Effect of phosphorus limitation and fatty acid addition on Oncorhynchus mykiss larvae

In aquatic freshwater ecosystems, phosphorus is the most limiting nutrient. Despite this, aquatic organisms need phosphorus for metabolism and biological structure. Algae convert inorganic nutrients (like P) in nutritional and biochemical substances. Therefore they are a good food source for herbivorous zooplankton. In a P limited environment, algae change their carbon to phosphorus ratio (C:P ratio) according to their environmental concentration. This means that they are not homeostatic whereas zooplankton contains a much more stable C:P ratio and is homeostatic. The C:P ratio of zooplankton is species specific. The ecological stoichiometry theory states that an individual fish would try to keep a constant nutrient ratio or, in other words, fish would be homeostatic according to their nutrient ratio. Fatty acids are important to aquatic ecosystems as high-energy, often-essential resources. Algae are able to synthesize ω3 and ω6 PUFAs. Zooplankton and fish can only convert one form of ω3 and ω6 PUFAs into another in limited amounts. Phytoplankton, zooplankton and fish are all not homeostatic according to the fatty acid concentration. A higher concentration of polyunsaturated fatty acids gives an increased condition of daphnids and fish. Little ecologically relevant research has been performed to investigate the effect of live prey food quality on the condition of fish larvae. Therefore, laboratory experiments were performed to investigate the effect of phosphorus depletion and fatty acid enrichment on juvenile rainbow trout. An experiment was performed to look at the development of fish eggs and larvae according to the P concentration and fatty acid spectrum before the actual experiment started. Rainbow trout eggs contained a very low P concentration. This concentration significantly increased after hatching whereas feeding larvae contained an even higher amount of P. According to the fatty acid concentration, Oncorhynchus mykiss larvae showed a decrease in concentration within the development. An exception is linolenic acid, which increased in concentration after first feeding. The O. mykiss larvae were fed with Artemia. High concentrations of the ω3 PUFA linolenic acid are present in Artemia. Since O. mykiss larvae are able to transform linolenic acid into EPA and DHA they probably store linolenic acid for later use. EPA and DHA are both important ω3 PUFAs. The effects of phosphorus depletion and fatty acid enrichment on juvenile rainbow trout were tested for seven days in cross experiments. Therefore, Scenedesmus obliquus was cultured in phosphorus deficient and phosphorus sufficient medium. These algae were fed for seven days to large cultures of Daphnia magna. In the same experiment, the two different quality daphnids were additionally enriched with a fatty acid mix, which contained 30% DHA and EPA in a ratio of 0.6. These four different Daphnia food qualities were fed to laboratory reared O. mykiss larvae. Two groups of rainbow trout larvae were fed with Artemia and starved, respectively as positive and negative control. Different analyses were performed of the rainbow trout larvae. The growth rate, RNA:DNA ratio, C:P ratio and fatty acid spectra were established to assess the effect of food quality of the different daphnids for the fish. Fish biochemical analyses were conducted with muscle tissue and the growth rate was calculated using the wet weight at the beginning and the end of the experiment. The starved fish showed in both experiments a lower condition with a significantly lower RNA:DNA and a significant negative growth rate. Starved fish also showed a lower fatty acid concentration compared to all other groups. Artemia fed larvae showed an increased condition in both experiments with a significantly higher growth rate and a higher RNA:DNA ratio. No clear significances were found according to the Daphnia fed rainbow trout. The O. mykiss larvae show a high variability in reaction within the different food treatments. Since ±23 days old rainbow trout larvae were used, there is a high possibility that the fish larvae were in a different developmental stage. The mean growth rates were not different comparing the four Daphnia groups within the experiments. However, between the experiments the growth rates showed a big difference. The mean growth rates of the rainbow trout larvae in experiment 1 were lower compared to experiment 2 (0.055 and 0.073, resp). Three possible reasons are suggested. Rainbow trout larvae in experiment 2 had higher PUFA concentrations (which can be used as energy source), the initial body weight was higher and (as a result of the higher body weight) the fish larvae were fed more. The RNA:DNA ratios were positively correlated with the growth rate whereas also the total HUFA concentration showed the same pattern. The total concentration of the fatty acids ALA, EPA and DHA showed a significant positive correlation with the growth rate and the RNA:DNA ratio. For the C:P ratios no clear pattern could be seen. The C:P ratio is expected to be homeostatic but the results of the experiments didn’t show homeostasis of the C:P ratio. From this can be concluded that O. mykiss larvae are not homeostatic according to the C:P ratio. Two hypotheses were presented. I hypothesized that the quality effect of phosphorus limitation on Daphnia would not be transferred to juvenile rainbow trout and HUFA enrichment of the daphnids of both food qualities would lead to a higher condition of the rainbow trout larvae. Both of them cannot be answered yet. More research needs to be done to investigate both effects on juvenile fish larvae.Chapter 1 Introduction 1 1.1 The role of phosphorus in the aquatic pelagic ecosystem 1 1.2 Homeostasis and stoichiometry 2 1.3 Variation between fish 4 1.4 The importance of fatty acids in the aquatic ecosystem 5 1.5 The effect of phosphorus and fatty acids on trophic levels 7 1.5.1 Scenedesmus obliquus 7 1.5.2 Daphnia magna 7 1.5.3 Oncorhynchus mykiss 8 1.6 Hypotheses 9 Chapter 2 Materials and methods 10 2.1 Experimental design 10 2.1.1 Analyses of the rainbow trout larvae 10 2.2 Prior experiment with different Daphnia species 12 2.3 Cultivation of the experimental organisms 14 2.3.1 Artemia 14 2.3.2 Scenedesmus obliquus 14 2.3.3 Daphnia magna 14 2.3.4 Oncorhynchus mykiss 15 2.4 Practical setup of the experiments 17 2.5 Analyses 18 2.5.1 Calculation of the specific growth rate 18 2.5.2 Carbon 18 2.5.3 RNA:DNA ratio 18 2.5.4 Fatty acid 19 2.5.5 Phosphorus 21 2.6 Repetitions and statistics 23 Chapter 3 Results 24 3.1 Outline 24 3.2 Change in P and fatty acids from eggs to larvae of O. mykiss 25 3.3 Experiment 1 28 3.3.1 C, P and fatty acid concentration of the prey organisms 28 3.3.2 Results from O. mykiss larvae 29 3.4 Experiment 2 34 3.4.1 C, P and fatty acid concentration of the prey organisms 34 3.4.2 Results from O. mykiss larvae 35 3.5 Correlations 40 Chapter 4 Discussion and conclusions 44 4.1 Phosphorus and fatty acid concentrations in rainbow trout eggs and larvae 45 4.2 Effects of food quality on rainbow trout larvae 47 4.3 Concluding remarks 52 References 54 Appendices 59 Acknowledgements 6