THE EFFECTS OF DIETARY ESSENTIAL FATTY ACID ENRICHMENT ON THE NUTRITION AND CONDITION OF RED KING CRAB (PARALITHODES CAMTSCHA TIC US) LARVAE By Asia Beder RECOMMENDED: Dr. Louise Copeman „ / Dr. Sherry T^mooe Dr. Ginny E ^ert Advisory Committee Chair Dr. Franz Mueter Chair, Graduate Program in Fisheries APPROVED: Dr. Joan Braddoek Eichelberger Dean of the Graduate, School THE EFFECTS OF DIETARY ESSENTIAL FATTY ACID ENRICHMENT ON THE NUTRITION AND CONDITION OF RED KING CRAB (PARALITHODES CAMTSCHA TICUS) LARVAE A THESIS Presented to the Faculty of the University of Alaska Fairbanks in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE By Asia Beder, B.S. Fairbanks, AK December 2015 Abstract Alaska red king crab (Paralithodes camtschaticus) is a commercially valuable crab species in Alaska; however, six of the nine red king crab stocks in Alaska are depleted and have failed to recover even after closures of the fisheries. Hatchery rearing is being explored as a possibility for rehabilitating these stocks. In Chapter 1 I review the importance of diet, particularly fatty acids (FAs), when raising crustacean larvae in hatcheries. Long-chain polyunsaturated FAs (PUFAs) are important in molting success, growth, ability to handle stress, and overall survival of crustaceans. Specifically, when the FAs docosahexaenoic acid (DHA, 22:6n-3), eicosapentaenoic acid (EPA, 20:5n-3), and arachidonic acid (AA, 20:4n-6) are added to the diet, crustacean survival increases. In Chapter 2 I performed a live-food enrichment experiment in which I tested the importance of dietary ratios of DHA, EPA and AA to red king crab larval growth, survival, and energy storage. Artemia nauplii that were fed to king crab larvae were enriched with four experimental diets, including 1) DHA, 2) DHA & EPA, 3) DHA & AA, and 4) a control diet that had the same total lipid per wet weight but was enriched with oleic acid (OA, 18:1n-9) and had very low PUFAs. Larval condition, assessed by viewing size and number of lipid droplets, was significantly greater at the glaucothoe stage for larvae fed PUFA enriched diets than larvae fed the control OA diet. Larvae that were fed diets enriched with DHA & EPA and DHA & AA had more and larger lipid droplets than larvae that received either the DHA or control (OA) diets. During the high mortality glaucothoe stage the total FAs per wet weight (^g/mg) was significantly lower in crabs fed the control diet. Further, during a salinity stress test glaucothoe previously fed the control OA diet recovered significantly slower than individuals previously fed PUFA-enriched diets, suggesting that PUFAs may improve performance during stress. These results advance our understanding of crustacean larval nutrition v and inform the feasibility of rehabilitating king crab stocks that crashed in the early 1980s throughout Alaska. vi Table of Contents Page Signature Page .............................................................................................................................i Title Page .................................................................................................................................. iii Abstract..................................................................................................................................... v T able of Contents..................................................................................................................... vii List of Figures ............................................................................................................................ ix List of Tables...............................................................................................................................x Abbreviations............................................................................................................................ xi Acknowledgments ...................................................................................................................xiii General Introduction ..................................................................................................................1 References..................................................................................................................................5 Chapter 1. A review of lipids and dietary fatty acid requirements for hatchery raised marine larval crustaceans .......................................................................................................................9 1.1 Abstract.................................................................................................................... 9 1.2 Introduction ........................................................................................................... 10 1.3 Lipids ......................................................................................................................10 1.4 Fatty Acids..............................................................................................................13 1.5 Enrichments of DHA, EPA and AA in Crustacean Larval Development ................15 1.6 Hatchery Stressors and AssessingCondition .........................................................18 1.7 Conclusion..............................................................................................................19 1.8 References............................................................................................................. 21 Chapter 2. The effects of dietary live-food essential fatty acid ratios on the condition, stress response and survival of red king crab larvae (Paralithodes camtschaticus).........................33 vii 2.1 Abstract 33 2.2 Introduction ........................................................................................................................34 2.3 Materials and Methods ....................................................................................................... 38 2.3.1 Broodstock and Larval Husbandry.....................................................................38 2.3.2 Larval Nutrition Experiment .............................................................................. 39 2.3.3 Survival and Visual Condition Assessment ........................................................39 2.3.4 Stress Test...........................................................................................................40 2.3.5 Weights and Biochemistry ................................................................................. 41 2.3.6 Statistical Analyses............................................................................................. 43 2.4 Results.................................................................................................................................43 2.4.1 Lipid Classes and Fatty Acids ............................................................................. 43 2.4.2 Growth .................................................................................................................52 2.4.3 Survival and Visual Condition Assessment ........................................................52 2.4.4 Stress Test ...........................................................................................................53 2.5 Discussion ...........................................................................................................................59 2.6 References .......................................................................................................................... 66 General Conclusions ............................................................................................................................. 77 References .............................................................................................................................................79 viii List of Figures Page Figure 1.1. Major lipid classes found in marine crustaceans........................................................ 11 Figure 2.1. Average (± SE) lipid per WWT (^g/mg) for crabs fed enriched diets ....................... 51 Figure 2.2. Relative proportions of (a) DHA, (b) EPA, and (c) AA for each diet by stage 54 Figure 2.3. Relative proportions of (a) DHA:EPA and (b) EPA:AA for diet by stage .................. 55 Figure 2.4. Principal component analysis for Artemia and crabs fed four different diets 56 Figure 2.5. Average (± SE) survival of larvae and juveniles for each diet treatment by stage .... 57 Figure 2.6. Average (± SE) number of lipid droplets for each diet by ontogenetic stage 57 Figure 2.7. Average (± SE) maximum lipid diameter (^m) for all diets from the Z1 to G stage . 58 Figure 2.8. Average (± SE) recovery time (seconds) by diet for fresh water stress test (n=30)... 58 ix List of Tables Page Table 1.1 Fatty acid profiles (% total FAs) of unenriched Artemia and copepods ......................... 16 Table 1.2. Percentage of major FAs in wild and cultured juvenile RKC ........................................ 18 Table 2.1. Biochemical composition of the enrichment emulsions (average ± SE, n=3) .............. 40 Table 2.2. Total fatty acids per WWT (^g/mg) and major fatty acids (>1.5%) in Artemia.......... 46 Table 2.3. Lipid class composition (% total lipids) of Artemia enriched for 24 h ...............47 Table 2.4. Total fatty acids per WWT (^g/mg) and major fatty acids (>1.5%) in Z1 and Z 4 ...... 48 Table 2.5. Total fatty acids per WWT (^g/mg) and major fatty acids (>1.5%) in RKC G ........... 49 Table 2.6. Total fatty