Olav Vadstein

Techniques for microbial control in the intensive rearing of marine larvae

The intensive cultivation conditions for marine larvae may easily cause microbial problems, resulting in poor growth and mass mortality, and techniques for improvement by enhancing environmental and larval factors should be developed. Establishment of a beneficial, protective microflora of marine larvae can be obtained by use of microbial matured water and probiotics. Microbial maturation of the rearing water before use in the larval tanks, can be obtained by running the water through a maturation unit that selects for non-opportunistic microflora. A diverse bacterial flora established by non-opportunists is believed to inhibit proliferation of opportunistic pathogenic bacteria in the water and the larvae. When used in the earliest developmental stages of marine fish larvae, enhanced growth and survival can be obtained. Also, the introduction of probiotic bacteria may promote the defence of the gut flora against pathogenic bacteria. Probiotic bacteria can be added directly to the water or administered to the larvae via live food, such as rotifers and Artemia. Further developments in these techniques are needed for improved control of bacterial number, growth and colonisation in the larval gut, as well as the identification of bacteria with probiotic effect on the host. Disinfection of eggs is a positive factor for control of growth and transfer of bacteria in larviculture. For marine fish eggs glutaraldehyde has suitable properties as a disinfectant, which improves hatching, development and survival of larvae. Because marine fish larvae have no specific immune system at hatching, non-specific defence is very important during the first developmental stages, and suitable techniques for stimulation of the non-specific immune defence may become important in future marine larval rearing. Enhanced viability of marine fish larvae and juveniles, has been obtained by treatment with FMI, which is a mannuronic acid polymer. Routes of administration were either into the water or via live food. A technique for incorporating immunostimulants in rotifers and Artemia for controlled transfer of immunostimulants to marine larvae, is described.

The use of immunostimulation in marine larviculture: possibilities and challenges

Abstract Inadequate microbial conditions are one of the main problems in rearing marine larvae. It is therefore an ultimate goal to develop methods for establishing microbial control at all stages of the cultivation process. In addition to measures improving environmental conditions, methods of improving the resistance of the larvae to bacterial infections need to be developed. One possibility is immunostimulation, which includes methods of enhancing the capacities of the specific and nonspecific immune systems. Although the information is limited, one must conclude that larval fish have a poorly developed immune system and that they primarily rely on nonspecific immune system. For specific defence, larvae have to rely on maternal immunity, which lasts only for a short period. Experiments have shown that maternal immunity may be manipulated by immunisation of the broodstock and that increased resistance to infections may be obtained. Direct immunostimulation of larvae must be aimed at the nonspecific part of the immune system, and several substances are known to have this ability. Experiments on nonspecific immunostimulation of fish suggest that the method has considerable potential for reducing losses in aquaculture, both during larval and on-growing stages. Reports on immunostimulation of larvae are, however, very limited. Further development of this method for larviculture will require the establishment of methods for the administration of the stimulant, and the adaptation of methods for detecting the response of the immune system. This last point is a particular challenge due to the small size and fragility of larvae. It is hypothesised that immunostimulation, together with other methods of achieving microbial control, will help to reduce the probability of microbial problems in larviculture. As a result, increased and more stable survival and growth are anticipated; resulting in the production of high-quality juveniles for the on-growth period.

Microbially matured water: a technique for selection of a non-opportunistic bacterial flora in water that may improve performance of marine larvae

Before transfer to larval incubators, water was membrane filtered to remove >95% of the bacteria and then transiently maintained in a biofilter that promoted recolonization of the water by non-opportunistic bacteria. The process is termed microbial maturation of the water. Hypothetically the bacterial flora in the matured water should protect the marine larvae from colonization and proliferation by opportunistic bacteria. Testing of the hypothesis demonstrated 76% higher survival of yolk sac larvae of Atlantic halibut (Hippoglossus hippoglossus) in matured than in membrane filtered water. Proliferation of opportunistic bacteria was observed in the rearing water after hatching of turbot eggs (Scophthalmus maximus), but to a less extent in the microbially matured water. In the early phase of first feeding of turbot larvae, the matured water induced qualitative differences in the gut microflora. Significantly higher initial growth rate of the turbot larvae in the matured water affected 51% higher average weight of 13 days old larvae than in membrane filtered water. Algal addition to the matured water enhanced the larval growth further. The experiments conducted supported the proposed hypothesis that microbial maturation selects for non-opportunistic bacteria, which protects the marine larvae from proliferation of detrimental opportunistic bacteria.

Heterotrophic, Planktonic Bacteria and Cycling of Phosphorus

As early as 1956, Rigler reported that heterotrophic bacteria were responsible for a large share of the uptake of inorganic phosphorus (P) in Toussant Lake (Rigler, 1956). Tracer experiments revealed that the bacteria sequestered two thirds of the phosphate, and Rigler stated that if they [bacteria] take up small increments of phosphorus received from inflowing water or from marginal vegetation, [bacteria] may compete with algae for this essential element.… If, in this process, they utilize inorganic phosphate, they would reduce the amount of phosphate available to algae and thus reduce the amount of organic matter produced by algae, (p. 560)

Colonization of Vibrio pelagius and Aeromonas caviae in early developing turbot (Scophthalmus maximus L.) larvae.

Polyclonal antisera made in rabbits against whole washed cells of Vibrio pelagius and Aeromonas caviae were used for detection of these bacterial species in the rearing water and gastrointestinal tract of healthy turbot (Scophthalmus maximus) larvae exposed to V. pelagius and/or Aer. caviae. The results demonstrated that this method is suitable for detection of V. pelagius and Aer. caviae in water samples and larvae at population levels higher than 103 ml−1 and 103 larva−1. Populations of aerobic heterotrophic bacteria present in the gastrointestinal tract of turbot larvae, estimated using the dilution plate technique, increased from approximately 4 × 102 bacteria larva−1 on day 3 post‐hatching to approximately 105 bacteria fish−1 16 days post‐hatching. Sixteen days after hatching, Vibrio spp. accounted for approximately 3 × 104 cfu larva−1 exposed to V. pelagius on days 2, 5 and 8 post‐hatching. However, only 103 of the Vibrio spp. belonged to V. pelagius. When larvae were exposed to Aer. caviae on day 2 post‐hatching, the gut microbiota of 5‐day old larvae was mainly colonized by Aeromonas spp. (104 larva−1), of which 9 × 103 belonged to Aer. caviae. Later in the experiment, at the time when high mortality occurred, 9 × 105Aer. caviae were detected. Introduction of V. pelagius to the rearing water seemed to improve larval survival compared with fish exposed to Aer. caviae and with the control group. It was therefore concluded that it is beneficial with regard to larval survival to introduce bacteria (V. pelagius) to the rearing water.

Surface disinfection of eggs from marine fish: evaluation of four chemicals

For surface disinfection of marine fish eggs Buffodine (1.06% free iodine), glutaraldehyde, chloramine-T and sodium hypochlorite (5% free chlorine) were tested using plaice (Pleuronectes platessa L.) as the main species for evaluation. Glutaraldehyde was the most promising candidate of the four chemicals tested. Good bactericidal effects without any documented negative effects on eggs and larvae were obtained at concentrations of 400–600 mg l−1 and contact times of 5–10 min. Replicated experiments under identical disinfection conditions revealed a clear correlation between the degree of successful surface disinfection and the initial bacterial load of the egg batch.

Pathways of Lipid Metabolism in Marine Algae, Co-Expression Network, Bottlenecks and Candidate Genes for Enhanced Production of EPA and DHA in Species of Chromista

The importance of n-3 long chain polyunsaturated fatty acids (LC-PUFAs) for human health has received more focus the last decades, and the global consumption of n-3 LC-PUFA has increased. Seafood, the natural n-3 LC-PUFA source, is harvested beyond a sustainable capacity, and it is therefore imperative to develop alternative n-3 LC-PUFA sources for both eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). Genera of algae such as Nannochloropsis, Schizochytrium, Isochrysis and Phaedactylum within the kingdom Chromista have received attention due to their ability to produce n-3 LC-PUFAs. Knowledge of LC-PUFA synthesis and its regulation in algae at the molecular level is fragmentary and represents a bottleneck for attempts to enhance the n-3 LC-PUFA levels for industrial production. In the present review, Phaeodactylum tricornutum has been used to exemplify the synthesis and compartmentalization of n-3 LC-PUFAs. Based on recent transcriptome data a co-expression network of 106 genes involved in lipid metabolism has been created. Together with recent molecular biological and metabolic studies, a model pathway for n-3 LC-PUFA synthesis in P. tricornutum has been proposed, and is compared to industrialized species of Chromista. Limitations of the n-3 LC-PUFA synthesis by enzymes such as thioesterases, elongases, acyl-CoA synthetases and acyltransferases are discussed and metabolic bottlenecks are hypothesized such as the supply of the acetyl-CoA and NADPH. A future industrialization will depend on optimization of chemical compositions and increased biomass production, which can be achieved by exploitation of the physiological potential, by selective breeding and by genetic engineering.

Characterization of the bacterial flora of mass cultivated Brachionus plicatilis

Bacterial density and composition in association of mass cultivated rotifers (Brachionus plicatilis, SINTEF-strain) was investigated, during experimental conditions identical to the procedures used for preparing rotifers as live food for marine cold water fish larvae. These procedures include cultivation, enrichment with squid meal and acclimation to low temperature by storage of the rotifer culture at 6 °C. Large variations were observed in the number of rotifer associated (1.8–7.6·103 colony forming units per rotifer−1) and free-living (0.6–25 107 cells ml−1) bacteria. An increase of 50–150% in the bacterial number was normally observed after feeding the rotifer with squid meal, but after three days of acclimation at 6 °C, the bacterial numbers decreased to the initial level.

Growth of turbot (Scophthalmus maximus L.) during first feeding in relation to the proportion of r/K-strategists in the bacterial community of the rearing water

A first feeding experiment with turbot was arranged as a two factorial design with filtered vs. microbially matured water as one factor, and with vs. without microalgae added to the tanks as the other. Application of microbially matured water had a highly positive effect on the growth of the larvae from day 5 after hatching and onwards. Algal addition gave the greatest effect on size from day 12, although both factors were highly significant. A positive interaction effect of combining matured water with the addition of microalgae was also observed. The positive effects may be related to the observed delay in colonization of the larvae in the early stages of first feeding. Evaluation of the water microflora by criteria based on the degree of maturation showed a lower proportion of opportunistic (i.e., r-selected) bacteria in tanks with matured water containing microalgae. It is suggested that rearing of turbot larvae in microbially matured water to which microalgae are added will lower the proliferation of opportunistic bacteria on the mucosal surfaces of the larvae, with more viable and fast-growing larvae as the result.

Control of the bacterial flora of Brachionus plicatilis and Artemia franciscana by incubation in bacterial suspensions

Abstract The accumulation of bacteria in Brachionus plicatilis and Artemia franciscana during a short-term incubation was quantified using immunocolony blot (ICB) and an enzyme-linked immunosorbent assay (ELISA). Four bacterial strains, isolated from turbot and halibut, were grazed effectively by both species when given at high concentrations (≥5×10 7 bacteria ml −1 ). B. plicatilis accumulated 21–63×10 3 bacteria per rotifer and A. franciscana up to 45×10 3 bacteria per metanauplius after 20–60 min of grazing. The composition of the bacterial microflora of the live food organisms changed drastically, as the bioencapsulated strains comprised up to 100% of the total count of colony-forming units. After incubation in the bacterial suspensions, B. plicatilis and A. franciscana were transferred to seawater with added microalgae ( Tetraselmis sp., 2 mg C l −1 ), to evaluate the persistence of the changed bacterial composition in conditions similar to those present in a first feeding tank. The bioencapsulated bacteria decreased in numbers, but in most cases remained present in both live food organisms after 24 h. It is possible, after a short-term incubation, to replace opportunistic (r-selected) bacteria present in the live food cultures with other bacteria, which persist as a dominant part of the bacterial flora of the live food for a relatively long period of time (4–24 h).