficacy varied between rotifer batches, probably depending on the residual organic matter load as well as the bacterial load in the culture water and in the rotifer s gut, which cannot be eliminated during the washing process. For one rotifer batch 50 ppm glutaraldehyde during 1 h was sufficient to eliminate the egg-associated microbiota, for another batch only 100 ppm during 2 h was effective. Table 3.4 Effectiveness of glutaraldehyde as a disinfectant for rotifer amictic eggs Concentration Exposure time Mobility after exposure to disinfectant Hatchability of treated amictic eggs Axenicity of treated amictic eggs MA plate count (log CFU ml -1 ) (Mean ± SD) 1h + 6h + 10 ppm 9h + 12h ± h ± h + 6h ± ppm 9h ± h ± h ± h ± ppm 2h ± h ± ppm 2h ± ppm 2h ± ppm 1h ± h

109 Chapter 3 Evaluation of nutritional effect of different food types in axenic rotifer cultures The effect of three different food types (Chlorella, wild-type (WT) yeast and mnn9 yeast mutant) on rotifer performance was evaluated based on the results of the second experimental series, in the absence of bacteria or when autoclave-killed MCs were added (Table 3.5). Rotifers fed on algae had the highest growth rate compared to those fed on yeast strains (p < 0.001). These results were highly reproducible. On the other hand, no significant difference was observed between the treatments with yeast strains (p > 0.05). Table 3.5 Growth rate over 5 days (mean ± SD, n = 4) of Brachionus plicatilis (clone 10) hatched from disinfected amictic eggs and fed three types of food: effect of food type (see Table 3.2 for experimental outline) Exp. Food type Treatment No bacteria Autoclaved MC Autoclaved MCR 2.1 Chlorella Wild-type yeast mnn9 yeast mutant 0.60 ± 0.02 b 0.25 ± a 0.28 ± 0.03 a 0.59 ± 0.02 b 0.26 ± 0.01 a 0.30 ± 0.04 a 0.57 ± 0.04 b 0.26 ± 0.03 a 0.29 ± 0.02 a 2.2 Chlorella Wild-type yeast mnn9 yeast mutant 0.69 ± 0.03 b 0.22 ± 0.02 a 0.26 ± 0.02 a 0.68 ± 0.05 b 0.24 ± 0.04 a 0.25 ± 0.04 a 0.69 ± 0.03 b 0.24 ± 0.02 a 0.28 ± 0.04 a 2.3 Chlorella Wild-type yeast mnn9 yeast mutant 0.66 ± 0.04 b 0.35 ± 0.07 a 0.33 ± 0.09 a 0.66 ± 0.02 b 0.36 ± 0.02 a 0.33 ± 0.10 a 0.66 ± 0.05 b 0.34 ± 0.03 a 0.30 ± 0.08 a Treatments with different superscripts in each experiment are significantly different from each other (Tukey test, p < 0.05). Evaluation of the probiotic/nutritional effect of microbial communities In the first series of experiments, the effect of different live MCs isolated either from normalperforming or from crashed rotifer cultures was evaluated. Fig. 3.1 represents the rotifer growth rates over five days for each food type. Comparisons were made between bacterial treatments versus control treatment (no bacteria). 97

110 Chapter 3 Exp ,0 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0,0 e Growth rat *** ** *** *** *** *** Chlorella WT yeast mnn9 Control Treatment 1 Treatment 2 Exp ,0 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0,0 Growth rate *** *** Chlorella WT yeast mnn9 Control Treatment 1 Treatment 2 Exp ,0 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0,0 Growth rate *** *** ** Chlorella WT yeast mnn9 Control Treatment 1 Treatment 2 * Exp ,0 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0,0 Growth rate *** *** Chlorella WT yeast mnn9 Control Treatment 1 Treatment 2 98

111 Chapter 3 Figure 3.1 Growth rate over 5 days (n = 4) of Brachionus plicatilis (clone 10) hatched from disinfected amictic eggs and fed three types of food: effect of the addition of live MCs (see Table 3.2 for more information on the treatments). Error bars indicate standard deviation. Asterisks denote significant differences compared to control treatment (Tukey test). Single asterisk (*) indicates p < 0.05; double asterisk (**) indicates p < 0.01; triple asterisk (***) indicates p < No significant stimulation of the growth rate by both types of MCs was found (p > 0.05) in the treatments where Chlorella was used as food source, except in experiment 1.1. In contrast, differences were seen when the two yeast strains were used as food. Growth rates were significantly improved (p < 0.001) when MCs from normal-performing cultures were added. The behaviour of MCs from crashed cultures was more variable, since they were collected from the cultures of different rotifer strains, and were utilized under two different forms: freshly-isolated or preserved at -80 C and regrown afterwards on MA. When comparing by origin of the preserved MCs, only the addition of MCR from L3 crashed culture showed a significant improvement (p < 0.05) in growth rate (Exp. 1.3, in the treatments where WT yeast or mnn9 was used). MC from L1 crashed culture was used in two forms. The fresh-isolated MC could stimulate the growth performance significantly (p < 0.001, Exp. 1.1, treatment 2), while no stimulation was found (p > 0.05) when that MC was preserved and regrown before use (Exp. 1.2, treatment 2). The experimental setup of the second series of experiments was similar to that of the first series. The only difference was that autoclave-killed instead of live MCs were added, allowing to investigate whether the change in growth rate observed in the first experimental series was solely the consequence of the extra nutrients added through the MCs. As shown in Fig. 3.2, no significant difference in growth rate was found (p > 0.05) either in the presence or absence of autoclave-killed MCs. This observation was reproducible for all three types of food and in all experiments. 99

112 Chapter 3 Exp ,0 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0,0 Growth rate Chlorella WT yeast mnn9 Control Treatment 1 Treatment 2 Exp ,0 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0,0 Growth rate Chlorella WT yeast mnn9 Control Treatment 1 Treatment 2 Exp ,0 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0,0 Growth rate Chlorella WT yeast mnn9 Control Treatment 1 Treatment 2 Figure 3.2 Growth rate over 5 days (n = 4) of Brachionus plicatilis (clone 10) hatched from disinfected amictic eggs and fed three types of food: effect of the addition of autoclave-killed MCs (see Table 3.2 for more information on the treatments). Error bars indicate standard deviation. No significant differences were found between the treatments (p > 0.05, Tukey test). 100

113 Chapter 3 Results of the interaction between food type and bacterial treatment on Brachionus growth rate on day 5, as analyzed by two-way ANOVA, are given in Table 3.6. A strong interaction (p < 0.001) was found in experiments 1.2, 1.3 and 1.4. However, no interaction was observed (p > 0.05) when dead MCs were added (Exp ). Table 3.6 Interaction between food type and bacterial treatment (p value) on Brachionus plicatilis growth rate over 5 days Interaction Experiment Food type * Bacterial treatment Food type * Bacterial treatment Exp Exp Exp Exp Exp Exp Exp Discussion Two disinfectants with strong biocidal property were used in this study. Glutaraldehyde is a disinfectant which displays a broad spectrum of activity and a rapid killing rate against the majority of microorganisms. It is capable of destroying all forms of microbial life including bacterial and fungal spores, tubercle bacilli and viruses. The mode of action of glutaraldehyde is based on the formation of intercellular bonds with the outer layers of bacterial cells or bacterial spores and, in this way, on the interference with the functionality of the cell wall (Scott and Gorman, 1991). Glutaraldehyde is widely used in disinfection of marine fish eggs before incubation. Salvesen and Vadstein (1995) found that glutaraldehyde was the most promising candidate of four disinfectants (Buffodine, glutaraldehyde, chloramine-t and sodium hypochlorite) tested on plaice eggs. Benzyldimethyldodecyl-ammonium bromide (BAB) belongs to the group of quaternary ammonium compounds (QACs). By binding to the phospholipid and protein layers, QACs impair the permeability of the cell membrane (Maris, 1995). 101

114 Chapter 3 While a good disinfectant should sterilize amictic eggs completely, it should not affect the viability of amictic eggs neither the mobility of hatched rotifers. In our study, none of the BAB treatments met both axenicity and zootechnical criteria. The amictic eggs ceased to hatch at high concentrations of BAB, probably due to toxicity. In addition, such high concentrations were still not sufficient to kill all the bacteria in the culture. For glutaraldehyde, appropriate conditions could be identified, allowing obtaining axenic amictic eggs that were still able to hatch after 3 h incubation and to develop further normally. Depending on the batch, rotifers should be exposed to ppm glutaraldehyde for 1-2 h at 28 C. Bacteria-free organisms are being used as a tool for studying microbiota-attributed functions. It allows studying the probiotic potential or pathogenicity of selected strains in a gnotobiotic environment (Martinez-Diaz et al., 2003; Marques et al., 2006). Germ-free organisms are also useful for the studies of nutritional requirements (Scott, 1983). Attempts to make axenic rotifer cultures from resting eggs were conducted by several authors using different kinds of disinfectants. Hagiwara et al. (1994) suggested soaking the resting eggs in 0.5 ppm and 0.25 ppm sodium hypochlorite solution for 60 min and 30 min, respectively. Douillet (1998) found that the disinfection of resting eggs was most effective at much higher concentration of sodium hypochlorite (0.5%) and shorter exposure time (3 min). Rombaut et al. (1999) tested the effect of merthiolate and glutaraldehyde. The latter was found to be effective at low concentration (0.05 ppm) and prolonged incubation time (6 h). Some authors went for another approach, using amictic eggs as material for starting an axenic culture. Most of them used antibiotic mixtures as disinfecting medium (Plasota et al., 1980; Hirayama and Funamoto, 1983; Dhert, 1996), which is not desirable in the light of antibiotic resistance among bacterial populations in rotifer cultures. Martinez-Diaz et al. (2003) tried out two disinfectants (PVP-Iodine and Hydrogen peroxide) and two antibiotic mixtures, 102

115 Chapter 3 starting either with adult females or amictic eggs. However, only the antibiotic treatment applied to amictic eggs was effective in eliminating the associated bacteria, while sustaining the viability of amictic eggs. Hence, the method applied in this study has several advantages. Firstly, axenic rotifer cultures can be obtained from amictic eggs of one single clonal strain, allowing for instance to obtain an axenic culture from a strain that does not produce resting eggs under lab conditions. It also avoids using resting eggs which are normally costly and may contain (if collected in the field) an uncharacterized rotifer mixture. Secondly, a non-antibiotic disinfectant (glutaraldehyde) is used, which eliminates the risk of antibiotic resistance build-up. Several studies investigated the effect of different diets on the growth as well as on the dietary value of rotifers to the fish larvae. Those studies indicate that green microalgae such as Chlorella sp. and Nannochloropsis sp. are the most suitable diets for rotifers. Moreover, rotifers fed on these algae can satisfy the nutritional demand of fish larval predators. Yeast can only be used at low concentrations to supplement algal requirement due to its deficiency in HUFA content (Caric et al., 1993; Tamaru et al., 1993; Sarma et al., 2001), or rotifers can be grown on yeast and then enriched with DHA or EPA (Dhert, 1996). In the present study, the mnn9 mutant which is defective in mannoprotein synthesis was chosen for evaluation of its nutritional effect on rotifer growth in comparison to the wild-type yeast. In a similar study done on Artemia nauplii, Marques et al. (2004) found a strong negative correlation between the yeast cell wall s mannoprotein content and Artemia performance. According to Telford (1970) and Coutteau et al. (1990), β-glucanase activity is detected in the digestive tract of Artemia, but no mannase activity was found. This finding coincided with the poor performance of Artemia fed on wild-type yeast, suggesting that the external mannoprotein layer of the yeast cell wall presents the major barrier to digestion. However, this was not the case for rotifers, as shown in the present study. Rotifer 103

116 Chapter 3 performance was statistically equal when a wild-type yeast strain or a mnn9 mutant was given as food. Biochemical studies on hydrolytic enzymes from the rotifer Brachionus plicatilis revealed the presence of β-1,3-glucanase and chitinase (Kleinow, 1993; Hara et al., 1997). There is no evidence of mannase activity in rotifers. However, as opposed to Artemia in which a mandibular grinding of food particles is unknown (Coutteau et al., 1990), the mastax organ which is present in rotifers (Dhert, 1996) may help to break the yeast cell wall, facilitating the access of digestive enzymes to the internal layers. Axenically grown rotifers were used in this study as a test model to reveal the role of indigenous microbiotas which were isolated from rotifer cultures. When yeast strains were given as food, rotifer growth performance was dependent on the MCs origin. Yet, the nature of MC had a strong influence. The effect on growth rate was significant and was reproducible when MCs were fresh-isolated, irrespective of the source where they were isolated from (i.e. normal-performing or crashed rotifer culture). In the other experiments with live MCs, all MCRs (regrown microbial communities) were not able to stimulate the growth of Brachionus (except in one case, when the MCR originated from L3 crashed culture), suggesting that regrown MCs have a less favourable effect or no effect on Brachionus even when low quality food (such as yeast cells) is used. In one particular case it was possible to directly compare the effect of a MC (Exp. 1.1, treatment 2) and the effect of the corresponding regrown MC (Exp. 1.2, treatment 2) (although these experiments were performed after each other, which is for logistical reasons unavoidable; the MC from exp. 1.1 would always need to be preserved in some way or another in attendance of the MCR of exp. 1.2). In this single case the MCR effect was not significant, while the effect of the corresponding MC was significant. Overall, it can be concluded that the presence of a MC is essential for good rotifer growth when suboptimal food is used. Yet, the source of MC attenuates the extent of the beneficial effect (direct use of MCs versus sub-cultured MCs). 104

117 Chapter 3 The results of the second experimental series seem to exclude the role of microbiota as an initial and additional source of nutrients for rotifers, although it has to be acknowledged that through autoclavation nutrients might be deprived from the cytoplasmic content of the microbial cells. It is possible that bacterial enzymes may help to improve the yeast digestibility, thus improving the rotifer growth, or that live bacteria have a direct effect on the rotifer metabolism. Bacterial enzymes, such as N-acetyl-β-glucosaminidase (chitinolytic activity) and β-glucosidase (cellulolytic activity), were detected in relatively high concentrations in the putative probiotic isolates (Rombaut, 2001), indicating a possible role in the digestion of yeast, which synthesizes chitin as a cell wall constituent (Smits et al., 1999). An additional reason for the beneficial effect of live bacteria might lay in their eventual capacity to grow on the yeast or Chlorella degradation product, generating new microbial biomass that can serve as food for rotifers. This nutrient recycling might also have a beneficial effect on the water quality. The third hypothesis is that bacterial cell wall material might induce a change in digestion competence in the rotifers, resulting in better growth rate. Associated microbiotas have been shown to affect a wide range of biological processes in the host organism, as revealed in the assay with gnotobiotic zebrafish (Rawls et al., 2004). Although the present experimental setup does not allow to clearly distinguish between the nutritional and probiotic effect of bacteria on rotifers, it does strongly suggest that the killed bacterial cells (some of them might have lost the cytoplasmic content) alone do not promote the growth of rotifers. In the present study, the MCs isolated from crashed rotifer cultures did not show any negative effect in the rotifer growth test, suggesting that the MCs associated with these particular crashed rotifer cultures were not responsible for the crash. This is in contradiction with the postulation of many authors, that the microbiota associated with rotifer production systems plays a major role in the instability and variability of the rotifer cultures (Hirayama, 1987; 105

118 Chapter 3 Gatesoupe et al., 1989; Gatesoupe, 1991; Skjermo and Vadstein, 1993; Harzevilli et al., 1997). Some bacterial strains such as Plavobacterium sp., Aeromonas sp. and Vibrio anguillarum were isolated from collapsing rotifer cultures and showed pathogenicity for the rotifer population (Harzevilli et al., 1997; Balompapueng et al., 1997). Hino (1993) formulated a more subtle hypothesis, suggesting that changes in the composition of the MC, and not the standing microbiota itself, are the cause of the collapse of rotifer cultures. Rapid succession in the MC was observed during batch culture of rotifers (Maeda and Hino, 1991). Shifts in the fingerprinting of the MC in the rotifer culture water, as revealed by means of denaturing gradient gel electrophoresis (DGGE), were found to be often associated with technical problems resulting in a reduced water quality in the rotifer production systems (Rombaut et al., 2001). The present results seem to support the view that the standing MCs, isolated either from a normal-performing rotifer culture or from a crashing rotifer culture, have no negative influence on the rotifer performance. Our experimental setup, however, does not allow to either support or negate the hypothesis that a rapid changing MC can be a facilitator of rotifer culture crashes. The results do suggest that regrowing a MC in a rich medium, such as Marine Agar (most probably resulting in a shift of MC s composition), is reducing the beneficial effect of that MC, both when Chlorella or yeast is used as the main food source. Future research, with other MCs isolated from especially freshly-crashed rotifer cultures, might further substantiate this hypothesis. Conclusions The glutaraldehyde treatment of amictic eggs represents a novel approach for obtaining axenic rotifers, as a disinfectant of non-antibiotic nature is used. This technique can be used for rotifer strains that do not produce resting eggs. Axenic rotifers obtained this way have shown to be an excellent tool for studying probiotic and nutritional function of 106

119 Chapter 3 microorganisms, as well as nutritional value of different food sources. It was proven that the presence of an indigenous microbiota is essential for the growth of rotifers, especially when low quality food (yeast) is offered. Such an effect could be obtained with a microbial community originating from a normal or a crashed culture, but not with a MCR, namely MC regrown in the lab on rich medium. The results further suggest that the MC isolated from one particular crashed rotifer culture was probably not responsible for the crash, as no negative effects were observed by adding this MC to axenic rotifer cultures. This of course does not exclude the possibility that the crashes observed in rotifer cultures are due to the presence of certain microorganisms. Acknowledgements This study was supported by a doctoral grant for candidates from developing countries (Bijzonder Onderzoeksfonds, grant number B/ DS502) given to the first author by Ghent University, Belgium, and in part by the EU ROTIGEN project QLRT ( The authors thank Serra M. and Roque A. (Spain) for providing rotifer strains, the Department of Genetics, Development and Molecular Biology, Aristotle University of Thessaloniki, Greece (a partner of Rotigen project), for identifying the rotifer clones. Special thanks go to Jean Dhont and Mathieu Wille for critical reading the manuscript. 107

120 Chapter 3 References Balompapueng, M.D., Hagiwara, A., Nishi, A., Imaizumi, K., and Hirayama, K. (1997) Resting egg formation of the rotifer Brachionus plicatilis using a semi-continuous culture method. Fish Sci 63: Caric, M., Sankonjire, J., and Skaramuca, B. (1993) Dietary effects of different feeds on the biochemical composition of the rotifer (Brachionus plicatilis Müller). Aquaculture 110: Coutteau, P., Lavens, P., and Sorgeloos, P. (1990) Baker's yeast as a potential substitute for live algae in aquaculture diets: Artemia as a case study. J World Aquaculture Soc 21: 1-8. Davis, D.A., and Arnold, C.R. (1997) Tolerance of the rotifer Brachionus plicatilis to ozone and total oxidative residuals. Ozone-Sci & Engin 19: Dhert, P. (1996) Rotifers. In: Lavens, P., Sorgeloos, P. (Eds.), Manual on the production and use of live food for aquaculture. FAO technical paper, pp Dougherty, E.C., Solberg, B., and Harris, L.G. (1960) Synxenic and attempted axenic cultivation of rotifers. Science 132: Douillet, P.A. (1998) Disinfection of rotifer cysts leading to bacteria-free populations. J Exp Mar Biol Ecol 224: Gatesoupe, F.J. (1982) Nutritional and antibacterial treatments of live food organisms: the influence on survival, growth rate and weaning success of turbot (Scophthalmus maximus). Ann Zootech 31: Gatesoupe, F.J. (1991) The effect of three strains of lactic bacteria on the production rate of rotifers, Brachionus plicatilis, and their dietary value for larval turbot, Scophthalmus maximus. Aquaculture 96: Gatesoupe, F.J., Arakawa, T., and Watanabe, T. (1989) The effect of bacterial additives on the production rate and dietary value of rotifers as food for Japanese flounder, Paralichthys olivaceus. Aquaculture 83: Hagiwara, A., Hamada, K., Hori, S., and Hirayama, K. (1994) Increased sexual reproduction in Brachionus plicatilis (Rotifera) with the addition of bacteria and rotifer extracts. J Exp Mar Biol Ecol 181: 1-8. Hara, K., Pangkey, H., Osatomi, K., Yatsuda, K., Hagiwara, A., Tachibana, K., and Ishihara, T. (1997). Some properties of β-1,3-glucan hydrolyzing enzymes from the rotifer Brachionus plicatilis. Hydrobiologia 358:

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124 Chapter 3 and survival of striped mullet (Mugil cephalus) and milkfish (Chanos chanos) larvae. Aquaculture 110: Tanasomwang, V., and Muroga, K. (1992) Effect of sodium nifurstyrenate on the reduction of bacterial contamination of rotifers (Brachionus plicatilis). Aquaculture 103: Telford, M. (1970) Comparative carbohydrase activities of some crustacean tissue and whole animal homogenates. Comp Biochem Physiol 34: Verdonck, L., Grisez, L., Sweetman, E., Minkoff, G., and Sorgeloos, P. (1997) Vibrios associated with routine production of Brachionus plicatilis. Aquaculture 149:

125 CHAPTER 4 Interference with the quorum sensing systems in a Vibrio harveyi strain alters the growth rate of gnotobiotically cultured rotifer Brachionus plicatilis Nguyen Thi Ngoc Tinh, Nguyen Dieu Linh, Thomas K. Wood, Kristof Dierckens, Patrick Sorgeloos, Peter Bossier (2007) Journal of Applied Microbiology 102

126 Interference with the quorum sensing systems in a Vibrio harveyi strain alters the growth rate of gnotobiotically cultured rotifer Brachionus plicatilis Nguyen Thi Ngoc Tinh, Nguyen Dieu Linh, Thomas K. Wood, Kristof Dierckens, Patrick Sorgeloos, Peter Bossier Journal of Applied Microbiology (In press: doi: /j x)

127 Chapter 4 Abstract The aim of this study is to evaluate the effect of Vibrio harveyi strains on the growth rate of the gnotobiotically cultured rotifer Brachionus plicatilis, and to establish whether quorum sensing is involved in the observed phenomena. Gnotobiotic Brachionus plicatilis sensu stricto, obtained by hatching glutaraldehyde-treated amictic eggs, were used as test organisms. Challenge tests were performed with eleven Vibrio harveyi strains and different quorum sensing mutants derived from the Vibrio harveyi BB120 strain. Brominated furanone [(5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone] as a quorum sensing inhibitor was tested in Brachionus challenge tests. Some Vibrio harveyi strains, such as strain BB120, had a significantly negative effect on the Brachionus growth rate. In the challenge test with MM77, an isogenic strain of BB120 in which the two autoinducers (HAI-1 and AI-2) are both inactivated, no negative effect was observed. The effect of single mutants was the same as that observed in the BB120 strain. This indicates that both systems are responsible for the growth-retarding (GR) effect of the BB120 strain towards Brachionus. Moreover, the addition of an exogenous source of HAI-1 or AI-2 could restore the GR effect in the HAI-1 and AI-2 non-producing mutant MM77. The addition of brominated furanone at a concentration of 2.5 mg l -1 could neutralize the GR effect of some strains such as BB120 and VH-014. In conclusions, two quorum sensing systems in Vibrio harveyi strain BB120 (namely HAI-1 and AI-2-mediated) are necessary for its GR effect in Brachionus plicatilis cultures. With some other Vibrio harveyi strains, however, the growth inhibition towards Brachionus does not seem to be related to quorum sensing. Interference with the quorum sensing system might help to counteract the GR effect of some Vibrio harveyi strains on Brachionus. However, further studies are needed to demonstrate the positive effect of halogenated furanone in non- 114

128 Chapter 4 gnotobiotic Brachionus cultures and eventually, in other applications of the aquaculture industry. Introduction The rotifer Brachionus plicatilis is being used as valuable and indispensable live food organisms in the industrial larviculture of many marine fish and shrimp species (Dhert et al., 2001; Liao et al., 2001; Lubzens et al., 2001; Shields, 2001; Marte, 2003). One of the constraints in rotifer culture is the large diversity of microbiota associated with this filterfeeding organism (Skjermo and Vadstein, 1993; Verdonck et al., 1997; Savas et al., 2005), which increases the risk of contaminating the fish larvae with opportunistic pathogens such as Vibrio sp., Aeromonas sp. and Pseudomonas sp. (Gatesoupe, 1990). Since the gastrointestinal microbiota of the early life stages of fish larvae is influenced by the microbiota associated with the live feed (Ringø and Birkbeck, 1999), the opportunistic bacteria associated with the enrichment process may cause detrimental effects when rotifers are fed to the fish larvae (Skjermo and Vadstein, 1993). The microbiota associated with rotifer production systems was considered to play a major role in the instability and variability of rotifer cultures (Hirayama, 1987; Skjermo and Vadstein, 1993; Harzevilli et al., 1997). Dhert et al. (2001) suggested that the problem of unexplained crashes in batch cultures can be partly solved by bacterial management. In a recent study, Tinh et al. (2006) demonstrated that a standing microbial community of a crash culture is not necessarily responsible for a rotifer crash, and that food type strongly interferes with the behaviour of the microbial communities in rotifer cultures. Conventional methods for microbial control are based on the use of antibiotics or bacteriostatic compounds that kill or inhibit the growth of bacteria. However, this approach has led to the development of bacterial resistance to antibiotics. The interference with the 115

129 Chapter 4 quorum sensing systems, a means of bacterial communication, has been advocated as a novel strategy to control pathogenic bacteria without interfering with their growth (Hentzer et al., 2003). Disruption of quorum sensing was suggested as a new anti-infective strategy in general (Finch et al., 1998) and with particular potential for use in aquaculture (Defoirdt et al., 2004). Quorum sensing is known as a mechanism by which bacteria coordinate gene expression in a density-dependent manner. This process depends on the production, release and detection of signal molecules called autoinducers (Miller and Bassler, 2001). By far, the most extensively investigated family of intercellular signaling molecules are the N-acyl homoserine lactones (AHLs). These molecules are associated with the quorum sensing processes in various human and plant pathogens, such as Pseudomonas aeruginosa (Rumbaugh et al., 2000), Erwinia carotovora, Agrobacterium tumefaciens (Whitehead et al., 2001), as well as Vibrio harveyi (Manefield et al., 2000) and other fish pathogens (Bruhn et al., 2005). Quorum sensing in Vibrio harveyi, a pathogen of many aquatic organisms (Gomez-Gil et al., 2004), is regulated via a multi-channel phosphorylation/dephosphorylation cascade. This bacterium produces and responds to the three signal molecules, HAI-1 (Harveyi Autoinducer 1), AI-2 (Autoinducer 2) and CAI-1 (Cholerae Autoinducer 1), which regulate the expression of genes, among others, responsible for bioluminescence (Bassler et al., 1993, 1994, 1997). HAI-1 is an AHL and was identified as N-(β-hydroxybutyryl)homoserine lactone (Cao and Meighen, 1989). AI-2 is a furanosyl borate diester (Chen et al., 2002), an universal signal that could be used by a variety of bacteria for communication among and between species (Cloak et al., 2002; Ohtani et al., 2002; Kim et al., 2003). Recently, a third quorum sensing component was discovered in Vibrio harveyi, which involves a Vibrio cholerae autoinducer CAI-1 (Henke and Bassler, 2004a). Application of quorum sensing antagonists is one of the techniques that have been investigated for bacterial control in aquaculture. Both natural and synthetic halogenated 116

130 Chapter 4 furanone compounds, which are secondary metabolites in the marine red alga Delisea pulchra (Manefield et al., 1999), have been investigated as promising quorum sensing antagonists. The unicellular alga Chlamydomonas reinhardtii also secretes substances that mimic the activity of AHL signal molecules and thus interferes with quorum sensing effects in naturally encountered bacteria (Teplitski et al., 2004). Rotifers, being an important element of the aquaculture food chain, can be used as an experimental in vivo system for studying the quorum sensing-mediated virulence in Vibrio harveyi. This investigation is facilitated by the establishment of methods to obtain gnotobiotic rotifer cultures either from disinfected resting eggs (Douillet, 1998; Rombaut et al., 1999) or from disinfected amictic eggs (Martinez-Diaz et al., 2003; Watanabe et al., 2005; Tinh et al., 2006), as they eliminate the effects of microbiota that is naturally present in the culture environment. In a first approach, we were interested in establishing whether Vibrio harveyi strains display any negative effect towards Brachionus. Second, we wanted to find out whether quorum sensing disruption can interfere with Vibrio Brachionus interactions. Materials and methods Rotifer strain Brachionus plicatilis (clone 10) was used in this study as test organisms. See Tinh et al. (2006) for the origin and identification. Preparation of food for rotifers The wild-type strain of baker s yeast (Saccharomyces cerevisiae) (BY4741; genotype Mat a; his 3 l; leu 2 0; met 15 0; ura 3 0) was used as food for the rotifers in the experiments. It was obtained from EUROSCARF (Institute of Microbiology, University of Frankfurt) in Germany. See Tinh et al. (2006) for the method of culturing and harvesting yeast. 117

131 Chapter 4 Axenic yeast was fed to the rotifers at a level of 2.4 x 10 5 cells per rotifer. Feeding was done twice (at the start of the experiment and 24 h after the challenge) under a laminar flow hood, using MultiGuard TM Barrier pipette tips (Sorenson BioScience, UT, USA). Method to obtain axenic rotifers: Amictic rotifer eggs were disinfected in 100 ppm of glutaraldehyde for 2 h at 28 C (Tinh et al. 2006). After disinfection, the dead rotifers and the amictic eggs were transferred to new sterile falcon tubes containing fresh 25 g l µm filtered and autoclaved seawater (FASW). The falcon tubes were placed on a rotor for incubation for 3 h to allow the amictic eggs to hatch. The culture was allowed to stand for 5 min to let most of the dead adult rotifers settle out. The sterile newly-hatched neonates were collected from the water column and distributed to sterile 50-ml falcon tubes (TRP, γ-irradiated) containing 20 ml of 25 g l -1 FASW, in order to obtain a density of 8 rotifers ml -1 at the start of the experiment. All the manipulations were performed under a laminar flow hood in order to maintain axenicity of the rotifer cultures. Axenicity test: See Tinh et al. (2006). Vibrio harveyi strains Eleven Vibrio harveyi strains and five mutants derived from BB120 strain were used in the challenge tests (Table 4.1). Ten V. harveyi strains (except BB120) had been isolated from shrimp hatcheries with disease outbreaks or from seawater at different locations, and were provided by the Department of Fishery Microbiology, University of Agricultural Sciences, Mangalore, India. They were identified as V. harveyi based on a series of biochemical tests, as described by Karunasagar et al. (1994). The BB120 strain and its mutants were obtained from the Department of Molecular Biology, Princeton University, New Jersey, USA. All the strains were preserved in 20% glycerol at -80 C. Before starting an experiment, the bacterial cultures were inoculated into fresh Marine Broth (Difco, Detroit, USA) and incubated at 28 C for 24 h 118

132 Chapter 4 on a shaker (120 rpm). After incubation, the bacterial suspensions were centrifuged at 4500 g for 10 min, subsequently the cells were resuspended with 25 g l -1 FASW and stored at 4 C before use (for maximum 3 h). Table 4.1 Vibrio harveyi strains used in this study Strain Characteristic Luminescence on Marine Agar Isolated in shrimp hatcheries in Ecuador Isolated from diseased shrimp in China - Isolated from dead amphipod in USA - VH-011 VH-012 VH-013 VH-014 VH-017 VH-021 VH-023 (LMG 04044) VH-039 (LMG 11659) VH-040 (LMG 11660) VH-042 (LMG 13949) BB120 Isolated from seawater in USA Isolated in shrimp hatcheries in Thailand Wild-type strain from which MM30, BB152, BB886, BB170 and MM77 are derived Dysfunctional AI-2 synthase Reference Karunasagar* Bassler et al. (1997) MM30 + Surette et al. (1999) BB152 Dysfunctional HAI-1 synthase + Bassler et al. (1994) BB886 Dysfunctional AI-2 receptor + Bassler et al. (1997) BB170 Dysfunctional HAI-1 receptor + Bassler et al. (1993, 1997) MM77 Dysfunctional HAI-1 and AI-2 synthase + Mok et al. (2003) *: These strains were provided by Dr. Indrani Karunasagar, Department of Fishery Microbiology, University of Agricultural Sciences, Mangalore, India. Preparation of cell-free washwater of MM30 and BB152 strains The MM30 and BB152 strains were grown in Marine Broth as described above (the optical density reached approximately 1 at 600 nm). After incubation, the cultures were centrifuged at 4500 g for 10 min and the pellets were re-suspended in 25 g l -1 FASW. The suspensions were centrifuged a second time after incubation for 30 min on a shaker (120 rpm). The supernatants 119

133 Chapter 4 were subsequently filter-sterilized over 0.22-µm Millipore filters (Bedford, MA, USA) and stored at -30 C until use (for maximum one month). Two milliliters of each washwater was added to 18 ml of rotifer culture water. Challenge tests The experiments took place in sterile 50-ml falcon tubes containing 20 ml of 25 g l -1 FASW. The falcon tubes were put on a rotor which was placed inside a temperature-controlled room (28 C, 2000 lx). Each treatment was performed in four replicates and each experiment was repeated twice. V. harveyi strains were added to the culture water after the first feeding, at 5 x 10 6 CFU ml -1. No bacteria were added in the control treatment. Rotifers were fed twice (see previous para), immediately after distribution of the newly-hatched rotifers (day 1) and 24 h after challenging with V. harveyi (day 2). Rotifer density was monitored daily until day 4 (72 h after challenge with V. harveyi). Furanone preparation (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone was synthesized as described by Ren and Wood (2004). The furanone was dissolved in absolute ethanol and stored at -30 C until use. It was added to the culture water following the addition of V. harveyi strains. Ethanol was added in the control treatment, corresponding to the amount added in the treatment with highest furanone concentration. Data collection and analysis Two sub-samples of 500 µl were withdrawn daily from each replicate to estimate the rotifer density. See Tinh et al. (2006) for the calculation of population growth rate. Parametric assumptions were evaluated using Shapiro-Wilk s test for normality and Levene s test for homogeneity of variances. For each experiment, the growth rates on day 4 were compared between treatments using one-way ANOVA, followed by a Tukey test. All the tests were performed using the SPSS program version

134 Chapter 4 Results Challenge tests with Vibrio harveyi strains Different V. harveyi strains were used in this study to verify their effect on the growth performance of B. plicatilis. Eleven strains were tested in the experiments (Table ), and their interference with Brachionus growth was different. Only three strains (VH-014, VH- 012, BB120) consistently caused a significant reduction in the growth rate (p < 0.05) of Brachionus during 72 h of challenge, while the other strains did not provoke any negative effect, although some of them were isolated from diseased shrimp. Table 4.2 Growth rate of Brachionus plicatilis (mean ± SD, n = 4) over 72 h: effect of challenge with Vibrio harveyi strains Treatment Experiment 1 Experiment 2 Control VH-014 VH-017 VH-021 VH-023 VH-039 VH-040 VH ± 0.08 b 0.19 ± 0.06 a 0.44 ± 0.05 b 0.46 ± 0.03 b 0.49 ± 0.07 b 0.41 ± 0.11 b 0.46 ± 0.03 b 0.43 ± 0.04 b 0.30 ± 0.03 b 0.08 ± 0.05 a 0.32 ± 0.08 b 0.24 ± 0.10 ab 0.35 ± 0.01 b 0.37 ± 0.06 b 0.27 ± 0.03 b 0.31 ± 0.05 b Treatments with different superscripts in each experiment are significantly different from each other (Tukey test, p < 0.05). All the strains were added at 5 x 10 6 CFU ml -1. Rotifers were fed with axenic yeast twice, at the start of experiment and after 24 h. Table 4.3 Growth rate of Brachionus plicatilis (mean ± SD, n = 4) over 72 h: effect of challenge with Vibrio harveyi strains Treatment Experiment 1 Experiment 2 Control VH-011 VH-012 VH ± 0.02 b 0.12 ± 0.06 ab 0.07 ± 0.05 a 0.16 ± 0.09 ab 0.31 ± 0.05 b 0.28 ± 0.08 b 0.07 ± 0.01 a 0.20 ± 0.07 ab Treatments with different superscripts in each experiment are significantly different from each other (Tukey test, p < 0.05). All the strains were added at 5 x 10 6 CFU ml -1. Rotifers were fed with axenic yeast twice, at the start of experiment and after 24 h. 121

135 Chapter 4 Challenge tests with Vibrio harveyi mutants The challenge tests were set up to determine whether the GR effect of V. harveyi towards Brachionus has any relation with the bacterial quorum sensing system. V. harveyi strain BB120 and its isogenic mutants, which are deficient in either of the two quorum sensing components (HAI-1 or AI-2-mediated), were added to Brachionus cultures. A mutation in either the HAI-1-mediated components (BB152 and BB170 strains) or the AI-2-mediated components (MM30 and BB886 strains) did not change the effect of V. harveyi (Table 4.4). In contrast, the deletion of both components (CAI-1-mediated component is still present) neutralized the GR effect of V. harveyi, since the Brachionus growth rate in the presence of the MM77 mutant was similar to that in the control treatment (p > 0.05). Table 4.4 Growth rate of Brachionus plicatilis (mean ± SD, n = 4) over 72 h: effect of challenge with Vibrio harveyi mutants Treatment Mutation in Experiment 1 Experiment 2 Control BB120 MM30 BB152 BB886 BB170 MM AI-2 synthase HAI-1 synthase AI-2 receptor HAI-1 receptor HAI-1 and AI-2 synthase 0.28 ± 0.04 c 0.18 ± 0.03 b 0.13 ± 0.02 ab 0.15 ± 0.05 ab 0.11 ± 0.05 ab 0.10 ± 0.04 a 0.25 ± 0.08 c 0.40 ± 0.09 c 0.22 ± 0.06 ab 0.15 ± 0.03 a 0.30 ± 0.05 b 0.16 ± 0.05 a 0.16 ± 0.03 a 0.39 ± 0.04 c Treatments with different superscripts in each experiment are significantly different from each other (Tukey test, p < 0.05). All the strains were added at 5 x 10 6 CFU ml -1. Rotifers were fed with axenic yeast twice, at the start of experiment and after 24 h. In a further series of experiments, the cell-free washwater of MM30 strain (AI-2-negative mutant) or BB152 strain (HAI-1-negative mutant) as an exogenous source of HAI-1 or AI-2 molecules, respectively, was added to the culture water concomitantly with the challenge with the MM77 strain (double mutant in HAI-1 and AI-2 synthase). We found that the addition of the washwater of the MM30 or BB152 strain could restore the GR effect of the MM77 strain (Table 4.5). 122

136 Chapter 4 Table 4.5 Growth rate of Brachionus plicatilis (mean ± SD, n = 4) over 72 h: effect of challenge with the MM77 strain (HAI-1 and AI-2 synthase mutant), with and without the addition of the washwater of the MM30 (AI-2 synthase mutant) or the BB152 (HAI-1 synthase mutant) strain Treatment Experiment 1 Experiment 2 Control MM77 MM77 + MM30 washwater MM77 + BB152 washwater 0.31 ± 0.04 b 0.27 ± 0.10 b 0.15 ± 0.02 a 0.12 ± 0.03 a 0.54 ± 0.04 b 0.51 ± 0.05 b 0.30 ± 0.06 a 0.28 ± 0.08 a Treatments with different superscripts in each experiment are significantly different from each other (Tukey test, p < 0.05). The MM77 strain was added at 5 x 10 6 CFU ml -1. Washwater was added at 10% of the culture volume. Rotifers were fed with axenic yeast twice, at the start of experiment and after 24 h. Effect of brominated furanone on the GR effect of Vibrio harveyi strain BB120 towards gnotobiotic Brachionus In this series of experiments, we investigated whether a brominated furanone [(5Z)-4-bromo- 5-(bromomethylene)-3-butyl-2(5H)-furanone] can be used as quorum sensing disrupting compound in Brachionus cultures. Furanone was added to the culture water at five different concentrations, with and without challenge with the wild-type V. harveyi strain BB120. Furanone added at 7.5 mg l -1 and 10 mg l -1 was shown to be toxic to Brachionus, as the growth rate was significantly reduced (p < 0.05) compared to that in the treatment without furanone (Table 4.6). Table 4.6 Growth rate of Brachionus plicatilis (mean ± SD, n = 4) over 72 h: effect of the addition of (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone Treatment Experiment 1 Experiment 2 Control 1 mg l -1 furanone 2.5 mg l -1 furanone 5 mg l -1 furanone 7.5 mg l -1 furanone 10 mg l -1 furanone 0.56 ± 0.12 b 0.38 ± 0.09 b 0.36 ± 0.10 ab 0.34 ± 0.07 ab 0.14 ± 0.08 a 0.14 ± 0.09 a 0.38 ± 0.10 b 0.35 ± 0.07 b 0.27 ± 0.05 ab 0.25 ± 0.09 ab 0.15 ± 0.05 a 0.10 ± 0.05 a Treatments with different superscripts in each experiment are significantly different from each other (Tukey test, p < 0.05). Rotifers were fed with axenic yeast twice, at the start of experiment and after 24 h. 123

137 Chapter 4 In the presence of BB120 strain, we found that the growth rate of Brachionus was highest in the treatment with 2.5 mg l -1 of furanone and was comparable to that in the control treatment (p > 0.05) (Table 4.7). A furanone concentration of 10 mg l -1 in the presence of BB120 strain resulted in total mortality of Brachionus within 24 h after the treatment. Table 4.7 Growth rate of Brachionus plicatilis (mean ± SD, n = 4) over 72 h: effect of challenge with the BB120 (wild-type) strain followed by the addition of (5Z)-4-bromo-5- (bromomethylene)-3-butyl-2(5h)-furanone Treatment Experiment 1 Experiment 2 Control BB120 BB mg l -1 furanone BB mg l -1 furanone BB mg l -1 furanone BB mg l -1 furanone BB mg l -1 furanone 0.51 ± 0.05 d 0.39 ± 0.07 bc 0.48 ± 0.04 cd 0.54 ± 0.09 d 0.34 ± 0.10 ab 0.26 ± 0.06 a ± 0.05 c 0.19 ± 0.03 b 0.25 ± 0.02 bc 0.32 ± 0.03 c 0.20 ± 0.03 b 0.05 ± 0.01 a 0 Treatments with different superscripts in each experiment are significantly different from each other (Tukey test, p < 0.05). The BB120 strain was added at 5 x 10 6 CFU ml -1. Rotifers were fed with axenic yeast twice, at the start of experiment and after 24 h. The densities of V. harveyi were determined at the start and at the end of these experiments by plating the water samples on TCBS agar (Thiosulfate Citrate Bile Salt Sucrose Agar; Biokar Diagnostics, France). No significant reduction (p > 0.05) in Vibrio growth was noticed in relation to control treatment after 72 h (Fig. 4.1), indicating that furanone, at the tested concentrations, did not interfere with the growth of V. harveyi. Vibrio count (log CFU ml -1 ) 7,0 6,0 5,0 4,0 3,0 2,0 1,0 0,0 a b 0 mg/l furanone a b 1 mg/l furanone a b 2.5 mg/l furanone a b 5 mg/l furanone a b 7.5 mg/l furanone a b 10 mg/l furanone 0 h 72 h 124

138 Chapter 4 Figure 4.1 Vibrio counts (log CFU ml -1 ) in the Brachionus culture water at 0 h and 72 h (n = 4). Error bars indicate standard deviation. Treatments with different letters are significantly different from each other (p < 0.05, Tukey test). In separate experiments, we tested the effect of furanone, added at 2.5 mg l -1, on the growth rate of Brachionus, which were challenged with the V. harveyi strains BB120, VH-012 and VH-014. These strains inhibited the growth of Brachionus in previous experiments. The alteration of GR effect was strain dependent (Table 4.8). Furanone could nullify the negative effect of the BB120 and VH-014 strains (p < 0.05). On the other hand, this compound did not show any effect towards the VH-012 strain (p > 0.05). Table 4.8 Growth rate of Brachionus plicatilis (mean ± SD) over 72 h: effect of challenge with Vibrio harveyi strains followed by the addition of 2.5 mg l -1 of (5Z)-4-bromo-5- (bromomethylene)-3-butyl-2(5h)-furanone Treatment Experiment 1 Experiment 2 Experiment 3 Control BB120 BB mg l -1 furanone VH-012 VH mg l -1 furanone VH-014 VH mg l -1 furanone 0.36 ± 0.05 b 0.15 ± 0.03 a 0.34 ± 0.06 b 0.11 ± 0.07 a 0.15 ± 0.10 a 0.11 ± 0.07 a 0.30 ± 0.01 b 0.47 ± 0.09 c 0.22 ± 0.03 ab 0.43 ± 0.07 c 0.18 ± 0.08 ab 0.21 ± 0.08 ab 0.12 ± 0.02 a 0.40 ± 0.01 bc 0.48 ± 0.09 bc 0.18 ± 0.02 a 0.41 ± 0.06 b 0.18 ± 0.05 a 0.23 ± 0.10 a 0.24 ± 0.05 a 0.52 ± 0.01 c Treatments with different superscripts in each experiment are significantly different from each other (Tukey test, p < 0.05). Vibrio harveyi strains were added at 5 x 10 6 CFU ml -1. Rotifers were fed with axenic yeast twice, at the start of experiment and after 24 h. Discussion In this study, gnotobiotically grown rotifers B. plicatilis were used as test organisms. In this way, the naturally occurring microbial communities could not interfere with the quorum sensing system of the tested Vibrio harveyi strains. In a preliminary study, which was aimed at standardizing the conditions for a challenge test in Brachionus, we found that a Vibrio harveyi (BB120 strain) density as high as 5 x 10 6 CFU ml -1 is sufficient to cause an observable effect in the Brachionus culture. This density is much higher compared to that 125

139 Chapter 4 reported by Defoirdt et al. (2005) for Artemia nauplii (10 4 CFU ml -1 ). We also found a suitable feeding regime in which Brachionus are fed only twice (at the start of the experiment and 24 h after the challenge) instead of being fed daily, since the GR effect was attenuated in the latter case (data not shown). Similarly, Defoirdt et al. (2005) tested two different feeding regimes (feeding once or twice) for Artemia franciscana nauplii, which were challenged with Vibrio harveyi BB120. The authors found that the virulence of the BB120 strain was significantly reduced if Artemia was fed twice. The above findings indicate that a good feeding regime may compensate for the negative effect caused by a bacterium on the test organism, which is consistent with the notion that Vibrio harveyi is an opportunistic pathogen. The Vibrio harveyi strains used in the study were isolated from different sources (seawater, diseased shrimp, and wild crustacean) at different geographical locations (Table 4.1). Four strains are bioluminescent on Marine Agar. However, there is no evidence of the presence of quorum sensing signal molecules in these strains (except BB120 strain). Although most of the strains were isolated from shrimp tanks with observed symptoms of disease, only three strains were shown to inhibit Brachionus growth (Table ). This suggests that the GR effect of Vibrio harveyi on Brachionus growth is strain-dependent. Vibrio harveyi was found to be an ubiquitous species in the aquacultural environments (Thompson et al., 2001). Different strains may produce different virulence factors, such as exotoxins (Manefield et al., 2000), siderophores (Lilley and Bassler, 2000), type III protein secretion (Henke and Bassler, 2004b), or metalloproteases (Mok et al., 2003), when invading the host. Vibrio sp. present in the rotifer culture tanks may be harmful to the rotifers but could also serve as food and contribute to their growth. Yu et al. (1990) reported that a Vibrio alginolyticus strain can cause the collapse of rotifer cultures. Balompapueng et al. (1997) found that Flavobacterium sp., Aeromonas sp. and Vibrio sp. isolated from the unstable collapsing rotifer cultures showed pathogenicity for the rotifer populations. On the other hand, in a study on the 126

140 Chapter 4 bioencapsulation of different bacteria belonging to Vibrionaceae in the gnotobiotic rotifer B. plicatilis, Martinez-Diaz et al. (2003) found that none of the tested bacteria negatively affected rotifer growth, rather they were used as food by the rotifers. However, it should be noticed that the food type used in that study (microalgae) is different from that used in our study (yeast). In a recent study, Tinh et al. (2006) stated that food type interferes with the performance of a microbial community, which is present in or added into a rotifer culture. In the other series of experiments, we tested the effect of mutants which are derived from strain BB120. The mutant strains are defective in either the HAI-1-mediated or the AI-2- mediated quorum sensing system, but not in the CAI-1-mediated component. All the mutants were able to reduce Brachionus growth rate significantly over 72 h, except the double-mutant MM77 (Table 4.4). Moreover, the addition of MM30 or BB152 washwater (as an exogenous source of HAI-1 or AI-2 autoinducers, respectively) restored the GR effect of the MM77 strain (Table 4.5). These data strongly suggest that the action of either the HAI-1-mediated or the AI-2-mediated channel of the quorum sensing system in the V. harveyi strain BB120 is sufficient to alter the growth rate of Brachionus. On the other hand, the CAI-1-mediated component alone is not responsible for the GR effect in this strain. Although there is evidence that all three quorum sensing channels are responsible for bioluminescence induction in V. harveyi (Henke and Bassler, 2004a), its GR effect towards Brachionus seems to be dependent on only two components of the quorum sensing system. The results found in Brachionus are different from those found by Defoirdt et al. (2005). These authors performed challenge tests in Artemia franciscana nauplii using the BB120 strain and the same mutants that were used in our study. They found that only the AI-2- mediated component, and not the HAI-1-mediated component, controls the virulence of BB120 strain towards Artemia. Possibly, pathogenic Vibrio spp. behave physiologically different in different host organisms, adapting in this way to the different environments and/or 127

141 Chapter 4 the immune system of the hosts. There are many studies concerning the importance of AHL and AI-2 autoinducers on the virulence of pathogenic bacteria. AHL signals are involved in the regulation of virulence factors in some human and plant-pathogenic bacteria. They were found to be produced by many strains of fish-pathogenic bacteria (Bruhn et al., 2005). AI-2 is found in many genera and was also reported to control virulence factor production in many species (Federle and Bassler, 2003). AI-2 signal was detected in Vibrio cholerae (Miller et al., 2002), Vibrio vulnificus (Kim et al., 2003) and Escherichia coli (Anand and Griffiths, 2003). AI-2 regulates starvation adaptation and stress resistance in Vibrio vulnificus and Vibrio angustum (McDougald et al., 2003) and toxin production in Clostridium perfringens (Ohtani et al., 2002). Finally, we investigated the effect of a brominated furanone as means of quorum sensing disruption. Halogenated furanones interfere with AHL-regulated biofilm formation and virulence in the human pathogen Pseudomonas aeruginosa (Hentzer et al., 2002, 2003), as well as with bioluminescence in Vibrio fischeri (Givskov et al., 1996). Ren et al. (2001, 2004) provided evidence that brominated furanone inhibited biofilm formation and swarming of Escherichia coli by interfering with AI-2 signaling. In a recent study, Defoirdt et al. (2006) found that brominated furanone blocked all three channels of the V. harveyi quorum sensing system. In our study, the growth rate of Brachionus over 72 h after the challenge with BB120 strain was proportional to the furanone concentration and reached a maximum in the presence of 2.5 mg l -1 of furanone (Table 4.6), indicating that this concentration of furanone is appropriate to completely block all quorum sensing pathways in the BB120 strain, and thus attenuate the GR effect of this strain towards Brachionus. On the other hand, furanone added at a concentration of 7.5 mg l -1 and higher appeared to be toxic for Brachionus. No reduction in Vibrio count was noticed compared to control treatment (Fig. 4.1), indicating that furanone did not interfere with the growth of V. harveyi. The same furanone compound was tested in 128

142 Chapter 4 gnotobiotic Artemia nauplii by Defoirdt et al. (2006). Artemia nauplii were only protected at a much higher furanone concentration (20 mg l -1 ), and the lowest concentration of furanone found to be toxic to Artemia (50 mg l -1 ) was higher than that for Brachionus, indicating that Brachionus are more sensitive to furanone. The effect of brominated furanone as a quorum sensing inhibitor was also tested for the Vibrio harveyi strains which reduced the growth rate in Brachionus. Interestingly, furanone added at 2.5 mg l -1 could neutralize the negative effect of the VH-014 strain, but not the effect of the VH-012 strain. These observations suggest that the GR effect in the VH-014 strain towards Brachionus might be regulated by a V. harveyi-type quorum sensing system, as in the case of the BB120 strain. The mechanism of alteration of Brachionus growth by the VH-012 strain remains to be elucidated. Halogenated furanones utilized at high concentrations can indeed have a toxic effect as was documented in mammalian cells. 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone, a by-product of drinking water disinfection, has been found to be mutagenic in bacteria and to be a potent carcinogen in rats (Vaittinen et al., 1995; Komulainen et al., 1997; Komulainen, 2004). This compound and its analog, mucochloric acid, are toxic to hepatocytes and gill epithelial cells in rainbow trout and to aquatic invertebrate Daphnia magna (Isomaa et al., 1995). In conclusion, our results indicate that both quorum sensing systems in some Vibrio harveyi strains are responsible for their GR effects towards Brachionus plicatilis, a live food organism for many aquaculture species. Therefore, disruption of both components may help to alleviate these effects of V. harveyi in Brachionus cultures. On the other hand, the importance of quorum sensing in non-gnotobiotic systems, and hence the beneficial effect of quorum sensing inhibitors in open Brachionus cultures, remain to be established. Finally, as Vibrio growth is not inhibited by halogenated furanone, the Vibrio number associated with 129

143 Chapter 4 Brachionus cultures is likely to remain unchanged, which might cause problems to their predators (such as fish larvae), unless quorum sensing inhibitors can also be utilized successfully at that stage of the aquaculture food chain. Acknowledgements We thank Dr. Indrani Karunasagar and Dr. Bonnie Bassler for kindly providing Vibrio harveyi strains and mutants. This study was supported by a doctoral grant for candidates from developing countries (Bijzonder Onderzoeksfonds, grant number B/ DS502) by Ghent University, Belgium. References Anand, S.K., and Griffiths, M.W. (2003) Quorum sensing and expression of virulence in Escherichia coli O157:H7. Int J Food Microbiol 85: 1-9. Balompapueng, M.D., Hagiwara, A., Nishi, A., Imaizumi, K., and Hirayama, K. (1997) Resting egg formation of the rotifer Brachionus plicatilis using a semi-continuous culture method. Fish Sci 63: Bassler, B.L., Greenberg, E.P., and Stevens, A.M. (1997) Cross-species induction of luminescence in the quorum-sensing bacterium Vibrio harveyi. J Bacteriol 179: Bassler, B.L., Wright, M., Showalter, R.E., and Silverman, M.R. (1993) Intercellular signalling in Vibrio harveyi: sequence and function of genes regulating expression of luminescence. Mol Microbiol 9: Bassler, B.L., Wright, M., and Silverman, M.R. (1994) Multiple signalling systems controlling expression of luminescence in Vibrio harveyi: sequence and function of genes encoding a second sensory pathway. Mol Microbiol 13: Bruhn, J.B., Dalsgaard, I., Nielsen, K.F., Buchholtz, C., Larsen, J.L., and Gram, L. (2005) Quorum sensing signal molecules (acylated homoserine lactones) in Gram-negative fish pathogenic bacteria. Dis Aquat Org 65:

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149 CHAPTER 5 N-acyl homoserine lactone degrading microbial enrichment cultures isolated from Penaeus vannamei shrimp gut and their probiotic properties in Brachionus plicatilis cultures Nguyen Thi Ngoc Tinh, R.A.Y.S. Asanka Gunasekara, Nico Boon, Kristof Dierckens, Patrick Sorgeloos, Peter Bossier Submitted

150 N-acyl homoserine lactone degrading microbial enrichment cultures isolated from Penaeus vannamei shrimp gut and their probiotic properties in Brachionus plicatilis cultures Nguyen Thi Ngoc Tinh, R.A.Y.S. Asanka Gunasekara, Nico Boon, Kristof Dierckens, Patrick Sorgeloos, Peter Bossier Submitted

151 Chapter 5 Abstract Three bacterial enrichment cultures (ECs) were isolated from the digestive tract of shrimp, by growing the shrimp microbial communities in a mixture of N-acyl homoserine lactone (AHL) molecules in six consecutive cycles. These ECs, characterized by DGGE analysis and subsequent rrna sequencing, degraded AHL molecules in the degradation assays. Apparently, resting cells of the ECs were also able to degrade all the three types of quorum sensing signal molecules in Vibrio harveyi in vitro. One of the strong AHL-degrading enrichment cultures, EC5, was tested in Brachionus experiments. In contrast to the results of in vitro experiments, EC5 was not able to degrade the V. harveyi AI-2 autoinducer in vivo, as evidenced by the negative effect of the V. harveyi HAI-1 mutant (in which only AI-2 and CAI-1 are functional) on the growth rate of Brachionus in the presence of this enrichment culture. However, EC5 could neutralize the negative effect of the AI-2 mutant (e.g. MM30) on Brachionus growth rate, suggesting in vivo interference with HAI-1 regulated metabolism. These AHL-degrading enrichment cultures need to be tested in other aquatic systems for their probiotic properties, preferably in combination with specific AI-2-degrading bacteria. Introduction Quorum sensing is a mechanism by which bacteria coordinate gene expression in a densitydependent manner. This process depends on the production, release and detection of chemical signal molecules called autoinducers (Miller and Bassler, 2001). Many bacterial processes are regulated by quorum sensing, including symbiosis, virulence, bioluminescence, antibiotic production, and biofilm formation (Lazdunski et al., 2004). Highly specific as well as universal quorum sensing languages exist which allow intra- and interspecies communication (Schauder et al., 2001). Gram-negative bacteria employ N-acyl homoserine lactones (AHLs) as autoinducers, while Gram-positive bacteria use oligopeptides to communicate (Miller and 137

152 Chapter 5 Bassler, 2001; Whitehead et al., 2001). By far, the most extensively investigated family of intercellular signalling molecules are the AHLs. AHLs are associated with the quorum sensing processes in various pathogens (Bruhn et al., 2005). Quorum sensing in Vibrio harveyi, a pathogen of many aquatic organisms (Gomez-Gil et al., 2004), is regulated via a multi-channel phosphorylation/dephosphorylation cascade. This bacterium produces and responds to three autoinducers, namely HAI-1, AI-2 and CAI-1, which regulate the expression of bioluminescence (Bassler et al., 1993, 1994, 1997; Chen et al., 2002) and other virulence factors (Henke and Bassler, 2004). HAI-1 (harveyi autoinducer 1) is an AHL and was identified as N-(β-hydroxybutanoyl) homoserine lactone (Cao and Meighen, 1989). AI-2 (autoinducer 2) is a furanosyl borate diester (Chen et al., 2002). It was shown to be a universal signal that could be used by a variety of bacteria for communication among and between species (Cloak et al., 2002; Ohtani et al., 2002; Kim et al., 2003). Recently, a third quorum sensing component was discovered in V. harveyi, which involves a Vibrio cholerae-like autoinducer CAI-1 (Henke and Bassler, 2004). CAI-1 has not been determined structurally, but the CAI-1-mediated quorum sensing pathway in Vibrio cholerae functions analogously to that of V. harveyi (Miller et al., 2002). The discovery of quorum sensing systems forms the basis of a novel strategy to control pathogenic bacteria without interfering with bacterial growth (Hentzer et al., 2003). Disruption of quorum sensing was suggested as a new anti-infective strategy in general (Finch et al., 1998), and in aquaculture particularly (Defoirdt et al., 2004). One of the approaches for quorum sensing disruption is the isolation of quorum sensing degrading bacteria. A number of bacteria are reported to utilize directly AHL molecules as sole sources of energy and nitrogen, thus, they can be used as potential quenchers of quorum sensing-regulated functions in pathogenic bacteria. A soil bacterium, Pseudomonas strain PAI-A, and a quorum-sensing bacterium, Pseudomonas aeruginosa strain PA01, can degrade 3-oxododecanoyl homoserine 138

153 Chapter 5 lactone and other long-acyl, but not short-acyl, as energy source for growth (Huang et al., 2003). Twenty five isolates which degrade N-hexanoyl homoserine lactone were isolated from a tobacco rhizosphere (Uroz et al., 2003). One of these isolates, Rhodococcus erythropolis strain W2, was used to quench quorum sensing-regulated functions of other microbes. Molina et al. (2003) reported that the introduction of the plasmid-borne aiia gene encoding a lactonase enzyme, into a rhizosphere isolate Pseudomonas fluorescens P3, can confer this isolate the ability to degrade AHLs. The transformant strain significantly reduced potato soft rot and tomato crown gall diseases which were caused by plant pathogenic bacteria. Unfortunately, the evidence of AHL-degrading bacterial isolates in the aquatic environment is still scarce. In the present study, three different microbial enrichment cultures (namely EC3, EC4 and EC5), isolated from the gut microbial communities of Pacific white shrimp Penaeus vannamei, were tested for their AHL-degrading property in vitro, as well as the ability to attenuate the quorum sensing-regulated negative effect of Vibrio harveyi in vivo, using gnotobiotic rotifers Brachionus plicatilis as test organisms. Materials and methods Isolation procedure Microbial communities (MCs) were collected from the digestive tract of healthy Pacific white shrimp juveniles Penaeus vannamei, maintained in culture on formulated feeds at Ghent University, Belgium. The digestive tract was removed from the shrimp body after dissection and was homogenized by means of a stomacher blender (Seward, UK). After homogenizing, the suspensions were centrifuged at 1600 g for 5 min, then the supernatant was preserved at - 80 C in 20% glycerol. 139

154 Chapter 5 These MCs were used as seed material for the isolation of AHL-degrading bacterial strains. Two hundred µl of MC suspension were inoculated into 20 ml of a minimal culture medium, which contained 9 g l -1 NaCl and 5 mg l -1 of AHL mixture (in equal weight for all compounds) (Table 5.1). The cultures were placed on a shaker (120 rpm) at 28 C. Table 5.1 AHL molecules used in this study AHL molecule N-Butyryl-DL-homoserine lactone N-Butyryl-DL-homocysteine thiolactone N-Hexanoyl-DL-homoserine lactone N-Heptanoyl-DL-homoserine lactone N-Octanoyl-DL-homoserine lactone Abbreviation C 4 -AHL C 4 -S-AHL C 6 -AHL C 7 -AHL C 8 -AHL The isolation was done in six consecutive cycles, each cycle lasted 48 h. At the end of each cycle, 200 µl of each sample was transferred to a new flask containing 20 ml of fresh medium. The cell densities at the start and at the end of each cycle were determined, by measurement of optical density (OD) at 550nm wavelength and by plating the samples on Marine Agar (Difco, Detroit, USA). Three enrichment cultures (ECs), originating from three different shrimp individuals, were obtained at the end of the sixth cycle. In addition, ten isolates (namely S301, S302, S303, S304, S403, S404, S501, S503, S506, and S508) were selected from these ECs, after several screening tests based on the ability to degrade AHL molecules. The ECs and the individual isolates were kept at -80 C in 20% glycerol for further characterization. Bacterial strains Chromobacterium violaceum strain CV026, a mini-tn5 mutant derived from the C. violaceum strain ATCC31532 (McClean et al., 1997), was used as AHL-reporter. This strain cannot produce AHL, but can detect and respond to a range of AHL molecules (with acyl side chain of four to eight carbons) by inducing the synthesis of the purple pigment violacein. Strain 140

155 Chapter 5 P3/pME6863, a transformant of a soil bacterium Pseudomonas fluorescens, was used as positive control in the degradation assays. Plasmid pme6863 carries the aiia gene from a soil bacterium Bacillus sp. A24 that encodes a lactonase enzyme (Molina et al., 2003). Vibrio harveyi strain BB120 and its mutants were obtained from the Department of Molecular Biology, Princeton University, New Jersey, USA (Table 5.2). Table 5.2 Vibrio harveyi strains used in this study Strain Characteristic Reference BB120 MM30 BB152 BB886 BB170 MM77 JMH612 JMH597 JAF375 Wild-type strain Dysfunctional AI-2 synthase Dysfunctional HAI-1 synthase Dysfunctional AI-2 receptor Dysfunctional HAI-1 receptor Dysfunctional HAI-1 and AI-2 synthase Dysfunctional AI-2 and CAI-1 receptor Dysfunctional HAI-1 and CAI-1 receptor Dysfunctional HAI-1 and AI-2 receptor Bassler et al. (1997) Surette et al. (1999) Bassler et al. (1994) Bassler et al. (1997) Bassler et al. (1993, 1997) Mok et al. (2003) Henke & Bassler (2004) Henke & Bassler (2004) Henke & Bassler (2004) Culture media Marine Broth (Difco, Detroit, USA) was used as a universal medium to grow bacterial enrichment cultures and Vibrio harveyi strains in the co-culture assay. P3/pME6863 strain was grown in Luria-Bertani (LB) medium, containing tryptone (BD, France, 1% w/v), yeast extract (Sigma, Germany, 0.5% w/v), and NaCl (0.4% w/v). CV026 strain was grown in LB medium supplemented with 20 mg l -1 of kanamycin, in order to maintain the plasmid carrying the gene responsible for violacein production. Preparation of cell-free washwater of Vibrio harveyi strains V. harveyi strains were grown in Marine Broth until the optical density (OD) reached approximately 1 at 600 nm. The culture was centrifuged at 4500 g for 10 min and the pellet was re-suspended in 0.22-µm filtered and autoclaved 20 g l -1 NaCl solution (ph = 7.0). The suspension was centrifuged a second time after incubation at 28 C for 30 min on a shaker 141

156 Chapter 5 (120 rpm). The supernatant was subsequently filter-sterilized over a 0.22-µm Millipore filter (Bedford, MA, USA) and stored at -30 C until use (for maximum one month). Denaturing Gradient Gel Electrophoresis (DGGE) Total DNA of the enrichment cultures was extracted using standard methods (Boon et al., 2000). 16S rrna gene fragments were amplified with the primers PRBA338fGC and P518r (Muyzer et al., 1993) and analyzed by DGGE with a denaturing gradient ranging from 45% to 60% (Boon et al., 2002). DNA sequencing 16S rrna gene fragments were cut out of the DGGE gel with a clean scalpel and added in 50 µl of PCR water. After 12 hours of incubation at 4 C, 1 µl of the PCR water was re-amplified with primer set P338F and P518r. Five µl of the PCR product was loaded on a DGGE gel (see previous para) and the most dominant band from each EC was sent out for sequencing. DNA sequencing of the ca. 180 bp fragments was carried out by ITT Biotech-Bioservice (Bielefeld, Germany). Analysis of DNA sequences and homology searches were completed with standard DNA sequencing programs and the BLAST server of the National Center for Biotechnology Information (NCBI) using the BLAST algorithm (Altschul et al., 1997) and the Ribosomal Database project (Cole et al., 2005). The sequences determined in this study have been submitted to the GenBank databases under accession numbers EF EF AHL degradation assays Cross-streak assay Cross-streak assay was performed on LB agar supplemented with 0.1 mg l -1 of N-Hexanoyl- DL-homoserine lactone (HHL), which was previously determined to be the minimum concentration of HHL for inducing the violacein production by the CV026 strain. The tested isolate was streaked at the center of the LB agar plate (two replicates for each isolate). After incubating the plates at 28 C for 24 h, CV026 culture was streaked four times 142

157 Chapter 5 to the left and right sides and perpendicular to the isolate s streak. P3/pME6863 strain was streaked on the positive control plates. Subsequently, the plates were incubated at 28 C for 24 h, followed by visual checking the degradation pattern, i.e. the non-appearance of purple pigmentation along the CV026 streaks. Microtiter assay The wells of a 96-well microplate were filled with two layers. The bottom layer consisted of 100 µl of semisolid LB agar (1% agar, ph = 6.5) supplemented with 1 mg l -1 of HHL. Fifty µl of suspension of an enrichment culture containing 3 x 10 6 CFU ml -1 was added to the well before the agar solidified. The top layer contained 50 µl of CV026 strain (10 6 CFU ml -1 ) in LB medium, which was added into each well after 24 h of incubation. Sixteen replicates were done for each enrichment culture. Eight replicates were done for each isolate. The negative control wells and positive control wells contained no bacteria or a suspension of P3/pME6863 strain in the bottom layer, respectively. The microplate was incubated at 28 C for 24 h after CV026 was added. The degradation of HHL molecule by the ECs was assessed by observation of the non-appearance of purple color in the wells. Correlation between the HHL concentration and the diameter of violacein-induced halos Before starting the degradation kinetics assay, a standard curve correlating the diameter of the purple-pigmented halo produced by the CV026 strain with the HHL concentration was established. An overnight-grown culture of CV026 was diluted in fresh LB medium to obtain an optical density of approximately 0.1 at 600 nm. Fifty µl of that suspension was spread on a LB agar plate. Ten µl of an HHL solution was subsequently applied to the center of the plate. Five concentrations were tested: 10, 5, 2.5, 1, and 0.5 mg l -1. Each concentration was done in triplicate. The diameters of the purple-pigmented halos produced by CV026 strain were measured after incubation of the plates at 28 C for 24 h. Assay to determine the degradation kinetics 143

158 Chapter 5 This assay was performed in 50-ml erlenmeyer s flasks containing 10 ml of LB medium supplemented with 5 mg l -1 of HHL. The enrichment cultures were inoculated into this medium at 10 6 CFU ml -1. Each treatment was done in triplicate. P3/pME6863 strain was inoculated into the positive control flasks. No bacteria were added in the negative control treatment. The flasks were put on a shaker (120 rpm). Degradation of HHL was assessed at 12 h, 24 h, 36 h and 48 h. At each sampling time, 1 ml of culture from each flask was 0.22-µm filtered. Subsequently, 10 µl of the filtrate was dropped in the center of an LB plate, on which 50 µl of a CV026 culture (with an optical density of approximately 0.1 at 600 nm) had been spread plated. The plate was put in an incubator at 28 C. The diameter of the purple-pigmented halo produced by CV026 strain was measured after 24 h incubation. The residual concentration of HHL in the culture filtrate was determined based on the standard curve. Degradation of Vibrio harveyi autoinducers by the enrichment cultures The cell-free washwater of the BB120 strain was prepared as described above. Three enrichment cultures (ECs) were inoculated separately into this washwater at a density of 10 6 CFU ml -1. Control treatment contained only cell-free washwater. Three replicates were done for each treatment. The cultures were incubated at 28 C with shaking (120 rpm). After 24 h and 48 h of incubation, the cultures were centrifuged at 4500 g for 10 min and subsequently 0.22-µm filter-sterilized to remove the EC cells. Two experiments were conducted. In the second experiment, 5 mg l -1 of AHL mixture (Table 5.1) was added into the BB120 washwater, prior to the inoculation of enrichment cultures. The levels of V. harveyi autoinducers remaining in the culture supernatants were quantified based on the ability to induce bioluminescence in the V. harveyi double mutants. JMH612, JMH597 and JAF375 strains were used as reporters for HAI-1, AI-2 and CAI-1, respectively. The reporter strains were grown in Marine Broth at 28 C with shaking (120 rpm) to an OD

159 Chapter 5 of approximately 1 and were diluted 1/5000 in fresh medium. Fifty µl of the diluted reporter cultures were mixed with 50 µl of the culture supernatants in 3-ml test tubes. The BB120 washwater incubated without ECs was used as control. The test tubes were incubated at 28 C for 3 h for the JAF375 reporter strain and 4 h for the JMH612 and JMH597 reporter strains. The luminescence intensity of the cultures was measured in relative light unit (RLU), by means of a Biocounter M2500 luminometer (Lumac, Netherlands). Co-culture assay with Vibrio harveyi mutants Co-culture assay was performed in the test tubes containing 10 ml of Marine Broth. A suspension of one of the enrichment cultures was inoculated at 10 6 CFU ml -1. At the same time, a suspension of BB120 strain or one of its mutants, namely BB152, MM30, and MM77 (see Table 5.2) was added to the medium at 10 5 CFU ml -1. The control tubes contained only V. harveyi strain. Each treatment was done in three replicates. The content of all the tubes were serially diluted and were plated using a spiral plater (Led Techno, Belgium) on TCBS (Thiosulfate Citrate Bile Salt Sucrose) agar for enumerating Vibrio density at 18 h of culture. The co-cultures, diluted according to the necessity, were measured for luminescence intensity using a Biocounter M2500 luminometer (Lumac, Netherlands), after 18 h incubation on a shaker (120 rpm) at 28 C. Relative log luminescence intensity (RLLI) was calculated by taking the ratio between the luminescence intensity (in log RLU) and the TCBS count (in log CFU ml -1 ) of the corresponding culture. Gnotobiotic rotifers as test organism Brachionus plicatilis (clone 10) was obtained from CIAD (Centro de Investigación en Alimentación y Desarrollo, Mazatlan Unit for Aquaculture) in Mexico and was confirmed to belong to the species Brachionus plicatilis sensu strictu (Papakostas et al., 2006). Amictic rotifer eggs were disinfected with 100 ppm of glutaraldehyde for 2 h at 28 C. The procedure for obtaining axenic rotifers is described in Tinh et al. (2006). 145

160 Chapter 5 Challenge tests to evaluate the in vivo effect of EC5 The challenge tests took place in sterile 50-ml falcon tubes containing 20 ml of 25 g l -1 filtered and autoclaved seawater (FASW). The falcon tubes were put on a rotor (4 rpm) which was placed inside a temperature-controlled room (28 C, 2000 lx). Each treatment was performed in four replicates and each experiment was repeated twice. An enrichment culture (EC5) was added to the culture water after the first feeding, at 5 x 10 6 CFU ml -1. V. harveyi strain BB120 or one of its mutants (MM30, BB152, BB886, BB170, and MM77) were added 3 h later, at the same density. No bacteria were added in the control treatment. Rotifers were fed twice with an axenic baker s yeast strain (Saccharomyces cerevisiae, wild-type strain), at the start of each experiment (day 1) and 24 h after challenging with V. harveyi (day 2). Rotifer density was monitored daily until day 4 (72 h after the challenge with V. harveyi). For the collection of samples and calculation of population growth rate, see Tinh et al. (2006). Data analysis Parametric assumptions were evaluated using Shapiro-Wilk s test for normality and Levene s test for homogeneity of variances. The data of luminescence induction of reporter strains were compared between treatments, using one-way ANOVA followed by Tukey test. Dunnett s T3 test was used for the sets of data which did not conform to the parametric assumptions. For the in vivo experiment, the Brachionus growth rates on day 4 were compared between pairs of treatments using independent-samples t-test. The difference between pair of treatments was considered significant if the p-value was below All the tests were performed using the SPSS program version Results Growth of enrichment cultures 146

161 Chapter 5 During the isolation process, the densities of shrimp microbial communities in minimal medium containing 5 mg l -1 of a mixture of AHL molecules were determined over six consecutive cycles. The bacterial density increased from 10 6 CFU ml -1 at the start of each cycle to approximately 10 8 CFU ml -1 at the end of each cycle (as determined by optical density measurement), or from 10 5 CFU ml -1 to 10 7 CFU ml -1 as determined by plating on Marine Agar. Degradation of HHL by the individual isolates Using the cross-streak assay performed on LB agar supplemented with 0.1 mg l -1 HHL, the capability of degrading this type of AHL molecule by ten individual isolates was assessed by visual inspection of the non-appearance of purple pigmentation produced by CV026 (Fig. 5.1). (a) (b) Figure 5.1 Cross-streak assay on LB agar plates supplemented with 0.1 mg l -1 HHL. (a) No degradation of HHL; (b) Degradation of HHL by the isolate. The ability to degrade HHL molecule was also evaluated in a microtiter assay using 96-well microplate. Table 5.3 summarizes the results of cross-streak assay and microtiter assay. 147

162 Chapter 5 Table 5.3 HHL degradation capability of different isolates Isolate S301 S302 S303 S304 S403 S404 S501 S503 S506 S508 + weak degradation ++ strong degradation EC where the isolate was selected from EC3 EC3 EC3 EC3 EC4 EC4 EC5 EC5 EC5 EC5 Cross-streak assay Microtiter assay Degradation of HHL by the enrichment cultures The ability of AHL degradation by the enrichment cultures was assessed qualitatively in a 96- well microplate. The enrichment cultures were grown in semisolid LB agar supplemented with 1 mg l -1 of N-Hexanoyl-DL-homoserine lactone (HHL). This concentration was determined previously (data not shown) to be the detection limit of HHL by the reporter strain Chromobacterium violaceum CV026 for this type of assay. The results showed that all the three enrichment cultures could degrade HHL below the detection limit after 24 h of incubation. The AHL degradation kinetics were evaluated in another assay. A standard curve was determined in advance, with the following equation: diameter of purple-pigmented halo = ln[hhl] (regression coeffient R 2 = ). According to Fig. 5.2, no chemical degradation was noticed in the negative control treatment, as the ph of LB medium was buffered at EC5 s degradation pattern was almost the same as that of the positive control strain, and these cultures could degrade HHL after 24 h to below 0.1 mg l -1 (which is the detection limit by the reporter strain CV026). EC4 had a slower degradation rate, as the residual HHL concentration only dropped below the detection limit after 48 h. 148

163 Chapter 5 6,0 5,0 [HHL], mg l -1 4,0 3,0 2,0 1,0 0,0 0h 12h 24h 36h 48h Time Negative control Positive control EC3 EC4 EC5 Figure 5.2 HHL degradation curves of control cultures and enrichment cultures (EC3, EC4, EC5), as indicated by the residual HHL concentration in the medium over 48 h. The error bars represent the standard deviation of three replicates. Blank LB medium was used as negative control. Strain P3/pME6863 was used as positive control. Degradation of Vibrio harveyi autoinducers by the enrichment cultures The capability of the ECs to degrade the V. harveyi autoinducers was investigated, by resuspending resting cells of the ECs in the cell-free washwater obtained from the BB120 strain. As BB120 is a wild-type strain, its washwater should contain all three autoinducers, namely HAI-1, AI-2 and CAI-1. In the absence of exogenous AHL molecules (experiment 1), the levels of luminescence induction in the indicator strains were decreased significantly in the presence of ECs for all three autoinducers, compared to the control treatment (p < 0.001) (Fig. 5.3), indicating that all the three ECs were able to degrade the V. harveyi autoinducers under the experimental conditions. Under the addition of exogenous AHLs (experiment 2), the degradation of V. harveyi autoinducers was also observed, although the degradation of AI- 2 and CAI-1 occurred to a less extent (p < 0.01 for AI-2, p < 0.05 for CAI-1) (Fig. 5.4). 149

164 Chapter 5 log RLU HAI-1 b b a a a a a a Control, 0h Control EC3 EC4 EC5 24 h 48 h log RLU AI-2 b b a a a a a a Control, 0h Control EC3 EC4 EC5 24 h 48 h log RLU CAI-1 b b a a a a a a Control, 0h Control EC3 EC4 EC5 24 h 48 h Figure 5.3 Induction of bioluminescence in the reporter strains (log RLU) by cell-free washwater from the Vibrio harveyi strain BB120, in the absence and presence of the enrichment cultures (Exp. 1). Error bars indicate standard deviation of three replicates. Treatments with different letters for the same sampling time are significantly different from each other (p < 0.001, Tukey test). 150

165 Chapter 5 log RLU HAI-1 b b a a a a a a Control, 0h Control EC3 EC4 EC5 24 h 48 h 7 6 b b AI-2 log RLU a a a a a a Control, 0h Control EC3 EC4 EC5 24 h 48 h log RLU b b a a a a a CAI-1 a Control, 0h Control EC3 EC4 EC5 24 h 48 h Figure 5.4 Induction of bioluminescence in the reporter strains (log RLU) by cell-free washwater from the Vibrio harveyi strain BB120, in the presence of 5 mg l -1 of exogenous AHL mixture (Exp. 2). Error bars indicate standard deviation of three replicates. Treatments with different letters for the same sampling time are significantly different from each other (p < 0.05, Tukey test). 151

166 Chapter 5 Co-culture assay with Vibrio harveyi strains This assay was performed in order to investigate whether the ECs have any effect on the production of bioluminescence when they are co-cultured with different V. harveyi mutants. log RLU / log TCBS count 2,0 1,8 1,6 1,4 1,2 1,0 0,8 0,6 0,4 0,2 0,0 c BB120 a BB120+EC3 bc BB120+EC4 ab BB120+EC5 c MM30 a MM30+EC3 b MM30+EC4 b MM30+EC5 b BB152 ab BB152+EC3 a BB152+EC4 ab BB152+EC5 b MM77 b MM77+EC3 a MM77+EC4 a MM77+EC5 Figure 5.5 Relative log luminescence intensity (RLLI) of pure cultures / co-cultures of Vibrio harveyi strains and ECs after 18 h incubation at 28 C. ECs were inoculated at 10 6 CFU ml -1. V. harveyi mutants were inoculated at 10 5 CFU ml -1. Error bars indicate standard deviation of three replicates. For each V. harveyi mutant, treatments with different letters are significantly different from each other (p < 0.05, Tukey test). According to Fig. 5.5, an obvious and significant reduction in RLLI (p < 0.05) was observed for the co-cultures with MM30 mutant (in which HAI-1 and CAI-1 are functional), by all the ECs. For the co-cultures of wild-type strain BB120 and other mutants, significant reduction in RLLI was also observed but not in all the treatments. In vivo effect of enrichment culture EC5 in Brachionus plicatilis cultures The effect of one of the enrichment cultures, EC5, was evaluated in Brachionus cultures, which were challenged with different V. harveyi mutants. All the V. harveyi mutants used in this study (except the double mutants MM77) have previously shown a negative effect on Brachionus growth rate (Tinh et al., 2007). According to the results shown in Table 5.4, the EC5 enrichment culture could only exert a neutralizing effect against the MM30 and BB

167 Chapter 5 mutants, which are dysfunctional in the AI-2-mediated quorum sensing system, in both experiments (p < 0.01). In another series of experiments, Brachionus were challenged with the double mutant MM77 and the washwater of the MM30 strain was added as an exogenous source of the HAI-1 and CAI-1 molecules (Table 5.5). It is obvious that in this case, EC5 was also able to neutralize the negative effect of the MM77 strain (p < 0.01). Table 5.4 Growth rate of Brachionus plicatilis (mean ± SD, n = 4) over 72 h: effect of challenge with Vibrio harveyi wild-type strain and single mutants, in the absence and presence of the enrichment culture EC5 Treatment Mutation in Experiment 1 Experiment 2 Control EC5 BB120 EC5 + BB120 MM30 EC5 + MM30 BB152 EC5 + BB152 BB886 EC5 + BB886 BB170 EC5 + BB ± ± ± ± 0.07 AI-2 synthase 0.15 ± 0.03* 0.34 ± 0.16* HAI-1 synthase 0.20 ± ± 0.16 AI-2 receptor 0.16 ± 0.05* 0.36 ± 0.12* HAI-1 receptor 0.13 ± ± ± ± ± ± ± 0.03* 0.26 ± 0.09* 0.09 ± ± ± 0.05* 0.22 ± 0.04* 0.06 ± ± 0.08 * Significant difference in growth rate between the paired treatments (p < 0.01, t-test). All the strains were added at 5 x 10 6 CFU ml -1. V. harveyi strains were added 3 h after the addition of EC5. Rotifers were fed with axenic yeast twice, at the start of experiment and 24 h after the challenge. Table 5.5 Growth rate of Brachionus plicatilis (mean ± SD, n = 4) over 72 h: effect of challenge with the MM77 strain (HAI-1 and AI-2 synthase mutant), with and without the addition of the MM30 (AI-2 synthase mutant) washwater, in the absence and presence of the enrichment culture EC5 Treatment Experiment 1 Experiment 2 Control EC5 MM77 EC5 + MM77 MM77 + MM30 washwater EC5 + MM77 + MM30 washwater 0.31 ± ± ± ± ± 0.02* 0.30 ± 0.10* 0.54 ± ± ± ± ± 0.06* 0.61 ± 0.02* 153

168 Chapter 5 * Significant difference in growth rate between the paired treatments (p < 0.01, t-test). All the strains were added at 5 x 10 6 CFU ml -1. The MM77 strain and the MM30 washwater were added 3 h after the addition of EC5. Two milliliters of MM30 washwater was added to 18 ml of rotifer culture water. Rotifers were fed with axenic yeast twice, at the start of experiment and 24 h after the challenge. Sequence analysis of enrichment cultures The DGGE pattern of the enrichment cultures (Fig. 5.6) shows that the three enrichment cultures are clearly different. The 16S rrna sequences of the dominant bands (arrow) were compared with those in the ribosomal RNA database. Most of the matched genera are Gramnegative, aerobic rod-shaped bacteria, which are widely distributed in oligotrophic environments. EC3 has 95.7% similarity with Pseudomonas sp., while EC4 resembles Sphyngomonas and Sphingopyxis sp. (97.1% similarity). The matched genera for EC5 are Rhizobium, Ensifer, Sinorhizobium and Aminobacter (96.2% similarity). Figure 5.6 DGGE pattern of the enrichment cultures. M: marker; lane 1: EC5; lane 2: EC4; lane 3: EC3. Arrows indicate the bands that are sequenced. The species to which the sequences have high homology are indicated in the text. 154

169 Chapter 5 Discussion To our knowledge, this is the first time AHL-degrading bacteria are isolated from aquatic animals. Our isolation procedure was based on the capability of microbial communities collected from shrimp gut to grow in a minimal medium containing a mixture of AHL molecules as carbon and nitrogen sources. These bacteria were able to proliferate in such a nutrient-poor environment. Our approach is similar to that used in the study of Uroz et al. (2003). AHL-degrading bacteria were isolated from a tobacco rhizosphere, using a minimal medium supplemented with ammonium sulfate as nitrogen source and N-hexanoyl homoserine lactone as carbon source. These HHL-degrading isolates were identified as members of the genera Pseudomonas, Comamonas, Variovorax and Rhodococcus. In our study, the results of 16S rrna sequence analysis show that all the ECs are inhabitants of oligotrophic environments. EC3 has 95.7% similarity with Pseudomonas sp., the members of which have an ability to metabolize a variety of diverse nutrients (Vamsee-Krishna et al., 2006) and many of them are found resistant to antibiotics (Miranda and Zemelman, 2002). Many bacterial isolates from this group are being used as biocontrol agents (Kamilova et al., 2006). The bacteria from Sphingomonas and Sphingopyxis genera, which show 97.1% similarity with EC4, are widely distributed in different land and water habitats. Several Sphingopyxis species have recently been isolated from the marine environment (Yoon et al., 2005; Yoon and Oh, 2005). Representatives of the Sphingopyxis group are known to possess biodegradative capabilities (Godoy et al., 2003; Sohn et al., 2004). The dominant group of EC5 closely matched Rhizobium and Aminobacter (96.2% similarity). Hu et al. (2003) found that an isolate of Agrobacterium tumefaciens, a member of Rhizobium, was among the bacteria which expressed the AHL degradation enzymes in the biofilms formed in a water reclamation system. Several isolates of Aminobacter group, which are capable of degrading insecticides and herbicides, have been isolated from agricultural soil in Northern Ireland and 155

170 Chapter 5 Canada (McDonald et al., 2005). The molecular analysis of the enrichment cultures characterized in our study shows that the ability to degrade AHL is probably more commonly present in a lot of different bacterial groups. During the past decade, several soil/plant isolates were described, which have the capability to degrade the AHL molecules secreted by pathogenic bacteria, and thus can be used as potential biocontrol agents in disease prevention. AHL-producing and AHL-degrading bacteria co-exist in every ecosystem, possessing different strategies to gain competitive advantages. Hu et al. (2003) found that in the bacterial biofilms developed in a water reclamation system, two groups of bacteria produced AHL signals, while three isolates belonging to Agrobacterium tumefaciens, Bacillus cereus and Ralstonia sp. expressed AHL-degrading enzymes. Bacillus thuringiensis suppressed the AHL-dependent virulence of a plant pathogen Erwinia carotovora, by interfering with the accumulation of AHL signals (Dong et al., 2004). An Acinetobacter sp. strain C1010, isolated from the rhizospheres of cucumbers, was able to degrade the AHL molecules produced by a phytopathogenic bacterium Burkholderia glumae (Kang et al., 2004). Leadbetter and Greenberg (2000) indicated that, a soil bacterium Variovorax paradoxus can grow on AHL signal molecules as the sole sources of energy and nitrogen. Several groups of AHL-degradation enzymes have been identified. These enzymes belong to either the acylase group, which breaks down the amide bond connecting the homoserine lactone ring to the acyl chain (Xu et al., 2003), or the lactonase group, which hydrolyzes the lactone ring (Dong et al., 2000, 2002; Lee et al., 2002). The enrichment cultures and the individual isolates which are characterized in our study were able to grow on AHL molecules as the sole sources of energy and nitrogen, thus they are expected to possess at least one type of AHL-degrading enzymes. We used two different methods, microtiter assay and degradation kinetics assay, to characterize the AHL degradation pattern of the enrichment cultures qualitatively and 156

171 Chapter 5 quantitatively, respectively. The latter assay allows us to see the change in degradation pattern with time by different enrichment cultures. HHL was used in these assays, as it is the single quorum sensing molecule which was secreted by the wild-type Chromobacterium violaceum strain (McClean et al., 1997). It should be noticed for this kind of assay, that the HHL degradation capacity of the ECs was tested against an LB medium background, which was different from the way in which they were isolated. It was observed that the degradation of HHL actually took place during the first 24 h for most of the ECs. The degradation rate was reduced afterwards (Fig. 5.2). The microtiter assay used in this study has lower sensitivity than the degradation kinetics assay (i.e. detection limit of 1 mg l -1 HHL for microtiter assay, as compared to 0.1 mg l -1 for the degradation kinetics assay, in the case CV026 is used as reporter strain). However, the former can be considered as an effective and reliable method for simultaneously screening a lot of AHL-degrading bacterial isolates in response to different AHL molecules. Several authors reported the use of different AHL-reporter strains. CV026 strain was described in the study of Uroz et al. (2003) as a biosensor strain, because of its capability to respond to a range of AHL molecules with short acyl side chain. Jafra and van der Wolf (2004) combined three indicator strains, CV026, green fluorescent protein (GFP)-labeled Escherichia coli strain JB534-MT102 and Agrobacterium tumefaciens strain NT1. Different bioassays based on different indicator strains have different sensitivity, depending on the molecular structure of the AHL molecules (Cha et al., 1998). In order to verify the ability of ECs to degrade the autoinducers produced by V. harveyi, the resting cells of ECs were inoculated into the cell-free supernatant obtained from the wild-type V. harveyi strain BB120. This is to assure the absence of further autoinducer production by the V. harveyi cells after a pre-determined incubation period. The culture supernatants, after incorporating the reporter strains, were incubated for different time intervals. The shorter 157

172 Chapter 5 incubation time needed for JAF375 (reporter strain for CAI-1) is probably due to the fact that the CAI-1 channel is activated at relatively low cell density (Henke and Bassler, 2004). In a subsequent assay, the ECs were grown in co-cultures with V. harveyi strains. V. harveyi was inoculated at lower density compared to the ECs, since the former is fast-growing bacteria. In these co-cultures, V. harveyi strains are constantly producing the quorum sensing signal molecules while ECs are degrading them. Also, a cell-to-cell contact and an increase in optical density (due to the bacterial growth) may interfere with the bioluminescence production, and also the degradation of autoinducers by the ECs. When performing the assay with the cell-free washwater from V. harveyi, the system is not that complex, thus the degradation capacity of the ECs can be easily evaluated. On the other hand, the co-culture assay is more advantageous to see the real interactions in the co-existing environment of both the ECs and V. harveyi. Interestingly, our enrichment cultures were capable of degrading all the three quorum sensing signal molecules produced by V. harveyi, which are not structurally identical. Since AI-2 and CAI-1 were not previously incorporated into the growth medium during the isolation, it is possible that these molecules were either simply absorbed or co-metabolized by the ECs in the nutrient-poor environment (washwater from BB120 culture). Co-metabolism is known as the transformation of a non-growth substrate by microorganisms in the obligate presence of a growth substrate (Dalton and Stirling, 1982). This is a very common phenomenon in the bacterial kingdom, when various xenobiotic or recalcitrant substances are mineralized by mixed communities or microorganisms (Hinteregger et al., 1992; Meade et al., 2002; Rozes et al., 2003; Ralebitso-Senior et al., 2003; Rentz et al., 2005). In our study, AI-2 and CAI-1 may serve as non-growth substrate which was utilized by ECs under nutrient-limited conditions. On the other hand, when exogenous AHL molecules were added, the ECs tended to degrade more AHL, hence, less AI-2 and CAI-1 were degraded (Fig ). 158

173 Chapter 5 One of the enrichment cultures (EC5) was finally evaluated on its effect in vivo, using gnotobiotically grown Brachionus plicatilis as a test model. It was recently demonstrated that both autoinducers HAI-1 and AI-2 are responsible for the growth-retarding (GR) effect of V. harveyi strain BB120 towards Brachionus (Tinh et al., 2007). In the present study, EC5 could only neutralize the GR effect of the mutants in which HAI-1 and CAI-1 are functional (MM30 and BB886), but not that of the mutants in which AI-2 and CAI-1 are functional (BB152 and BB170). This finding was different from that of the in vitro experiments, which showed that EC5 could break down all the three autoinducers in V. harveyi. To explain the difference between in vitro and in vivo experiments, the following hypotheses are proposed. The degradation of AHL (HHL) was observed in the presence of a nutrient-rich background (i.e. LB medium), as indicated in the degradation assays. These data were confirmed by in vivo experiments, under conditions that other nutrients were around (e.g. digested yeast cells). On the other hand, AI-2 was probably only degraded in a minimal medium (i.e. V. harveyi washwater), where nutrients became a limiting factor, and not against a multi-nutrient background. This explanation reconciles the in vitro and in vivo data for AI-2. If this would be confirmed by further experiments, AI-2 degradation as a strategy for combating infections would not be an option, unless strains with an AI-2-specific degradation capability could be isolated. In conclusion, all the enrichment cultures isolated in this study showed a strong degradation towards AHL molecules, as revealed in the degradation kinetic assay, against a multi-nutrient background of the LB medium. They might have a tendency to degrade other quorum sensing signal molecules (e.g. V. harveyi AI-2 and CAI-1 autoinducers) only when growing in a minimal medium, most likely by co-metabolism. Under in vivo conditions, i.e. with nutrients present, it is likely that only the degradation of AHL takes place. Consequently, the AHL-degrading enrichment cultures may not be effective against pathogens which regulate 159

174 Chapter 5 their virulence via a multi-channel quorum sensing system, such as V. harveyi. The potential of these AHL-degrading enrichment cultures in microbial control needs to be tested with target animals, i.e. fish or shellfish larvae, preferably in combination with AI-2-specific degrading bacteria. Acknowledgement We thank Dr. Bonnie Bassler for kindly providing the Vibrio harveyi mutant strains, and Tom Defoirdt for the help in using the Biocounter M2500 luminometer. This study was supported by a doctoral grant of the Research Fund BOF of Ghent University, Belgium (grant number B/ DS502), awarded to the first author. References Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D.J. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: Bassler, B.L., Greenberg, E.P., and Stevens, A.M. (1997) Cross-species induction of luminescence in the quorum-sensing bacterium Vibrio harveyi. J Bacteriol 179: Bassler, B.L., Wright, M., Showalter, R.E., and Silverman, M.R. (1993) Intercellular signalling in Vibrio harveyi: sequence and function of genes regulating expression of luminescence. Mol Microbiol 9: Bassler, B.L., Wright, M., and Silverman, M.R. (1994) Multiple signalling systems controlling expression of luminescence in Vibrio harveyi: sequence and function of genes encoding a second sensory pathway. Mol Microbiol 13: Boon, N., de Windt, W., Verstraete, W., and Top, E.M. (2002) Evaluation of nested PCR- DGGE (denaturing gradient gel electrophoresis) with group-specific 16S rrna primers for the analysis of bacterial communities from different wastewater treatment plants. FEMS Microbiol Ecol 39:

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180 CHAPTER 6 An N-acyl homoserine lactone-degrading microbial community improves the survival of first-feeding turbot larvae (Scophthalmus maximus L.) Nguyen Thi Ngoc Tinh, Vu Hong Nhu Yen, Kristof Dierckens, Patrick Sorgeloos, Peter Bossier Submitted

181 An N-acyl homoserine lactone-degrading microbial community improves the survival of firstfeeding turbot larvae (Scophthalmus maximus L.) Nguyen Thi Ngoc Tinh, Vu Hong Nhu Yen, Kristof Dierckens, Patrick Sorgeloos, Peter Bossier Submitted

182 Chapter 6 Abstract In this study, the technique of administering N-acyl homoserine lactone (AHL)-degrading enrichment cultures (ECs) as biocontrol agent in turbot larviculture has been investigated. EC3 and EC5, originating from the microbial community of Penaeus vannamei shrimp gut, were incorporated into first-feeding turbot larvae through addition to the rearing water and/or bioencapsulation in rotifers, prior to their feeding to the turbot larvae. Both ECs were able to colonize the larval gut and to persist up to five days after their addition was discontinued. However, only EC5, administered in both ways, was effective in improving turbot larvae survival under the experimental conditions, i.e. when the survival of turbot larvae was compromised through the daily addition of AHL molecules (1 mg l -1 ). The latter treatment reduced the survival to 5.9% or 10.4% dependent on the experiment (while in the control treatment, the survival was 35% and 92.1%, respectively). Through the addition of EC5, the effect of AHL could be nullified. There was a strong negative correlation between the residual AHL concentration in the water and the larval survival on the last day. These experiments demonstrate that a low concentration of AHL could have a negative effect on turbot larval survival, apparently through the prevailing microbial community, while EC5 can counteract this. Introduction Turbot (Scophthalmus maximus L.) is an important aquaculture species in Southern Europe and in Shandong province in China. One of the most critical aspects in turbot farming is its highly variable survival during the larval rearing phase. Research on this critical phase has focused on the nutritional requirements of larvae during early exogenous feeding, together with the microbial characteristics of the intensive rearing environment (Shields, 2001). Major bacterial colonization of the gut of turbot larvae coincides with the start of feeding (Munro et 167

183 Chapter 6 al., 1994). Most bacterial species isolated from the intestinal tract of larval turbot belong to Vibrionaceae (Nicolas et al., 1989; Gatesoupe, 1990; Munro et al., 1994; Blanch et al., 1997). Live food organisms, especially rotifers, were shown to be the main source of bacterial colonization of turbot larval gut (Gatesoupe, 1990; Keskin and Rosenthal, 1994; Munro et al., 1994). Efforts have been made to reduce the bacterial load associated with rotifers before feeding to turbot larvae, by rinsing of rotifers (Keskin and Rosenthal, 1994) or exposure of rotifers to ultraviolet radiation (Munro et al., 1999). The microbial environment of turbot larviculture can be controlled by manipulating the r/k-strategists proportion of the bacterial community. Salvesen et al. (1999) reported that, a lower proportion of r-selected bacteria in the tanks with microbially matured water containing microalgae resulted in higher percentage of viable and fast-growing larval turbot. An alternative approach for microbial management of turbot larviculture involved the selection of beneficial bacteria (probiotic bacteria). These bacteria were isolated from the rearing environment of turbot larvae (Huys et al., 2001; Hjelm, 2004a,b) or from rotifer cultures (Gatesoupe, 1994). Several bacterial strains, when introduced to the rearing water or bioencapsulated in the rotifers, were retrieved in high numbers in larval gut, and were able to improve the survival rates of first-feeding turbot larvae (Gatesoupe, 1994, 1997; Makridis et al., 2000). Recently, disruption of quorum sensing was suggested as a new strategy for microbial control in aquaculture (Defoirdt et al., 2004). In the present study, we investigated the use of two enrichment cultures of N-acyl homoserine lactone (AHL)-degrading bacteria in controlling the overall microbial activity in fish larvae, thus, aiming at improving the survival of turbot larvae in their first-feeding period. Materials and methods Source of bacteria and growth conditions 168

184 Chapter 6 The enrichment cultures EC3 and EC5, mixtures of AHL-degrading bacteria, were isolated previously at the Laboratory of Aquaculture & Artemia Reference Center (ARC) Ghent University, Belgium (see Chapter 5). They were made resistant to 100 mg l -1 rifampicin and were preserved in 20% glycerol at -80 C. The PCR-DGGE profile shows that the rifampicinsensitive (RS) and rifampicin-resistant (RR) enrichment cultures have the same dominant bands (Fig. 6.1). Before the experiment started, 200 µl of the stock cultures were inoculated into Nine Salt Solution (NSS, see composition in Tinh et al., 2006) supplemented with 113 mg l -1 of sodium acetate and 4 mg l -1 of ammonium chloride (C/N ratio = 20). The enrichment cultures (ECs) were grown in this medium for two weeks. Every two days, fresh medium was added (fed-batch culture) and the optical density (OD) of the culture was determined using a spectrophotometer (Thermo Spectronic). Two days before the experiment started, the ECs were acclimatized to 16 C, which is the temperature of the rearing water during the experiment. Figure 6.1 PCR-DGGE profile of the enrichment cultures used in this study. M: marker; 1: EC5-RR; 2: EC5-RS; 3: EC3-RR; 4: EC3-RS. 169