UNIVERSITY OF PRINCE EDWARD ISLAND

Nutritional condition and quality assessment of the American , Homarus americanus, at different stages of the moult cycle

A Thesis

Submitted to the Graduate Faculty

in Partial Fulfillment of the Requirements

for the Degree of

Master of Science

In the Department of Pathology and Microbiology

Faculty of Veterinary Medicine

University of Prince Edward Island

Michael A. Ciaramella

Charlottetown, P. E. I.

August 2011

© 2011 M. Ciaramella

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ii SIGNATURE PAGE

Gt'Q^OvJ

REMOVED ACKNOWLEDGEMENTS

I would like to thank my supervisors Dr. Andrea Battison and Dr. Barbara Homey for the help and support they have provided me with over the past two years. I would additionally like to thank the members of my supervisory committee Dr. Fred Kibenge, Dr. Jean Lavallee and Dr. Dave Speare for their guidance in conducting my MSc research and the thesis writing. An additional thanks to all those who have helped along the way including everyone at the Lobster Science Centre and Atlantic Veterinary College for being there to support me through my program.

I gratefully acknowledge the Atlantic Veterinary College Lobster Science Centre, Innovation PEI, the Department of Pathology and Microbiology, and NSERC the financial support received to conduct my MSc research and present it around the world.

Finally I would like to thank all the friends I have made along the way, my fellow graduate students at the University, and my family for all their support!

v Table of Contents 1. General Introduction 1

1.1. The , Homarus americanus 1

1.1.1. General Background 1

1.1.2. Growth 2

1.1.3. Quality 4

1.1.4. Economic Importance 5

1.2. Moult Staging 6

1.2.1. Drach Staging 6

1.2.1. Pleopod Staging 8

1.3. Energy Reserves 8

1.4. Hemolymph Biochemistry 12

1.4.1. Circulating Metabolites 12

1.4.2. Hemolymph Osmolality 14

1.5. Research Objectives 15

1.6. References 17

2. Practical Optimisation of the Dataset 23

2.1. Abstract 23

2.2. Introduction 23

2.3. Materials and Methods 26

2.3.1. Population Monitoring for Optimisation of Sample Collection 26

2.3.2. Collection of Lobster Samples 26

2.3.3. Laboratory Processing of Collected 30

2.3.4. Statistical Analyses of sampling data 31

2.4. Results 31

2.4.1. Population Monitoring for Optimisation of Sample Collection 31

2.4.2. Collection of Lobster Samples 34

2.4.3. Laboratory Processing of Lobsters Collected 35

2.5. Discussion 36

2.6. References 40

VI 3. Evaluation of Fluctuations in Tissue Water Content of the American Lobster Homarus americanus during the Moult Cycle 43

3.1. Abstract 43

3.2. Introduction 43

3.3. Materials and Methods 45

3.3.1. Homogenisation of Lobster Tissues 45

3.3.2. Lyophilisation of Lobster Tissues 46

3.3.3. Calculation of Percent Water in Tissue Samples 46

3.3.4. Statistical Analysis 47

3.4. Results 47

3.4.1. Lyophilisation Study 47

3.4.2. Analysis of Tissue Water Content Data 48

3.4.3. Statistical Analysis 49

3.5. Discussion 49

3.6. References 53

4. Fluctuations in Glycogen Stores of the Hepatopancreas and Muscle Tissues of the American Lobster Homarus americanus during the Moult Cycle 55

4.1. Abstract 55

4.2. Introduction 56

4.3. Materials and Methods 61

4.3.1. Optimisation of Tissue and Oyster Glycogen Standard Digestion 61

4.3.2. Evaluation of the Glucose Oxidase Method for Glucose Determination in Lobster Tissues 62

4.3.3. Evaluation of the Hexokinase Method for Glucose Determination in Lobster Tissues 63

4.3.4. Calculation of Oyster Glycogen Equivalents 63

4.3.5. Validation of Glucose Determination Using the Hexokinase Assay 64

4.3.6. Assessment of Background Glucose in Lobster Tissues 65

4.3.7. Relative Distribution of Tissue Glycogen at Different Stages of the Moult Cycle 66

4.3.8. Statistical Analysis of Glycogen Data 67

4.4. Results 67

4.4.1. Optimisation of Tissue and Standard Glycogen Digestion 67

VII 4.4.2. Evaluation of the Glucose Oxidase Method for Glucose Determination in Lobster Tissues 68

4.4.3. Evaluation of the Hexokinase Method for Glucose Determination in Lobster Tissues 69

4.4.4. Validation of Glucose Determination Using the Hexokinase Assay 70

4.4.5. Assessment of Background Glucose in Lobster Tissues 73

4.4.6. Relative Distribution of Tissue Glycogen at Different Stages of the Moult Cycle 74

4.5. Discussion 83

4.6. References 97

5. Fluctuations in Stored Lipid of the Hepatopancreas and Muscle Tissues of the American Lobster Homarus americanus during the Moult Cycle 102

5.1. Abstract 102

5.2. Introduction 102

5.3. Materials and Methods 105

5.3.1. Comparison of Different Extraction Methods for the Determination of Lipid Concentrations in Lobster Tissues 105

5.3.2. Precision of the Folch Centrifugation Method for Lipid Extraction 107

5.3.3. Long Term Stability of Hepatopancreas Tissue Samples Stored at -80°C 107

5.3.4. Assessment of Lipid Concentrations in Lobster Tissues Representing Different Moult Categories 108

5.3.5. Statistical Analysis of Lipid Data 108

5.4. Results 109

5.4.1. Comparison of Different Extraction Methods for the Determination of Lipid Concentrations in Lobster Tissues 109

5.4.2. Precision of the Folch Centrifugation Method for Lipid Extraction 110

5.4.3. Long Term Stability of Hepatopancreas Tissue Samples Stored at -80°C Ill

5.4.4. Assessment of Lipid Concentrations in Lobster Tissues Representing Different Moult Categories Ill

5.5. Discussion 119

5.6. References 127

6. Biochemical Analysis of Hemolymph Metabolites during the Moult Cycle in the American Lobster, Homarus americanus 130

6.1. Abstract 130

VIII 6.2. Introduction 131

6.3. Materials and Methods 136

6.3.1. Biochemical Analysis of Hemolymph Metabolites and Osmolality During the Moult 136

6.3.2. Statistical Analysis of Hemolymph Biochemistry and Osmolality Data 136

6.4. Results 137

6.4.1. Evaluation of Hemolymph Biochemical Parameters for the Primary 2009 Moult Cohort 137

6.4.2. Evaluation of Hemolymph Biochemical Parameters for the 2009 and 2010 Moult Cohorts 137

6.4.3. Evaluation of Year Effects on Hemolymph Biochemical Parameters 143

6.4.4. Correlations between the Hemolymph Biochemical Parameters 144

6.5. Discussion 145

6.6. References 152

7. Correlations among the Changes in Physiology and Biochemistry of the Tissues and Hemolymph in the American Lobster, Homarus americanus, during the Moult Cycle 156

7.1. Introduction 156

7.2. Materials and Methods 157

7.3. Results 158

7.3.1. Relative Concentrations of Water, Lipid and Glycogen in the Hepatopancreas across the Five Moult Categories 158

7.3.2. Relative Concentrations of Water, Lipid and Glycogen in the Muscle Tissues across the Five Moult Categories 159

7.3.3. Examination of the Correlations between Tissue Water Content and Biochemical Composition 161

7.3.4. Examination of the Correlations between Tissue Energy Reserves and Metabolite Concentrations in the Hemolymph 162

7.4. Discussion 169

7.5. References 176

8. Summary and Conclusions .' 178

8.1. References 186

9. Appendices 189

Appendix A Homogenisation and Lyophilisation of tissue samples 189

IX Appendix B Folch centrifugation method of total lipid extraction from hepatopancreas ....190

Appendix C Folch centrifugation method of total lipid extraction from muscle 191

Appendix D Glucose oxidase method for glucose determination 192

Appendix E Hexokinase method for glucose determination 193

Appendix F Solution Preparations 194

x List of Figures

Figure 2.1: Map of lobster fishing areas surrounding Prince Edward Island 28 Figure 2.2: Clipping of a pleopod for determination of moult stage 29 Figure 2.3: Measurement of carapace length 29 Figure 2.4: Hemolymph extraction 29 Figure 2.5: Hemolymph protein concentrations in LFA 26A Georgetown, PE, 2009 and 2010 .. 32 Figure 2.6: Percent landings from different moult categories (LFA 26A), Georgetown, PE, 2009 33 Figure 2.7: Graphical representation of the nutritional status samples collected 35 Figure 3.1: Lyophilisation study for the determination of dehydration time 47 Figure 3.2: The average tissue water content of the five moult categories examined 49 Figure 4.1: Diagram of a chitin molecule 57 Figure 4.2: Glucose standard curves generated with the glucose oxidase method over time... 69 Figure4.3: Glucose standards run with the hexokinase method overtime 70 Figure 4.4: Linearity of oyster glycogen standard curves 73 Figure 4.5: Total hepatopancreas glycogen in lobsters from the 2009 primary moult cohort... 76 Figure 4.6: Standardised hepatopancreas glycogen in lobsters from the 2009 primary moult cohort 76 Figure 4.7: Standardised pincher claw muscle glycogen in lobsters from the 2009 primary moult cohort 77 Figure 4.8: Standardised crusher claw muscle glycogen in lobsters from the 2009 primary moult cohort 77 Figure 4.9: Standardised tail muscle glycogen in lobsters from the 2009 primary moult cohort 78 Figure 4.10: Standardised hepatopancreas glycogen in lobsters from all moult cohorts collected in 2009 and 2010 80 Figure 4.11: Standardised pincher claw muscle glycogen in lobsters from all moult cohorts collected in 2009 and 2010 80 Figure 4.12: Standardised crusher claw muscle glycogen in lobsters from all moult cohorts collected in 2009 and 2010 81 Figure 4.13: Standardised tail muscle glycogen in lobsters from all moult cohorts collected in 2009 and 2010 81 Figure 4.14: The effects of year on glycogen concentrations in the hepatopancreas 82 Figure 4.15: The effects of year on glycogen concentrations in the tail muscle 82 Figure 5.1: Comparison of lipid extraction procedures 109 Figure 5.2: Total lipid concentrations in the hepatopancreas for the 2009 primary moult cohort 112 Figure 5.3: Standardised lipid concentrations in the hepatopancreas for the 2009 primary moult cohort 113

XI Figure 5.4: Standardised lipid concentrations in the pincher claw muscle for the 2009 primary moult cohort 114 Figure 5.5: Standardised lipid concentrations in the crusher claw muscle for the 2009 primary moult cohort 115 Figure 5.6: Standardised lipid concentrations in the tail muscle for the 2009 primary moult cohort 115 Figure 5.7: Standardised lipid concentrations in the hepatopancreas for all moult cohorts from the 2009 and 2010 samplings 117 Figure 5.8: Standardised lipid concentrations in the tail muscle for all moult cohorts from the 2009 and 2010 samplings 117 Figure 5.9: Standardised lipid concentrations in the pincher claw muscle for all moult cohorts from the 2009 and 2010 samplings 118 Figure 5.10: Standardised lipid concentrations in the crusher claw muscle for all moult cohorts from the 2009 and 2010 samplings 118 Figure 5.11: Evaluation of year effect on lipid concentrations in the pincher claw muscle 119 Figure 6.1: Variations in hemolymph total protein concentrations during the moult for all lobsters from 2009 and 2010 138 Figure 6.2: Variations in hemolymph uric acid concentrations during the moult for all lobsters from 2009 and 2010 139 Figure 6.3: Variations in hemolymph triglyceride concentrations during the moult for all lobsters from 2009 and 2010 141 Figure 6.4: Variations in hemolymph cholesterol concentrations during the moult for all lobsters from 2009 and 2010 141 Figure 6.5: Variations in hemolymph glucose concentrations during the moult in all lobsters from 2009 and 2010 142 Figure 6.6: Variations in hemolymph osmolality during the moult for all lobster from 2009 and 2010 142 Figure 6.7: Comparison of hemolymph total protein concentrations between collection years 143 Figure 6.8: Comparison of measured hemolymph osmolality between collection years 144 Figure 7.1: Relative composition of the hepatopancreas 158 Figure 7.2: Relative composition of the pincher claw muscle 159 Figure 7.3: Relative composition of the crusher claw muscle 160 Figure 7.4: Relative composition of the tail muscle 160 Figure 7.5: Regression analysis of hemolymph cholesterol versus lipid concentrations in the hepatopancreas 166 Figure 7.6: Regression analysis of hemolymph triglyceride versus lipid concentrations in the hepatopancreas 166 Figure 7.7: Regression analysis of total hemolymph protein versus lipid concentrations in the hepatopancreas 167

XII List of Tables

Table 2.1: Summary of moult categories as defined by pleopod staging and shell hardness .... 27 Table 2.2: Table of sampling seasons and expected moult categories present 33 Table 2.3: Summary of sample size for each moult category 34 Table 4.1: Summary of solutions used in the glycogen assays 62 Table 4.2: Summary of glycogen assay sample sizes 66 Table 4.3: The effect of incubation time on glycogen digestion 68 Table 4.4: The effect of tissue amount on digestion of glycogen 68 Table 4.5: Precision of hexokinase assay on tissue samples and oyster glycogen standards 71 Table 4.6: Stability of tissue glycogen following long term storage 72 Table 4.7: Stability of lyophilised tissue samples following long term storage 72 Table 4.8: Evaluation of baseline glucose concentrations in tissue samples 74 Table 4.9: Regression equations for oyster glycogen curves used for the calculation of tissue glycogen 75 Table 5.1: Comparison of manual vs. electronic homogenisation of tissue samples 110 Table 5.2: Precision of lipid extractions in four lobster tissues 110 Table 5.3: Stability of tissue samples after long term storage at -80°C Ill Table 6.1: Pearson correlations between measured hemolymph biochemical parameters .... 145 Table 7.1: Correlations between tissue water content and metabolic reserves 161 Table 7.2: Pearson correlations between tissue energy reserves and hemolymph biochemical parameters for males 164 Table 7.3: Pearson correlations between tissue energy reserves and hemolymph biochemical parameters for females 165 Table 7.4: Assessment of nutritional condition biomarkers by moult category and sex 168

XIII List of Abbreviations

ACC Care Committee ALMQ Atlantic Lobster Moult and Quality AMG Amyloglucosidase AVC Atlantic Veterinary College AVCLSC Atlantic Veterinary College Lobster Science Centre B&D Bligh and Dyer CB Sodium Citrate Buffer CCM Crusher Claw Muscle CHOL Cholesterol EH Electric Homogenisation EPM Early Pre-moult FoC Folch Centrifugation FoF Folch Filtration GLUC Glucose Gin Glycogen Index GOx Glucose Oxidase GPH Glass Piston Homogeniser G-6-P Glucose-6-phosphate HK Hexokinase HP Hepatopancreas HI Heat Inactivation IM Inter-moult LFA Lobster Fishing Area LIM Late Inter-moult Un Lipid Index LPM Late Pre-moult MH Manual Homogenisation Min Minute MPM Mid Pre-moult NS Nutritional Status OD Optical Density OG Oyster Glycogen OSM Osmolality PCM Pincher Claw Muscle PM Pre-moult PoM Post-moult Rl Refractive Index RT Room Temperature TG Triglycerides TM Tail Muscle TP Total Protein UA Uric Acid WC Water Content

XIV 1. General Introduction

1.1. The American lobster, Homarus americanus

1.1.1. General Background

There are four major families of decapod that are referred to as lobster.

These include the clawed in the family Nephropidae and the unclawed species in the Palinuridae, Synaxidae, and Scyllaridae families (Phillips et al., 1980). The

American lobster, Homarus americanus, belongs to the family Nephropidae. Its natural habitat extends along the eastern coast of North America from North Carolina to

Newfoundland (Herrick, 1909). The wide geographical distribution of the American lobster attests to its diverse nature, adaptive capabilities and diverse feeding habits.

Homarus americanus are poikilotherms, meaning they are unable to regulate their own body temperature and therefore it mimics that of the external environment (Nybakken and Bertness, 2005). They are also osmoconformers (Dall, 1970), thus their internal osmotic pressure changes in response to alterations in the external medium to maintain the hemolymph isosmotic to their environment (Robertson, 1960). This causes lobsters to be highly susceptible to environmental manipulations (Passano,

1960), which can cause alterations in natural biochemical and physiological processes.

Lobsters are predators and opportunistic scavengers and will feed on a variety of crustaceans, fish, mollusks, echinoderms, cnidarians, algae and marine grasses. The majority of their diet consists of various and mollusk species (Lawton and

Lavalli, 1995). Their diet is highly influenced by food availability (Castille and Lawrence,

1 1989) as well as life history stage (Conklin, 1980) and can exhibit alterations in response to season (Lawton and Lavalli, 1995). Diet and nutrition are fundamental aspects in maintaining good health.

1.1.2. Growth

Growth in crustaceans is very different from that observed in vertebrates. The continual growth observed in most vertebrate species is impossible in crustaceans due to their hard calcerous exoskeleton. The exoskeleton of crustaceans must be shed through a moulting process for the organism to grow (Aiken, 1980). Moulting is a natural physiological occurrence, during which the formation of a new shell takes place beneath the old one. Immediately following the extrusion of the exoskeleton, a process termed ecdysis, water is absorbed quickly to expand the new shell (Waddy et al., 1995; Mykles, 1980). In the period following ecdysis, mineralisation of the new shell begins (Travis, 1955a). During the moulting process, pronounced changes in biochemistry and physiology take place, which make it a costly yet crucial period in the organism's life cycle.

As a result of the intermittent growth cycles of crustaceans, there are long periods with no change in size observed. This period is known as the inter-moult (IM) and can occupy as much as 65% of the moult cycle (Waddy et al., 1995). The IM is followed by the pre-moult (PM), a brief period referred to as ecdysis and a post-moult (PoM) recovery period. From this point on, moult will refer to the complete cycle including

2 preparatory and recovery stages, while ecdysis refers to the actual stage within the moult cycle that the old shell is exuviated.

The moult has been examined in many decapod crustaceans. Through these studies, specific moult stages have been characterised (Waddy et al., 1995). The different stages have been devised based on the physiological, biochemical, and structural changes taking place in preparation for the moult, as a result of ecdysis and in response the PoM recovery phase. These changes can be categorized into four generalized stages including IM, PM, ecdysis and PoM.

The IM period represents the "normal state" of the organism where no drastic physiological or biochemical changes occur and energy reserves are accumulated for use in the following moult. The PM would include all changes taking place in preparation for ecdysis. Degradation and resorption of the old exoskeleton, accumulation of organic reserves, and formation of a new epicuticle represent a few of the major changes taking place at this time (Passano, 1960; Aiken, 1980; Parrish and

Martinelli-Liedtke, 1999). During ecdysis, the shell is exuded and expansion of the newly formed cuticle occurs via ingestion and absorption of water (Mykles, 1980;

Mykles, 1981). Following ecdysis are the PoM or recovery phases when mineralisation of the shell takes place and replacement of water with new tissue growth occurs

(Passano, 1960; Aiken, 1980; Parrish and Martinelli-Liedtke, 1999). A more in-depth classification of moult staging will be discussed in Section 1.2.

3 1.1.3. Quality

Historically, Atlantic Canada has a reputation for providing a high quality product to its buyers. However, in southwest Nova Scotia between 2000 and 2005, the lobsters caught were of very poor quality. During this time, the meat yields were low and water content was high, which is very different from the typical "well-meated" lobster often observed in that area. Quality refers primarily to the amount of meat present and is inversely proportional to tissue water content in lobsters. Poor quality is generally related to the lobsters' moult cycle. In the PM preparatory stages directly preceding ecdysis, metabolic reserves are utilised and replaced with water (Baumberger and

Olmsted, 1928; Dall and Smith, 1987) causing an increase in tissue water content. At ecdysis, copious amounts of water are ingested for the expansion of the newly formed shell. The water remains until the lobster is able to replace it with muscle.

Consequently, decreased quality is noted in the later pre-moult and PoM lobsters.

Quality will slowly increase over time during the PoM recovery phase when the lobster is regenerating lost muscle mass and filling in the new shell (Retzlaff et al., 2007).

Muscle atrophy is another major physiological process that occurs during the moult in crustaceans (Mykles, 1992). A decrease in the size of muscle fibers is observed mainly in the claw muscles (Mykles, 1992) to facilitate a reduction in muscle mass and allow for easier exuviation of the old shell. This atrophy of the muscle tissues contributes to the decrease in quality that is often associated with the moult.

4 Behavioral changes can also occur within a population in response to the moult. In the

later PM stages feeding will stop resulting in a period of inanition (Dall, 1990), which

extends into the PoM. The period of inanition immediately preceding and following

ecdysis suggests that energy reserves will be integral to survival at that time. Stored

reserves are the sole form of energy available during the starvation period and are

relied upon to survive the energetically expensive physiological changes that take place

during ecdysis. Thus, these reserves may also play an important role in quality by

dictating what is available for the generation of new tissue.

1.1.4. Economic Importance

Lobsters have been fished since the early seventeenth century, but the industry was

not established until the late 18th to early 19th centuries (Herrick, 1909). Over the

years, drastic changes have occurred in the industry with decreases in mean size and

overall catch over time. Coinciding with the decreasing catch was an increase in price

(Herrick, 1909). In 2009, 56,554 metric tonnes of lobster were landed in Atlantic

Canada valuing over $495 million (DFO, 2010a). In the same year, over 43,900 metric tons valued at over $300 million were landed in the United States (NOAA, 2011). In

Canada, the lobster fishing industry employed over 31,000 fishermen in 2007 (DFO,

2010b) in addition to over 25,000 Canadians employed through the industry as plant workers and exporters (Weston, 2009). Thus, the lobster industry contributes significantly to both the Canadian and American economies. This warrants a better understanding of general biology, physiology and health assessment to better manage and conserve this lucrative resource.

5 1.2. Moult Staging

1.2.1. Drach Staging

Moulting is a natural phenomenon in the life cycle of decapod crustaceans and plays an

integral role in the growth and survival of the organism. The moult cycle of H.

americanus has been studied and divided into categories or stages based on significant

physiological, morphological, biochemical, and behavioral changes that occur

throughout the moult process (Waddy et al., 1995). The currently accepted categories

for the moult are the Drach stages, which are labeled A through E, with a number of

subdivisions in stages A through D (Waddy et al., 1995). Stage E, or ecdysis, is the only

one that is not subdivided as this stage is very short and consists of the actual

exuviation of the old exoskeleton (Waddy et al., 1995). During ecdysis, the

thoracoabdominal membrane cracks facilitating escape from the shell (Waddy et al.,

1995). At this time, the exoskeleton is soft and vulnerable to predation, confining the

lobster to its shelter as it enters into the PoM recovery phase of the cycle.

The first two stages of the PoM are A and B, which together comprise approximately

10% of the moult cycle. In stage A, the lobsters quickly absorb water to expand the

new soft exoskeleton to its new size, which can be as much as a 50% increase (Waddy

et al., 1995). Stage A is divided into three sub-stages (A-A2), which correspond to

physiological changes taking place such as water absorption and the start of shell

mineralisation (Waddy et al., 1995). The B stages (B-B2) represent the main period of

shell calcification and hardening. The lobster then enters the C stages (C-C4). During

the earlier C stages (C-C3), the calcification of the newly formed shell continues. This

6 stage also represents the onset of new tissue formation to replace the water absorbed

in stage A and fill the new space obtained through expansion of the new shell. Once

the lobster enters the C4 stage of the moult cycle, it is no longer considered a PoM

lobster but is considered to be in the IM stage. This IM stage is the longest stage,

taking up about 65% of the cycle during which energy stores that were lost during the

moult are replenished (Waddy et al., 1995).

At the end of the IM (C4) stage, the lobster then enters the D stages. This represents

the moult preparatory period, which is subdivided into five different stages (D-D4). It is

during these PM stages that the synthesis of the new cuticle takes place. This

represents the only point in the lobster life cycle that pleopods can be used as an

external means of determining moult stage. In stages D through D4/ a new epicuticle is

secreted beneath the old one, the formation of a pigmented layer begins, extensive

absorption of calcium from the old shell takes place, and the onset of water absorption

occurs (Waddy et al., 1995). In the later stages of the PM, feeding behavior changes

with a gradual decrease in active feeding before it ceases all together (Dall, 1990).

During this time metabolic reserves are relied upon as the sole source of energy. Also

during the D stages, muscle tissue, primarily in the claws, begins to atrophy to allow for an easier escape from the old shell (Mykles, 1992; Mykles, 1999; Mykles and Skinner,

1990; Haj et al., 1996; Renato de Oliveira Cesar, Jose et al., 2006).

7 1.2.1. Pleopod Staging

Pleopod staging is a common technique used to distinguish the stage of a lobster

within the moult preparatory stages (D stages); it is based on the morphological

changes in cuticle formation within the pleopods (Aiken, 1973). The pleopods are the

swimming appendages located on the ventral side of the abdomen or tail of the

lobster. These appendages are relatively thin, which facilitates staging methods by

allowing the visual assessment of cuticle formation. However, because this method

relies on the morphological changes in cuticle excretion taking place it is only viable

during the D stages of the moult cycle (Aiken, 1973). Pleopods lacking any signs of

cuticle formation are considered stage zero, which encompasses all IM and PoM

periods. A crude method of distinguishing between the IM and PoM categories is

through shell hardness. The shell during the IM will be hard as it is completely

chitinized and mineralized at this time. In the PoM, chitinization and mineralisation is

still taking place, making the shell more flexible and less rigid (Waddy et al., 1995).

1.3. Energy Reserves

Energy reserves in an organism have two main applications. The first is ensuring a steady flow of energy for use in maintaining normal biochemical and physiological function (Gurr et al., 2002). The second is a more specialized reserve that can be

mobilised in times of need (Gurr et al., 2002). Stored metabolic reserves such as lipid and glycogen will dictate the overall condition of an organism. Nutritional condition as defined for crustacean species is "the extent to which it has accumulated reserves of nutrients to allow normal physiological function and growth (Moore et al., 2000)."

8 Crustaceans, unlike vertebrate species, do not show external physical signs of health or

condition throughout their life cycle because of the plastic state of their external

skeleton (Moore et al., 2000). This makes the assessment of health and condition very

difficult and is the reason for the interest in developing methods for assessing health

and nutrition in crustacean species (Moore et al., 2000; Dall, 1974; Vinagre and Silva,

1992).

The most commonly accepted indices of nutritional state in crustaceans are the

protein, lipid and carbohydrate concentrations in various tissues, most notably the

muscle and hepatopancreas. The muscle and hepatopancreas have been thoroughly

identified as main organs of storage for these metabolites (Parrish and Martinelli-

Liedtke, 1999; Parvathy, 1971; Barclay et al., 1983; Dall, 1981).

Although it was initially believed that the main source of energy in crustaceans was

protein (New, 1976; Comoglio et al., 2008), this is not found to be the case in all

decapods. Lipids have been identified as the primary source of energy during periods

of starvation in the false Southern king Paralomis granulose (Comoglio et al.,

2005) and the juvenile Eriocheir sinensis (Wen et al., 2006).

Carbohydrates were determined to be the main source of energy in the Pacific white Litopenaeus vannamei (Sanchez-Paz et al., 2007), the isopod Limnoria lignorum

(George, 1966) and the juvenile giant freshwater Macrobrachium rosenbergii

(Clifford and Brick, 1983). The specific energy reserve utilised during starvation will vary greatly depending on the species, geographic distribution, recent feeding habit,

9 diet and developmental stage of the organism (Vinagre and Silva, 1992; Barclay et al.,

1983; Clifford and Brick, 1983; Sanchez-Paz et al., 2006).

Lipid refers to a substance soluble in non-aqueous organic solvents but not in water

(Gurr et al., 2002). There are many classes of lipids, which can vary greatly between species, tissue, and cell types (Urich, 1994a). Its role in the structure and maintenance of cellular membranes, the formation of hormones, and a high nutritional capacity highlight the primary biological functions and importance of lipids in biological systems

(Bollenbacher et al., 1972; Kaplan et al., 2003). Triacylglycerols are the primary form of lipids used for storage, with wax esters being used as well in some marine species. In vertebrate species, lipids are typically stored in specialized tissue deposits known as adipose tissue. Many marine fish and crustaceans lack adipose tissues and lipids are stored primarily within other body tissues (Gurr et al., 2002).

Although lipid is a very compact and energy efficient reserve, providing 39 kJ/g, it is not without disadvantages (Urich, 1994a). The hydrophobic nature of lipids causes problems with the rapid transport from storage to areas of utilisation. Lipid metabolism can also only be carried out in the presence of oxygen (Urich, 1994a). The disadvantages of lipid suggest that in certain circumstances other reserves may be preferentially utilised. Periods of stress are likely examples of these circumstances because this would require energy quickly and oxygen may be limited.

Glycogen represents another metabolic reserve that is important in vertebrate species for the proper function and maintenance of organs (Verri et al., 2001). A glycogen

10 molecule is essentially composed of long chains of branched glucose molecules that

can be hydrolyzed and released into cellular and extracellular fluid for the rapid

generation of energy through glycolysis or the pentose phosphate pathways (Chang

and O'Connor, 1983; Matsui et al., 1996). Although this reserve offers a less energy

efficient alternative at 17 kJ/g (Urich, 1994a) it can generate a much quicker response

during acute stress or energy demand.

The quantity of stored energy reserves can provide insight into the feeding habits and

physiological processes of energy utilisation and storage. Nutrition in crustaceans has

been minimally studied and is just recently coming to the forefront of crustacean

biology. Acquiring knowledge of what is considered a "normal" nutritional state in

crustaceans is integral to the development of successful plans for conservation and

optimisation of the extremely lucrative lobster fishing industry in Atlantic Canada and

other fisheries around the world. Effective measures of nutritional state in crustaceans

will also be useful in conducting ecological studies (Dall, 1974; Moore et al., 2000) on

moult frequency, growth at moult, food availability and overall health in wild

populations.

The biochemical composition of tissues in decapod crustaceans will vary over time due to changing biotic factors such as maturation, reproduction, and food availability (Rosa and Nunes, 2003). In some crustaceans such as the Hawaiian , it has been found that the glycogen content in the abdominal muscle was the best measure of

nutritional state (Parrish and Martinelli-Liedtke, 1999). However, other studies have

11 identified the hepatopancreas as the most reliable tissue to measure energy reserves

(Dall, 1974). This alludes to the diverse nature of crustaceans and warrants the

development of nutritional and health assessment techniques for individual species.

Patterns in energy mobilisation can also be observed in which the period of inanition is

segmented into periods of mobilisation of a single energy reserve followed by a change

to another (Cuzon et al., 1980; Vinagre and Silva, 1992; Stuck et al., 1996; Wen et al.,

2006). During these periods of inanition, the organism relies heavily on stored energy

reserves, which allow for survival and help with physiological changes taking place

during that time. The availability of lipid and glycogen stores prior to the moult can

dictate the amount of growth obtained by manipulating moult increment and frequency. It may also have an impact on the ultimate survival of that individual. Thus, the ability to assess energy reserves of lobster populations non-lethally will enable

better monitoring of populations and conservation planning. This information could

potentially act as a means of predicting growth and survival of a population as it will be greatly affected by stored metabolic reserves.

1.4. Hemolymph Biochemistry

1.4.1. Circulating Metabolites

Biochemical analysis of the blood in vertebrate species is used in both veterinary and human medicine as a means of assessing health and internal physiology (Marks, 1978;

Mercaldo-Allen, 1991; Dufour, 2003). These data are commonly referred to as

12 biochemical panels and report the concentrations of a variety of biochemical

components in the blood or urine (Marks, 1978).

Parameters commonly measured include various metabolites, electrolytes and

enzymes. Biochemistry panels can include compounds less commonly found in the

blood or urine. The presence of these can be indicative of general or specific problems

with internal anatomy or physiology (Marks, 1978), such as abnormalities in organ

function and tissue damage. The concentrations can then be compared to their

respective reference values, based on measurements from a population of healthy

individuals. Abnormalities observed may be useful in the identification of problems in

nutrition and health.

Variations in what would be considered a normal reference interval for an individual

parameter will vary greatly depending on species, sex and life cycle of the organism.

Available reference values for many crustacean species are limited, making the results

of biochemical analysis of hemolymph samples difficult to interpret (Noga, 2000). Thus far, knowledge of hemolymph biochemical changes attributed to the moult cycle is

limited.

Enhanced knowledge of the natural changes in hemolymph biochemistry and the factors that affect hemolymph metabolite concentrations could be useful as a means of assessing overall health and nutrition. Proteins are long chains of covalently bonded amino acids, which represent the most abundant macromolecules in living cells

(Lehninger et al., 1993b). They can act as a significant energy reservoir in some

13 crustaceans (Barclay et al., 1983; Neiland and Scheer, 1953). Uric acid is a waste product of the oxidative degradation of amino acids and nucleotides (Urich, 1994b;

Lehninger et al., 1993a) and thus is indicative of protein catabolism. The lipid metabolites present in the hemolymph include triglycerides (TG) and cholesterol

(CHO). Triglycerides are neutral lipids commonly used for storage and the generation of energy. Cholesterol plays an important role in the structure of membranes and the formation of hormones (Gurr et al., 2002). Glucose (GLUC) represents the primary hemolymph sugar in circulation and is important in maintaining regular organ function in crustaceans (Radford et al., 2005).

The development of reference intervals for various hemolymph metabolites would allow for better understanding and monitoring of crustacean health and nutrition.

However, the complexity of the crustacean life cycle, the marked changes in physiology and biochemical composition throughout many life history stages and the pronounced physiological differences between vertebrate and invertebrate species can make the development of standard procedures and reference intervals for biochemical analysis in crustaceans difficult.

1.4.2. Hemolymph Osmolality

Osmolality is the measure of osmotic pressure in the hemolymph, which is a measure of dissolved and particulate matter in a fluid. Changes in osmotic pressure are dictated through the active or passive regulation of particulate concentrations within body fluids (Robertson, 1960). The active regulation of osmolality in crustaceans has been

14 suggested to play an integral role in the absorption of water at ecdysis (Baumberger and Olmsted, 1928; Wilder et al., 2009). The absorbed water functions as a mechanism for expansion, while the hydrostatic pressure generated by the high water content of crustaceans allows for movement even in the fragile state of a PoM lobster. Muscle contraction in PoM crustaceans causes a change in hydrostatic pressure and allows movement immediately following the moult (Taylor and Kier, 2003), making the lobster less susceptible to predation and aiding in the commencement of feeding.

Methods of osmotic regulation vary among crustaceans, especially between marine and freshwater species. Marine crustaceans typically exhibit isosmotic or hyposmotic equilibrium in which osmolyte concentrations are equal to or slightly less than the surrounding medium. In freshwater species, the osmotic gradient is hyperosmotic, meaning hemolymph osmolytes are higher than those in the surrounding medium

(Baumberger and Olmsted, 1928; Robertson, 1960).

1.5. Research Objectives

The availability of metabolic reserves plays an important role in the PoM recovery time and ultimately dictates quality in the lobster. The above average water temperatures observed in southwest Nova Scotia in 2003-2005 (Retzlaff et al., 2007) have been suggested as a reason for the poor quality catches in those years. The problem was believed to be caused by a shift in the lobsters moult cycle triggered by changing water temperatures, as was reported in the western rock lobster, , in

Western (Caputi et al., 2010)

15 The poor quality lobsters observed in southwest Nova Scotia raised awareness of the fact that very few methods have been developed for the assessment of health and nutrition in the live lobster. To attain a better understanding of lobster health and nutrition, physiological and biochemical changes will be examined in H. americanus throughout different stages of the moult cycle. Changes examined will include the natural fluctuations in water content and lipid and glycogen stores as well as the biochemical analysis of various hemolymph metabolites.

This study ultimately aims to define the moult-induced changes in energy stores (lipid and glycogen) and their specific role during the moult cycle. The identification of the primary metabolic reserve utilised during the moult will be compared to changes in hemolymph metabolite concentrations to discern any patterns or correlations that may exist between them. Patterns and correlations can then be assessed for their significance or relevance in predicting metabolic stores and used for the development of a non-lethal means of assessing nutrition in the lobster.

16 1.6. References

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Aiken, D.E., 1973. Proecdysis, setal development, and molt prediction in the American lobster (Homarus americanus). J. Fish. Res. Board Can. 30,1337-1344.

Barclay, M.C., Dall, W., Smith, D.M., 1983. Changes in lipid and protein during starvation and the moulting cycle in the tiger prawn, esculentus Haswell. J. Exp. Mar. Biol. Ecol. 68(3), 229-244.

Baumberger, P.J., Olmsted, J.M.D., 1928. Changes in the osmotic pressure and water content of during the molt cycle. Physiol. Zool. Vol. l(No. 4), 531-544.

Bollenbacher, W.E., Borst, D.W., O'Connor, J.D., 1972. Endocrine regulation of lipid synthesis in Decapod crustaceans. Am. Zool. 12, 381-384.

Caputi, N., Melville-Smith, R., de Lestang, S., Pearce, A., Feng, M., 2010. The effect of climate change on the western rock lobster (Ponulirus cygnus) fishery of Western Australia. Can. J. Fish. Aquat. Sci. 67, 85-96.

Castille, F.L., Lawrence, A.L., 1989. Relationship between maturation and biochemical composition of the gonads and digestive glands of the Penaeus aztecus Ives and Penaeus setiferus (L.). J. Crustacean Biol. 9(2), 202-211.

Chang, E.S., O'Connor, J.D., 1983. Metabolism and Transport of Carbohydrates and Lipids, in: Bliss, Dorothy H. and Mantel, Linda H. (Ed.), The Biology of Crustacea Vol. 5: Physiological regulation. Academic Press, Inc., pp. 263-287.

Clifford, H.C.I., Brick, R.W., 1983. Nutritional physiology of the freshwater shrimp Macrobrachium rosenbergii (de Man). 1. Substrate metabolism in fasting juvenile shrimp. Comp. Biochem. Physiol. A 74(3), 561-568.

Comoglio, L., Gosdsmit, J., Amin, O., 2008. Starvation effects of physiological parameters and biochemical composition of the hepatopancreas of the southern Lithodes santolla (Molina 1782). Rev. Biol. Mar. Oceanogr. 43(2), 345-353.

Comoglio, L., Smoiko, L., Amin, O., 2005. Effects of starvation on oxygen consumption, ammonia excretion and biochemical composition of the hepatopancreas on

17 adult males of the False Southern king crab Parolomis granulosa (Crustacea, ). Comp. Biochem. Physiol. B 140(3), 411-416.

Conklin, D.E., 1980. Nutrition, in: Cobb, Stanley J. and Phillips, Bruce F. (Ed.), The Biology and Management of Lobsters. Academic Press, Inc., New York, NY, pp. 277-300.

Cuzon, G., Cahu, C, Aldrin, J.F., Messager, J.L., Stephan, G., Mevel, M., 1980. Starvation effect on metabolism of Penaeus japonicus. Proc. World Maricult. Soc. 11, 410- 423.

Dall, W., Smith, D.M., 1987. Changes in protein-bound and free amino acids in the muscle of the tiger prawn Penaeus esculentus during starvation. Mar. Biol. 95, 509-520.

Dall, W., 1981. Lipid absorption and utilization in the Norwegian lobster, (L.). J. Exp. Mar. Biol. Ecol. 50, 33-45.

Dall, W., 1990. Moulting and Growth, in: Russell, F.S. (Ed.), Advances in Marine Biology. London, New York, Academic Press, pp. 213-250.

Dall, W., 1974. Indices of nutritional state in the western rock lobster, (Milne Edwards). I. Blood and tissue constituents and water content. J. Exp. Mar. Biol. Ecol. 16(2), 167-180.

Dall, W., 1970. Osmoregulation in the lobster Homarus americanus. J. Fish. Res. Board Can. 27,1123-1130.

DFO (Department of Fisheries and Oceans), 2010b. Commercial Fisheries: Licences, Atlantic Region. 2011(June/08), 1. http://www.dfo- mpo.gc.ca/stats/commercial/licences-permis/licences-permis-atl-eng.htm

DFO (Department of Fisheries and Oceans), 2010a. Commercial Fisheries: Landings, Seafisheries. 2011(June/08), 1. http://www.dfo- mpo.gc.ca/stats/commercial/sea-maritimes-eng.htm

Dufour, R.D., 2003. Sources and Control of Preanalytical Variation, in: Kaplan, L.A., Pesce, Amadeo J. and Kazmierczak, Steven C. (Eds.), Clinical Chemistry: Theory, Analysis, Correlation, 4th ed. Mosby, St. Louis, Missouri, pp. 64-82.

George, R.Y., 1966. Glycogen content in the wood boring isopod, Limnoria lignorum . Science 153(741), 1262-1264.

18 Gurr, M.I., Harwood, J.L., Frayn, K.N., 2002. Lipid Biochemistry, 5th ed. Blackwell Science Ltd., Maiden, MA, USA.

Haj, A.J.E., Clarke, S.R., Harrison, P., Chang, E.S., 1996. In vivo Muscle Protein Synthesis Rates in the American Lobster Homarus Americanus During the Moult Cycle and in Response to 20-Hydroxyecdysone. J. Exp. Biol. 199, 579-585.

Herrick, F.H., 1909. Natural History of the American lobster. Bull. Bur. Fish. 29,169-192.

Kaplan, L.A., Naito, H.K., Pesce, A.J., 2003. Classification and Descriptions of Proteins, Lipids, and Carbohydrates, in: Anonymous, Clinical Chemistry: Theory, Analysis, Correlation, 4th ed. Mosby, St. Louis, Missouri, pp. 1024-1042.

Lawton, P., Lavalli, K.L., 1995. Postlarval, Juvenile, Adolescent, and Adult Ecology, in: Factor, J.R. (Ed.), Biology of the Lobster Homarus americanus. Academic Press, Inc., New York, NY, pp. 47-88.

Lehninger, A.L., Nelson, D.L., Cox, M.M., 1993a. Amino Acid Oxidation and the Production of Urea, in: Anonymous , Principles of Biochemistry, 2nd ed. Worth Publishers, Inc., New York, NY, pp. 506-541.

Lehninger, A.L., Nelson, D.L., Cox, M.M., 1993b. Amino Acids and Peptides, in: Anonymous , Principles of Biochemistry, 2nd ed. Worth Publishers, Inc., New York, NY, pp. 111-133.

Marks, V., 1978. Laboratory Tests, in: Williams, D.L, Nunn, R.F., and Marks, V. (Eds.), Scientific Foundations of Clinical Biochemistry. William Heinemann Medical Books Ltd., Chicago, IL, pp. 1-12.

Matsui, M., Kakut, M., Misaki, A., 1996. Fine structural features of oyster glycogen: mode of multiple branching. Carbohydr. Polym. 31, 227-235.

Mercaldo-Allen, R., 1991. Changes in the blood chemistry of the American lobster, Homarus americanus, H. Milne Edwards, 1837, over the molt cycle. J. Shellfish Res. 10(1), 147.

Moore, L.E., Smith, D.M., Loneragan, N.R., 2000. Blood refractive index and whole- body lipid content as indicators of nutritional condition for penaeid (Decapoda: ). J. Exp. Mar. Biol. Ecol. 244(1), 131-143.

Mykles, D.L., 1999. Proteolytic Processes Underlying Molt-lnduced Claw Muscle Atrophy in Decapod Crustaceans. Am. Zool. 39, 541-551.

19 Mykles, D.L., 1992. Getting out of a Tight Squeeze: Enzymatic Regulation of Claw Muscle Atrophy in Molting. Am. Zool. 32(3), 485-494.

Mykles, D.L., Skinner, D.M., 1990. Atrophy of Crustacean Somatic Muscle and the Proteinases That Do the Job. A Review. J. Crustacean Biol. 10(4), 577-594.

Mykles, D.L., 1981. Ionic requirements of transepithelial potential difference and net water flux in the perfused midgut of the American lobster, Homarus americanus. Comp. Biochem. Physiol. A 69, 317-320.

Mykles, D.L., 1980. The mechanism of fluid absorption at ecdysis in the American lobster, Homarus americanus. J. Exp. Biol. 84, 89-101.

Neiland, K.A., Scheer, B.T., 1953. The influence of fasting and of sinus gland removal on body composition of Hemigrapsus nudus. (Part V of the hormonal regulation of metabolism in crustaceans). Physiol. Comp. Oecol. 3, 321-326.

New, M.B., 1976. A review of dietary studies with shrimp and prawns. 9, 101-144.

NOAA, 2011. FishWatch - U.S. Facts. 2011(June/08). http://www.nmfs.noaa.gov/fishwatch/species/amer lobster.htm

Noga, E.J., 2000. Hemolymph biomarkers of crustacean health, in: Fingerman, Milton and Nagabhushanam, Rachokonda (Ed.), Recent Advances in Marine Biotechnology: Immunobiology and pathology. Science Publishers, Inc., Enfield, NH, pp. 125-163.

Nybakken, J.W., Bertness, M.D., 2005. Marine Biology: An Ecological Approach, 6th ed. Pearson/Benjamin Cummings, New York, NY.

Parrish, F.A., Martinelli-Liedtke, T.L., 1999. Some Preliminary Findings on the Nutritional Status of the Hawaiian Spiny Lobster (Panulirus marginatus). Pac. Sci. 53(4), 361-366.

Parvathy, K., 1971. Glycogen storage in relation to the moult cycle in the two crustaceans Emerita asiatica and Ligia exotica. Mar. Biol. 10, 82-86.

Passano, L.M., 1960. Molting and its Control, in: Waterman, T.H. (Ed.), The Physiology of Crustacea: Volume I Metabolism and Growth. Academic Press, Inc., New York, NY, pp. 473-536.

20 Phillips, B.F., Cobb, J.S., George, R.W., 1980. General Biology, in: Cobb, Stanley J. and Phillips, Bruce F. (Ed.), The Biology and Management of Lobsters. Academic Press, Inc., New York, NY, pp. 2-82.

Radford, C.A., Marsden, I.D., Davison, W., Taylor, H.H., 2005. Haemolymph glucose concentrations of juvenile rock lobsters, edwardsii, feeding on different carbohydrate diets. Comp. Biochem. Physiol. A 140, 241-249.

Renato de Oliveira Cesar, Jose, Zhao, B., Malecha, S., Ako, H., Yang, J., 2006. Morphological and biochemical changes in the muscle of the marine shrimp Litopenaeus vannamei during the molt cycle. Aquaculture 261, 688-694.

Retzlaff, A., Claytor, R., Petrie, B., Frail, C, Tremblay, J., Pezzack, D., Lavallee, J., 2007. Variation in moult timing and market quality in the American lobster {Homarus americanus). Cat. No. Fsl01-3/2006E, 22-26.

Robertson, J.D., 1960. Osmotic and Ionic Regulation, in: Waterman, T.H. (Ed.), The Physiology of Crustacea: Volume I Metabolism and Growth. Academic Press, Inc., New York, NY, pp. 317-339.

Rosa, R., Nunes, M.L., 2003. Biochemical composition of deep-sea decapod crustaceans with two different benthic life strategies off the Portuguese south coast. Deep- Sea Res. Pt. 1 50,119-130.

Sanchez-Paz, A., Garcia-Carreno, F., Hernandez-Lopez, J., Muhlia-Almazan, A., Yepiz- Plascencia, G., 2007. Effect of short-term starvation on hepatopancreas and plasma energy reserves of the Pacific white shrimp (Litopenaeus vannamei). J. Exp. Mar. Biol. Ecol. 340(2), 184-193.

Sanchez-Paz, A., Garcia-Carreno, F., Muhlia, A., Adriana, Peregrino-Uriarte, A.B., Hernandez-Lopez, J., Yepiz-Plascencia, G., 2006. Usage of energy reserves in crustaceans during starvation: Status and future directions. Insect Biochem. Mol. Biol. 36, 241-249.

Stuck, K.C., Watts, S.A., Wang, S.Y., 1996. Biochemical Responses during starvation and subsequent recovery in postlarval Pacific white shrimp, Penaeus vannamei. Mar. Biol. 125, 33-45.

Taylor, J.R.A., Kier, W.M., 2003. Switching skeletons: Hydrostatic support in molting crabs. Science 301(5630), 209-210.

21 Travis, D.F., 1955a. The moulting cycle of the spiny lobster, Latreille. II. Pre-ecdysial histological and histochemical changes in the hepatopancreas and integumental tissues. Biol. Bull. Vol. 108(No. 1), 88-112.

Urich, K., 1994b. Small Nitrogenous Compounds, in: Anonymous, Comparative Animal Biochemistry. Springer-Verlag, New York, NY, pp. 403-462.

Urich, K., 1994a. Lipids, in: Anonymous , Comparative Animal Biochemistry. Springer- Verlag, New York, NY, pp. 562-623.

Verri, T., Mandal, A., Zilli, L., Bossa, D., Mandal, P.K., Ingrosso, L., Zonno, V., Vilella, S., Ahearn, G.A., Storelli, C, 2001. D-Glucose transport in decapod crustacean hepatopancreas. Comp. Biochem. Physiol. A 130, 585-606.

Vinagre, A.S., Silva, R.S.M., 1992. Effects of starvation on the carbohydrate and lipid metabolism in crabs previously maintained on a high protein or carbohydrate- rich diet. Comp. Biochem. Physiol. A 102(3), 579-583.

Waddy, S.L., Aiken, D.E., De Kleijn, D. P. V., 1995. Control and Growth of Reproduction, in: Factor, J.R. (Ed.), Biology of the Lobster Homarus americanus. Academic Press, Inc., San Diego, CA, pp. 217-266.

Wen, X., Chen, L., Ku, Y., Zhou, K., 2006. Effect of feeding and lack of food on the growth, gross biochemical and fatty acid composition of juvenile crab, Eriocheir sinensis. Aquaculture 252, 598-607.

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Wilder, M.N., Huong, D.T.T., Jasmani, S., Jayasankar, V., Kaneko, T., Aida, K., Hatta, T., Nemoto, S., Wigginton, A., 2009. Hemolymph osmolality, ion concentrations and calcium in the structural organization of the cuticle of the giant freshwater prawn Macrobrachium rosenbergii: Changes with the molt cycle. Aquaculture 292,104-110.

22 2. Practical Optimisation of the Dataset

2.1. Abstract

A total of 88 lobsters were collected from lobster fishing area (LFA) 26A out of

Georgetown, Prince Edward Island for the nutritional status (NS) project. Sampling was carried out in May through September of 2009 and 2010 using standard commercial wire lobster traps. Five distinct moult categories were devised based on pleopod staging and shell hardness: late inter-moult (LIM), early pre-moult (EPM), mid pre- moult (MPM), late pre-moult (LPM) and post-moult (PoM). Later pre-moult lobsters did not trap well, as mirrored in the Atlantic Lobster Moult and Quality (ALMQ) monitoring project, most likely due to alterations in feeding behaviors. The ALMQ data also revealed two moulting cohorts within the population (ALMQ, 2011). Seventeen of the 88 lobsters collected to assess nutritional status were a part of a secondary moult cohort. This brought to light the potentially confounding factor of cohort variability considered during data analysis.

2.2. Introduction

The lobster fishery in Canada and the United States is divided into several fishing zones. In the United States, the fishery is divided into 6 lobster management areas, of which the Maine zone is further divided into seven management zones. The fishery in

Atlantic Canada is segmented into 41 different lobster fishing areas (LFAs) (Cobb and

Castro, 2006). Within each of the fishing zones and LFAs, management practices are set in place with respect to local tradition and the stock characteristics such as the

23 moult cycle. Management practices regulate fishing season, quantity and types of traps, and legal size to avoid overexploitation and allow for proper conservation of the populations (Cobb and Castro, 2006).

Biological variations can exist among lobsters within the different LFAs in Atlantic

Canada. Crustaceans such as the lobster can exhibit shifts in physiological and behavioural characteristics in response to environmental factors, including temperature, salinity, light intensity and food availability (Nery and Santos, 1993;

Chang, 1995; Mikami and Greenwood, 1997; Wahle and Fogarty, 2006). Thus, spatial and temporal variations in environmental conditions can result in behavioural (Such as feeding and moulting) and physiological (Such as biochemical composition) differences among populations. Such changes have previously been noted in the American lobster

(Stewart and Li, 1969; Dove et al., 2005). These differences in environmental conditions can play a significant role in the timing and success of the moult as well as the specific physiological changes that occur throughout the moult (Wahle and Fogarty,

2006).

The morphological and physiological changes occurring during the moult have previously been characterised (Waddy et al., 1995) and formal moult stages have been established on the basis of these data. The pleopods of the lobster have been identified as a fairly accurate means of visually assessing moult stage throughout the moult preparatory period (Aiken, 1980). The development of pleopod staging,

24 discussed in Section 1.2.1, has allowed for a fairly simple and non-lethal means of

monitoring the pre-moult portion of the moult cycle in the lobster.

The changes in physiology that take place during the moult can affect the quality of the

lobster (See Section 1.1.3). The biological variation among populations plays an

integral role in the development of management practices. Some fishing seasons in

the different LFAs attempt to target periods when the highest quality product can be

obtained. The highest quality products are often associated with the inter-moult

period and early in the moult preparatory stages before significant muscle atrophy

occurs and tissue water content is low. Within these categories lobsters are still

considered to be fully meated and of good quality for consumption.

In 2004, the Atlantic Veterinary College Lobster Science Centre (AVCLSC) started

monitoring lobster populations through pleopod staging and hemolymph protein

concentrations as part of the Atlantic Lobster Moult and Quality (ALMQ) project1. The

hemolymph protein data revealed good correlations with moult stage, identifying it as

a potential means of assessing moult in wild lobster populations (ALMQ, 2011).

Hemolymph protein concentrations have previously been shown to increase preceding the moult followed by a rapid decrease post moult (Bursey and Lane, 1971; Travis,

1955b; Spindler-Barth, 1976; Mercaldo-Allen, 1991). This method of assessing moult in

1 The ALMQ project is a 5 year (2007-2012) monitoring project being carried out by the AVCLSC. It monitors moult stage and blood protein within various lobster populations overtime in several LFAs surrounding NB, IMS, and PE.

25 crustaceans can be useful in monitoring annual changes in the moult cycle and in ensuring collection of lobsters in specific moult categories.

This chapter addresses the basis of sample collection and procedures used for obtaining the lobsters collected for the nutritional status (NS) aspect of the ALMQ project. Population monitoring data were examined to assess the temporal aspects of the moult cycle and aid in collection of actively moulting lobsters. These data also allowed for the screening of the lobsters collected in an attempt to eliminate non- target variation in biochemical analyses of the tissue and hemolymph samples.

2.3. Materials and Methods

2.3.1. Population Monitoring for Optimisation of Sample Collection

Data from the ALMQ project were used to monitor the moult cycle in the LFA 26A lobster population. A Brix refractive index refractometer was used to determine the

Brix indices as a representation of hemolymph protein concentrations. The hemolymph protein concentrations were compared to sampling effort and moult stage

(pleopod stage) data. These data were compiled to give a general representation of trends in the moult cycle of lobsters from LFA 26A. All protocols for the ALMQ and NS projects were approved by the Animal Care Committee of the University of Prince

Edward Island.

2.3.2. Collection of Lobster Samples

Lobsters were collected from five moult categories devised based on pleopod staging and shell hardness (Table 2.1). The intent was to collect 10 males and 10 females from

26 each category. Lobsters were collected from LFA 26A (Figure 2.1) in the

Northumberland Strait from May through September of 2009 and 2010 with the scientific fishing license (SG-PEI-10-063) obtained through Fisheries and Oceans Canada

(DFO). Standard wire commercial lobster traps were baited with mackerel and submerged overnight before hauling.

Table 2.1: Summary of moult categories as defined by pleopod staging and shell hardness. Traditional Drach moult stages are depicted along with corresponding pleopod stage shell hardness and the devised nutritional status moult category in which each would fall. Table was adapted from (Aiken, 1973 and 1980).

Drach Moult Pleopod Shell Hardness Nutritional Status Stage Stage Moult Category c4 0 Hard Late inter-moult (LIM) Do' 1.0 Hard Do" 1.5 Hard Early pre-moult (EPM) Do" 2.0 Hard Do'" 2.5 Hard Di' 3.0 Hard - Medium Di" 3.5 Hard - Medium Mid pre-moult (MPM) Di'" 4.0 Hard - Medium

D2' 4.5 Hard - Medium

D2" 5.0 Medium Late pre-moult (LPM)

D3 5.5 Medium A 0 Soft B 0 Soft Ci 0 Soft-Medium Post-moult (PoM) c2 0 Medium c3 0 Medium

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WB^a/' "X2 L **fc» <-^^«sr-, - W^S * I a* jk > Monctof^^ » "•26 A 1 ^ m A. — Amttdi-ct J • 1 J| O *-viiiif*,iv»n*«

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Figure 2.1: Map of lobster fishing areas surrounding Prince Edward Island. LFAs surrounding Prince Edward Island (PE), CAN. LFA 26A extends from Borden to East Point. All sampling was done out of the Georgetown Harbour (Image Credit: DFO 2007)

Lobsters were tagged using numbered zip ties and the tip of one of the first non- modified pleopods was clipped with a pair of surgical scissors (Figure 2.2). Pleopod stage was determined via direct light microscopy (Aiken, 1973). Physical data including carapace length (Figure 2.3), sex, and shell hardness were recorded for each lobster.

Hemolymph was collected (3 mL) with a 22 gauge needle and 3cc syringe from the ventral abdominal sinus (Figure 2.4). Approximately 3-4 drops of hemolymph was placed into each of the Brix and refractive index (Rl) pocket refractometers to measure the Brix index (PAL-1 from Atago, Bellevue, WA) and the Rl (PAL-RI from Atago,

Bellevue, WA). The rest of the hemolymph was used to fill a 1.5 mL boil-proof microfuge tube (Progene) and was then centrifuged (5 min. @ 6,500 rpm). The supernatant (plasma) was placed into a new microfuge tube and the cell pellet and

28 plasma samples were stored in a refrigerated cooler. Both these samples and the lobsters were transported back to the AVCLSC in coolers.

Figure 2.2: Clipping of a pleopod for determination of moult stage. Pleopods are located on the ventral abdomen of the lobster and are used for moult staging (Photo Credit: M. Burton).

Figure 2.4: Hemolymph extraction. Extraction of lobster hemolymph from the ventral abdominal sinus (Photo Credit: M. Burton).

Figure 2.3: Measurement of carapace length. Carapace length of a sample lobster being measured with calipers. The carapace length extends from the notch of eyestalk to the posterior edge of the carapace ( Photo Credit: M. Burton).

29 2.3.3. Laboratory Processing of Lobsters Collected.

Lobsters were stored, live, in a dedicated aquatic holding facility (re-circulating Instant

Ocean, 4.0 ± 0.4°C and 29.73 ± 1.04 parts per thousand) and necropsies were performed within 24 hours of collection. Injuries or abnormalities in the physical appearance of the lobsters were noted. Sex and weight (ValorTM 2000 Ohaus, Pine

Brook, NJ, USA) were recorded and 1 mL of hemolymph was drawn using a 22 gauge needle and 3cc syringe from the abdominal cavity. A vial of ciliate media (Appendix F) and two vials of tryptic soy broth with salt (TSB*) (Appendix F) each received three drops of hemolymph. Ciliate cultures were incubated at 4°C. One TSB* sample was held at 15°C and the other at 28°C. Samples were checked for the presence of ciliates

(weekly for 4 weeks) or bacterial growth (daily for 1 week), respectively. Hemolymph

Rl was determined a second time with a handheld manual refractometer (PUR-1410 from Shilac).

Lobsters were euthanized with potassium chloride (KCI 6mL/kg) (Battison et al., 2000) before the hepatopancreas (HP), pincher claw muscle (PCM) and crusher claw muscles

(CCM) were removed and photographed. The remaining HP, PCM, CCM and a portion of the TM were placed into sterile sample bags, flash frozen on dry ice and stored at

-80°C.

30 2.3.4. Statistical Analyses of sampling data

Kruskall-Wallis and Mann-Whitney nonparametric analyses were used for determination of statistically significant differences in Brix values within and between different months. All statistical analyses were performed with Minitab® statistical software version 15.1, 2006 and significance is reported at p < 0.05.

2.4. Results

2.4.1. Population Monitoring for Optimisation of Sample Collection

The mean hemolymph protein concentrations monitored through the ALMQ project in

2009 and 2010 showed a statistically significant increase from May to June in both years. In July of both years there was a significant drop in the mean hemolymph protein concentrations from those observed in May and June. In August of 2009 the mean protein concentration dropped again then increased in September, while the

2010 samples showed an increase from July through to September. Statistical analysis of the data revealed significant differences between years in every month except

September and significant differences between protein concentrations of all months within each year with the exception of July and September of 2009 (Figure 2.5).

31 16 E3 2009 15 • 2010 S" 14 b 2- 13 c a 12 o £ 11 •

fio C e 1 9 d d 2 8 1 S3 ft 7 I Jul Aug 6 May Jun Sep Month

Figure 2.5: Hemolymph protein concentrations in LFA 26A Georgetown, PE, 2009 and 2010. Average hemolymph protein (Brix) by month in lobsters from 2009 and 2010. Asterisks indicate significant differences in mean protein values between years and different letters indicate significant differences between months within one year. Significance at p < 0.05, Mann- Whitney. Data supplied through the ALMQ project.

Table 2.2 represents the moult categories the lobsters in LFA 26A are expected to be in over the course of one year. Figure 2.6 shows the percentage of lobsters caught each month through the ALMQ project that were in the five moult categories examined.

Two moult cohorts were observed within the LFA 26A population. The data indicated that the primary moult occurred in July with the active preparatory and recovery periods in May through September, respectively, and the secondary moult beginning in

September. Each cohort was denoted by an increase in the percent of pre-moult lobsters caught, which was observed in June and in September (Figure 2.6).

32 Table 2.2: Table of sampling seasons and expected moult categories. General representation of the LFA 26A lobster populations progression through the moult cycle. Open fishing season and Nutritional status sample collections are represented by the appropriate green boxes. "Weather" indicates conditions unsuitable for fishing and ice designates times of the year when ice cover inhibits fishing.

Month Sampling Conditions Nutritional Status Moult Category January ^^^^^^Hei^Hl^^^l IM1 February ^^^^^B^^^^^^l IM March IM April ^^^^H^^^^^H IM May Fishing Season UM2/EPM3 June Fishing Season EPM/MPM4 July DFO Permits MPM/ LPM5/PoM6 August DFO Permits PoM September DFO Permits PoMand IM October HH^^SS^^^H IM November IM December ^^I^^SJS^^^HI IM ^nter-moult, 2late inter-moult, early pre-moult, "mid pre-moult, 5late pre-moult, and 6post-moult.

May Jun Jul Aug Sep Oct Month

Figure 2.6: Percent landings from different moult categories (LFA 26A), Georgetown, PE, 2009. Percent of monthly catch in the devised moult categories of late inter-moult (LIM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM). Data supplied by the ALMQ project. The data shows the primary moult in July with a secondary moult preparation in September.

33 2.4.2. Collection of Lobster Samples

Lobster samples collected in 2009 and 2010 for the NS project resulted in the collection of all the male lobsters needed. The target of ten females in the MPM and LPM categories was not achieved (Table 2.3). Within the study dataset, the majority of

lobsters collected followed the general trend in moult in which progression from LIM to LPM occurred from May through June followed by PoM lobsters being caught in July through September. There were 17 lobsters in the 2009 and 2010 samplings

representing the earlier moult categories (LIM, EPM, MPM, and LPM) that were considered to be members of the secondary moulting cohort. Pre-moult lobsters caught in August and September after or during the collection of most PoM lobsters represent the secondary moult cohort (Figure 2.7). The one PoM lobster caught in May also represents a secondary moult cohort, most likely from previous year.

Table 2.3: Summary of sample size for each moult category. Number of lobsters collected representing the five devised moult categories for the 2009 and 2010 sampling seasons.

2009 2010 Total Moult Category Male Female Male Female Male Female LIM1 10 10 0 0 10 10 EPM2 10 9 0 0 10 9 MPM3 9 4 1 4 10 8 4 LPM 4 1 i 6 0 ! 10 1 PoM5 10 10 0 0 10 10 ^ate inter-moult, 2early pre-moult, mid pre-moult, late pre-moult, and 5post-moult

34 Sep- O O O O GBDO O ••

Aug- ODD CEO O

5 Jul-

May- mmmm mmm — • O

Year 2009 2010 2009 2010 2009 2010 2009 2010 2009 2010 Category UM EPM MPM LPM PoM

Figure 2.7: Graphical representation of the nutritional status samples collected. Summary of the Nutritional Status lobsters collected from each month in the five moult categories from 2009 and 2010. Moult categories include late inter-moult (UM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM). The closed circles represent lobsters from the primary moult cohort and the open circles represent a secondary moult cohort.

2.4.3. Laboratory Processing of Lobsters Collected

Twelve out of 88 of the TSB* samples at 15°C and 11/88 TSB* samples at 28°C showed positive bacterial growth of Gram negative rods. There was no ciliate growth observed in any of the samples. All protein data for the NS samples are reported in chapter 6.

Tissue selection for histology was part of another project and will not be addressed here. Lobsters NS 64 and 69 (females) were removed from the dataset as abnormalities were observed in the biochemistry panels. This is discussed in Chapter

7. Thus, the dataset is represented by 86 lobsters unless otherwise stated.

35 2.5. Discussion

Moult in the lobster is cyclical and dictated largely by season, thus the different moult categories are prominent at different times of the year. To obtain lobsters from each of the five target moult categories, the sampling period was extended beyond that of the normal fishing season in LFA 26A, which is from May 1st to June 30th. Permits were obtained from DFO to extend the fishing period through to September in both 2009 and 2010. Poor weather conditions and ice cover inhibited the extension of the sampling season to the months of November through April, when all boats are out of the water.

Even with the extended sampling season lobsters, especially females, in the MPM and

LPM were not caught. Fishing was suspended for two weeks in July of 2009 for boat maintenance, which may have negatively affected the collection of LPM lobsters as they would be predicted to be most prevalent during this time. The sampling was extended to 2010 in an attempt to complete the sample collection of 10 lobsters from each sex in each moult category for this study. At the end of the 2010 sampling, the female data sets, especially the LPM, were still incomplete. During the LPM category, feeding slows and ultimately stops (Dall, 1990). With no feeding taking place, the lobsters are less likely to venture into the baited traps in search of food. The difference in trapability between males and females suggests that there is some behavioural difference between the sexes which requires further examination.

36 The different stages of the moult cycle are identified by pronounced changes in physiology and behaviour (Waddy et al., 1995). The target moult categories in this project were defined to encompass the different moult stages of lobsters to observe trends during the moult cycle.

Data from the 88 NS lobsters along with the data from the ALMQ population monitoring studies (ALMQ, 2011), were used to assess temporal fluctuations in the moult cycle. All lobsters were collected from LFA 26A to avoid changes in physiology based on spatial variations due to environmental constraints (Dove et al., 2005).

Hemolymph protein and percent catch data collected for the ALMQ project showed good agreement with moult category. The good agreement was noted in the gradual increase in hemolymph protein concentrations from May through June followed by a decrease in July when the moult occurs. These data correspond to the moult stages of the lobsters collected through the NS project. This allowed for a generation of a model of the moult cycle in the LFA 26A lobster population. This model was used for the assessment of NS samples collected and enabled the identification of confounding factors, such as season, within the dataset.

Examination of NS sample collection revealed that the lobsters were most likely from two separate moulting cohorts. The collection of LIM, EPM and MPM lobsters in May and June represent the first cohort followed by the moult in July and subsequent collection of PoM lobsters in July, August and September. All of the study LPM and some of the LIM, EPM and MPM lobsters were collected in August and September,

37 which represent the secondary cohort that will likely remain in the EPM stage over winter and proceed through the moult early in the following spring (Waddy et al.,

1995). The presence of a secondary cohort is supported by the catch data collected through the ALMQ project, which shows a second increase in percent catch of lobsters in the pre-moult categories in September.

The presence of a secondary cohort could indicate differences in moulting behavior within the population. This could also mean that physiological differences exist between lobsters moulting in the different cohorts. However, evidence for such variations is not currently available. Physiological differences between cohorts are poorly understood and insufficient data were collected during this project to assess such changes. As these cohorts progress through the moult, seasonal changes in biochemical composition of the hemolymph and tissue (Rosa and Nunes, 2003a and

2003b) could potentially induce variations between the cohorts. While there data were segregated according to moult stage, caution must be taken when interpreting the biochemical results of groups including both moult cohorts to avoid misinterpretation of the data. Attempts to better understand the physiological variations that may exist between cohorts are necessary.

Procedures for sample collection and processing were designed to produce data that most closely represented the values of lobsters from a natural population. Lobsters were collected directly through trapping with traditional baited commercial wire traps left out overnight, rather than lobsters being purchased directly from a retail supplier.

38 The hemolymph biochemical composition, especially in regards to GLUC and lactate, can change significantly during periods of stress such as handling and emersion

(Telford, 1968a; Paterson et alv 1997; Bergmann et al., 2001; Lorenzon et al., 2007).

These stressors are unavoidable but were minimized by collecting the hemolymph samples immediately following removal of lobsters from the traps. The subsequent rapid centrifugation of the hemolymph was performed to separate the plasma from the cells and avoid clotting and subsequent release of cellular components that could alter the biochemistry results. Lobster fishing area 26A was chosen for collecting samples for this study because of its proximity to the AVCLSC, which meant transport and handling times would be minimal in comparison to other sites.

Examination of the samples collected revealed the general trends in moult cycle that occur annually within the LFA 26A lobster population. Potential confounding factors such as spatial and temporal variations in environmental conditions and stress were addressed and sampling procedures designed to minimize their effects on the data.

These data could be useful for future studies assessing moult in lobsters to help optimise sampling procedures and collect lobsters in specific moult categories to avoid unnecessary stress on the population. The identification of two moult cohorts within the population also brought to light some interesting questions and a potential confounding factor to be accounted for when assessing natural changes in physiology during the moult.

39 2.6. References

Aiken, D.E., 1980. Molting and Growth, in: Cobb, Stanley J. and Phillips, Bruce F. (Ed.), The Biology and Management of Lobsters: Volume I Physiology and Behavior. Academic Press, Inc., New York, NY, pp. 91-164.

Aiken, D.E., 1973. Proecdysis, setal development, and molt prediction in the American lobster (Homarus americanus). J. Fish. Res. Board Can. 30,1337-1344.

ALMQ, 2011. Atlantic Lobster Moult and Quality Project. 2011(April 13). http://www.lobsterscience.ca/moult/navMenu.htm

Battison, A., MacMillan, R., MacKenzie, A., Rose, P., Cawthorn, R., Horney, B., 2000. Use of injectable potassium chloride for euthanasia of American lobsters (Homarus americanus). Comp. Med. 50(5), 545-550.

Bergmann, M., Taylor, A.C., Moore, G.P., 2001. Physiological stress in decapod crustaceans (Munida rugosa and Liocarcinus depurator) discarded in the Clyde Nephrops fishery. J. Exp. Mar. Biol. Ecol. 259, 215-229.

Bursey, C.R., Lane, C.E., 1971. Ionic and protein concentration changes during the molt cycle of Penaeus duorarum. Comp. Biochem. Physiol. A 70, 155-162.

Chang, E.S., 1995. Physiological and biochemical changes during the molt cycle in decapod crustaceans: an overview. J. Exp. Mar. Biol. Ecol. 193, 1-14.

Cobb, S.J., Castro, K.M., 2006. Homarus Species, in: Phillips, B.F. (Ed.), Lobster: Biology, Management, Aquaculture and Fisheries. Blackwell Publishing Ltd., Oxford, UK, pp. 310-339.

Dall, W., 1990. Moulting and Growth, in: Russell, F.S. (Ed.), Advances in Marine Biology. London, New York, Academic Press, pp. 213-250.

Dove, A.D.M., Sokolowski, M.S., Bartlett, S.L., Bowser, P.R., 2005. Spatio-temporal variation in serum chemistry of the lobster, Homarus americanus Milne- Edwards. J. Fish Dis. 28, 663-675.

Lorenzon, S., Giulianini, P.G., Martinis, M., Ferrero, E.A., 2007. Stress effect of different temperatures and air exposure during transport on physiological profiles in the American lobster Homarus americanus. Comp. Biochem. Physiol. A 147(1), 94- 102.

40 Mercaldo-Allen, R., 1991. Changes in the blood chemistry of the American lobster, Homarus americanus, H. Milne Edwards, 1837, over the molt cycle. J. Shellfish Res. 10(1), 147.

Mikami, S., Greenwood, J.G., 1997. Influence of light regimes on phyllosomal growth and timing of moulting in orientalis (Lund) (Decapoda: Scyllaridae). Mar. Freshw. Res. 48, 777-782.

Nery, L.E.M., Santos, E.A., 1993. Carbohydrate metabolism during osmoregulation in Chasmagnathus granulata Dana, 1851. Comp. Biochem. Physiol. B 106(3), 747- 753.

Paterson, B.D., Grauf, S.G., Smith, R.A., 1997. Haemolymph chemistry of tropical rock lobsters () brought onto a mother ship from catching dinghy in Torres Strait. Mar. Freshw. Res. 48, 835-838.

Rosa, R., Nunes, M.L., 2003b. Nutritional quality of red shrimp, Aristeus antennatus (Risso), pink shrimp, Parapenaeus longirostris (Lucas), and Norway lobster, Nephrops norvegicus (Linnaeus). J. Sci. Food Agr. 84, 89-94.

Rosa, R., Nunes, M.L., 2003a. Biochemical composition of deep-sea decapod crustaceans with two different benthic life strategies off the Portuguese south coast. Deep-Sea Res. Pt. 1 50,119-130.

Spindler-Barth, M., 1976. Changes in the chemical composition of the common shore crab, Carcinus maenas, during the molting cycle. J. Comp. Physiol. B 105,197- 205.

Stewart, J.E., Li, M.F., 1969. A study of lobster (Homarus americanus) ecology using serum protein concentration as an index. Can. J. Zool. 47, 21-28.

Telford, M., 1968a. The effects of stress on blood sugar composition of the lobster, Homarus americanus. Can. J. Zool. 46, 819-826.

Travis, D.F., 1955b. The molting cycle of the spiny lobster, Panulirus argus Latreille. III. Physiological changes which occur in the blood and urine during the normal molting cycle. Biol. Bull. 109, 484-503.

Waddy, S.L, Aiken, D.E., De Kleijn, D. P. V., 1995. Control and Growth of Reproduction, in: Factor, J.R. (Ed.), Biology of the Lobster Homarus americanus. Academic Press, Inc., San Diego, CA, pp. 217-266.

41 Wahle, R.A., Fogarty, M.J., 2006. Growth and Development: Understanding and Modelling Growth Variability in Lobsters, in: Phillips, B.F. (Ed.), Lobster: Biology, Management, Aquaculture and Fisheries. Blackwell Publishing Ltd., Oxford, UK, pp. 1-44. 3. Evaluation of Fluctuations in Tissue Water Content of the American Lobster Homarus americanus during the Moult Cycle

3.1. Abstract

The hepatopancreas (HP), pincher claw muscle (PCM), crusher claw muscle (CCM) and

tail muscle (TM) samples collected from lobsters in various moult categories for the

nutritional status (NS) project were homogenised and then freeze dried for three days.

The difference between the wet and dry tissue weights was calculated to determine

the percent water in each tissue sample. There was a significant increase in percent

water content in the post-moult (PoM) category in all tissues, corresponding with the

influx of water at ecdysis for expansion of the new cuticle and an increase in size.

Tissue specific trends in pre-moult water content were observed, showing a decrease

followed by hydration just before the moult in all tissues except the TM.

3.2. Introduction

Growth in crustaceans is complex with long periods of tissue generation following a

short but rapid increase in size obtained at ecdysis. The large increase in size is

achieved through expansion of a newly formed cuticle by rapid uptake of water (DeFur

et al., 1985) in the immediate PoM. This process results in the fluctuations in tissue

water content observed in the crab Pachygrapsus crassipes (Baumberger and Olmsted,

1928), the lobster Homarus vulgaris (Lowndes and Panikkar, 1941), the prawn Penaeus indicus (Read and Caulton, 1980), the crab Munida subrugosa (Romero et al., 2006) and the prawn P. monodon (Suneetha et al., 2009) during the moult cycle. The active

43 regulation of hemolymph osmotic pressure has been suggested to be integral in the uptake of water during the moult as observed in P. crassipes (Baumberger and

Olmsted, 1928) and the freshwater prawn M. rosenbergii (Wilder et al., 2009).

The rapid expansion of the new shell and increase in percent tissue water alters the meat content in the lobster (See Section 1.1.3). Poorly-meated lobsters caught in the late pre-moult and subsequent moult recovery stages present a decrease in quality, which negatively impacts market value. Quality in response to moult has been assessed in the snow crab fishery in Eastern Canada and shows a high correlation between moult category and meat yield (Dufour et al., 1997).

The hemolymph of marine decapods and the external environment are generally isosmotic or in osmotic equilibrium (Robertson, 1960). Osmotic regulation to maintain an isosmotic state entails the control of total particulate and ionic concentrations in the hemolymph to ensure they are equal to that of the external medium (Robertson,

1960). In crustaceans, two mechanisms of ionic regulation have been identified. The first is through the differential excretion of ions in the urine by the antennal glands and the second is through controlled uptake of ions via the gills (Robertson, 1960).

Active regulation of osmotic pressure has not been readily observed in the American lobster Homarus americanus as they are considered to be osmoconformers (Dall,

1970). Osmoconformers maintain their hemolymph isosmotic to the external medium and typically lack the ability to actively regulate osmotic pressure. However, in H. americanus, the specific particulate composition of the hemolymph contrasts with

44 what would be expected under passive equilibrium, suggesting they are capable of actively controlling the concentrations of various osmolytes (Robertson, 1960). This indicates that although they conform to the osmotic pressure of the surrounding medium, they are able to actively regulate which osmolytes make up the hemolymph osmolality.

In H. americanus, the increase in water content at ecdysis has been shown to occur through oral ingestion of water (Mykles, 1980), similar to what is seen in the blue crab,

Calinectes sapidus (Neufeld and Cameron, 1994). The ingested water is then absorbed into the hemolymph space through the gut by means of a localised osmotic gradient

(Mykles, 1980), which relies heavily on the active transport of Na+ (Mykles, 1981).

This chapter reports the changes in tissue water content of the lobsters representing the five devised moult categories; late inter-moult (LIM), early pre-moult (EPM), mid- pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM). The trends in tissue water content are interpreted in relation to physiological and behavioural changes necessary for successful exuviations of the old exoskeleton and expansion of a new one to achieve an increase in size.

3.3. Materials and Methods

3.3.1. Homogenisation of Lobster Tissues

The HP, PCM, CCM and TM samples collected as described in section 2.3.3 were removed from storage at -80°C and minced with a razor blade. The tissue was placed into a pre-weighed 15 mL plastic screw cap vial, weighed (TE-64 Sartorius Goettingen,

45 Germany) and then thawed at room temperature (RT). Each tissue was homogenised

(OMNI International TH homogeniser with a stainless steel EZ Coupling G5-75, 5mm diameter and 75 mm length, generator probe, Kennesaw, GA). Distilled water was added to each sample as needed to ensure complete homogenisation. Vials were capped and frozen at -80°C.

3.3.2. Lyophilisation of Lobster Tissues

The HP, PCM, CCM and TM from ten sample lobsters were chosen, two from each moult category, and homogenised (Section 3.3.1) for the lyophilisation study. The cap was removed from each vial of frozen tissue and the tops were covered with gauze held in place with a rubber band. These samples were placed into glass lyophilisation canisters then onto a lyophiliser (Labconco Corporation Model 75035, Kansas City,

MO). The samples were weighed daily until no change in weight was observed. The gauze and rubber band were removed and the dry samples were then re-capped and stored at -80°C. Based on the results the NS tissue samples were subsequently lyophilised for a minimum of 3 days with the lyophilisation procedures described above.

3.3.3. Calculation of Percent Water in Tissue Samples

Water content was calculated as: ((Wet-Dry)/ Wet) * 100 = %W where "Wet" is the weight of the vial containing the wet tissue, "Dry" is the weight of the vial with dry tissue after lyophilisation and %W is the percent water content of the tissue sample.

46 3.3.4. Statistical Analysis

Kruskall-Wallis and Mann-Whitney nonparametric statistical analyses were carried out on all the data to address differences in water content between moult categories, sex and cohort. Minitab® statistical software version 15.1, 2006 was used for analyses and significance is reported at p < 0.05.

3.4. Results

3.4.1. Lyophilisation Study

Samples placed on the lyophiliser showed a drastic decrease in water content after one day (Figure 3.1). Samples were left on the lyophiliser for 3 days for observation and little change in sample weight was observed after the initial sampling on day one.

1

« 3 (A (A +

3

i ,—_ i 1 1 0 12 3 Days Figure 3.1: Lyophilisation study for the determination of dehydration time. Tissue weights measured over the course of three days on the lyophiliser (n=40). The four tissues and five moult categories are equally represented with two tissues from each of the five moult categories lyophilised.

47 3.4.2. Analysis of Tissue Water Content Data

Water content in the various tissues was evaluated for the lobsters in the different moult categories. The percent water of all the tissues examined shows a significant increase in the post-moult (PoM) category (Figure 3.2). The average percent water in the PoM category of the HP, PCM, CCM, and TM tissues, represented as median, mean

± SD, were 69.13, 69.62 ± 4.58; 82.29, 82.48 ± 1.43; 82.48, 82.59 ± 2.17; and 80.38,

80.50 ± 1.07, respectively. There were no significant changes in %W during the LIM through LPM of the HP and PCM. Significant changes were observed in the LIM and the pre-moult categories in the CCM and TM. In the CCM there was a significant increase in the LPM (79.05, 78.92 ± 0.56) from the EPM and MPM (77.68, 77.99 ± 1.02 and 77.67, 78.23 ± 1.98, respectively). In the TM there was a significant decrease between the LIM (78.33, 78.40 ± 0.86) and the MPM and LPM categories (77.44, 77.66

± 0.79 and 77.29, 77.10 ± 1.13, respectively).

48 90

85 ri ab a a b '" ab 80 ^ b bF^V E^'- DLIM 2^ 75 H 4-» I c 0EPM § 70 u i m nMPM

ra 65 //•/ 5 /s, /// ELPM 60 f;>M: &• /Si ! EPoM 55 rfiw $ 1 50 ZL_i IS* i LA HP PCM CCM TM

Tissue

Figure 3.2: The average tissue water content of the five moult categories examined. Summary of the average percent water in the late inter-moult (LIM, n=20), early pre-moult (EPM, n=19), mid pre-moult (MPM, n=18), late pre-moult (LPM, n=ll) and post-moult (PoM, n=19) moult categories for the four tissues examined: hepatopancreas (HP), pincher claw muscle (PCM), crusher claw muscle (CCM) and tail muscle (TM). The error bars show ± SE. Different letters indicate significant differences between moult stages within tissues (p < 0.05, Mann-Whitney).

3.4.3. Statistical Analysis

Significant differences in tissue water content were observed with Mann-Whitney

analyses between the different moult categories (Figure 3.2). There were no statistically significant differences observed between sex and moult cohorts (data not shown).

3.5. Discussion

The tissue water content of H. omericonus varies during the moult cycle as is observed in many decapods (Baumberger and Olmsted, 1928; Lowndes and Panikkar, 1941; Read

49 and Caulton, 1980; Romero et al., 2006; Suneetha et al., 2009). The increased water content is expected as it allows expansion of the new cuticle to increase in size at ecdysis. This expansion is integral to the growth cycle of decapod crustaceans.

The significant increase in percent water content in the CCM during the LPM suggests that water uptake begins in this tissue prior to ecdysis. The significant increase could be a means of stretching the softened shell to aid in the exuviation of the claw muscle.

However, the increase is very small and could reflect an increase in muscle atrophy and metabolism, which causes a decrease in muscle mass and metabolic reserves. The lost muscle mass and metabolic reserves may then be replaced with water.

A slight but not significant increase in water content was seen in the LPM category of the HP and PCM. The onset of the water influx in the LPM, prior to ecdysis, has been observed previously in H. americonus (Mykles, 1980). It has been suggested that the early intake of water will cause the carapace to crack allowing escape from the old shell (DeFur et al., 1985). A larger sample size could eliminate some of the variation observed and give a better depiction of the trends in water content of the LIM and pre- moult categories. The decrease in water content of the TM as pre-moult progresses suggests a differential water loss in the muscle tissues. A decrease of water content in the tail muscle could potentially aid in exuviation of the tail from the old shell at ecdysis by decreasing its overall size.

The changes in tissue water content can also be attributed to changes in metabolic reserves. As reserves are metabolised they are replaced by water (Baumberger and

50 Olmsted, 1928; Dall and Smith, 1987). An increase in metabolic reserves, such as lipid and glycogen, during the LIM, EPM and MPM categories, has been observed in preparation of the moult in crustaceans (Passano, 1960). These reserves will help meet the metabolic requirements of the moult and would result in a relative decrease in tissue water content, potentially explaining the observations in the HP and TM. As the

HP is generally considered to be the primary site of energy storage in crustaceans

(Heath and Barnes, 1970) fluctuations in water content due to alterations in metabolic reserves are expected. Further examination of this phenomenon is required to verify that the trends observed are significant. The depletion of these reserves following ecdysis would in turn result in a relative increase in water content as the metabolic reserves are used and replaced again by water (Baumberger and Olmsted, 1928; Dall and Smith, 1987).

Inability to detect a statistically significant change in water content of the HP is most likely due to the small sample sizes and high variation in the data. This variation is most likely due to the opportunistic nature of the lobsters and the variable food supply in natural environments. Food availability dictates the amount of reserves available for storage (Johnston et al., 2003; Romero et al., 2006), which would affect water content.

Greater sample sizes could eliminate some of the variation and allow for significant trends in tissue water content to be elucidated in all tissue samples.

Although no significant differences were observed between moult cohorts and sex within the moult categories, the small samples sizes representing the different cohorts

51 and the few lobsters in the LPM category did not allow for reliable statistical analysis to be carried out. Further analyses of the effects of cohort in all moult categories and sex, especially in the LPM, are warranted.

Analysis of water content in lobster tissues revealed trends similar to those observed in other decapod crustaceans with a significant increase in the PoM (Baumberger and

Olmsted, 1928; Lowndes and Panikkar, 1941; Read and Caulton, 1980; Romero et al.,

2006; Suneetha et al., 2009). The significant increase in the PoM water content confirms the role of water in expanding the shell to maximize potential growth in the lobster and alludes to moult induced changes in quality or meat content (Dufour et al.,

1997). The association between water content and quality or meat yield stresses the importance of proper monitoring and management practices. Such practices will allow for the appropriate modifications to fishing protocols and ensure that the highest quality product is marketed.

52 3.6. References

Baumberger, P.J., Olmsted, J.M.D., 1928. Changes in the osmotic pressure and water content of crabs during the molt cycle. Physiol. Zool. Vol. l(No. 4), 531-544.

Dall, W., Smith, D.M., 1987. Changes in protein-bound and free amino acids in the muscle of the tiger prawn Penaeus esculentus during starvation. Mar. Biol. 95, 509-520.

Dall, W., 1970. Osmoregulation in the lobster Homarus americanus. J. Fish. Res. Board Can. 27,1123-1130.

DeFur, P.L., Mangum, C.P., McMahon, B.R., 1985. Cardiovascular and ventilatory changes during ecdysis in the blue crab Rathbun. J. Crustacean Biol. 5(2), 207-215.

Dufour, R., Bernier, D., Brethes, J., 1997. Optimisation of meat yield and mortality during snow crab ( opilio O. Fabricius) fishing operations in Eastern Canada. Canadian Technical Report of Fishery and Aquatic Sciences 2152,1-31.

Heath, J.R., Barnes, H., 1970. Some changes in biochemical composition with season and during the moulting cycle of the common shore crab, Carcinus maenas (L). J. Exp. Mar. Biol. Ecol. 5(3), 199-233.

Johnston, D.J., Calvert, K.A., Crear, B.J., Carter, C.G., 2003. Dietary carbohydrate/lipid ratios and nutritional condition in juvenile southern rock lobster, . Aquaculture 220, 667-682.

Lowndes, A.G., Panikkar, N.K., 1941. A note on the changes in water content of the lobster (Homarus vulgaris M.-EDW.) during moult. J. Mar. Biol. Assoc. U. K. 25(1), 111-112.

Mykles, D.L., 1981. Ionic requirements of transepithelial potential difference and net water flux in the perfused midgut of the American lobster, Homarus americanus. Comp. Biochem. Physiol. A 69, 317-320.

Mykles, D.L., 1980. The mechanism of fluid absorption at ecdysis in the American lobster, Homarus americanus. J. Exp. Biol. 84, 89-101.

Neufeld, D.S., Cameron, J.N., 1994. Mechanism of the net uptake of water in moulting blue crabs (Calinectes sapidus) acclimated to high and low salinities. J. Exp. Biol. 188,11-23.

53 Passano, L.M., 1960. Molting and its Control, in: Waterman, T.H. (Ed.), The Physiology of Crustacea: Volume I Metabolism and Growth. Academic Press, Inc., New York, NY, pp. 473-536.

Read, G.H.L., Caulton, M.S., 1980. Changes in mass and chemical composition during the moult cycle and ovarian development in immature and mature Penaeus indicus Milne Edwards. Comp. Biochem. Physiol. A 66, 431-437.

Robertson, J.D., 1960. Osmotic and Ionic Regulation, in: Waterman, T.H. (Ed.), The Physiology of Crustacea: Volume I Metabolism and Growth. Academic Press, Inc., New York, NY, pp. 317-339.

Romero, CM., Lovrich, G.A., Tapella, F., 2006. Seasonal changes in dry mass and energetic content of Munida subrugosa (Crustacea: Decapoda) in the Beagle Channel. J. Shellfish Res. 25(1), 101-106.

Suneetha, Y., Sreenivasula Reddy, P., Jyothi, N., Srinivasulu Reddy, M., 2009. Studies on the analysis of proximal changes during molting process in the Penaeid prawn, . World J. Zool. 4(4), 286-290.

Wilder, M.N., Huong, D.T.T., Jasmani, S., Jayasankar, V., Kaneko, T., Aida, K., Hatta, T., Nemoto, S., Wigginton, A., 2009. Hemolymph osmolality, ion concentrations and calcium in the structural organization of the cuticle of the giant freshwater prawn Macrobrachium rosenbergii: Changes with the molt cycle. Aquaculture 292, 104-110.

54 4. Fluctuations in Glycogen Stores of the Hepatopancreas and Muscle Tissues of the American Lobster Homarus americanus during the Moult Cycle

4.1. Abstract

Glycogen concentrations in the hepatopancreas (HP), pincher claw muscle (PCM), crusher claw muscle (CCM) and tail muscle (TM) were determined indirectly by enzymatic digestion to glucose (GLUC) with amyloglucosidase (Rhizopus sp.). Methods for the determination of GLUC concentrations were validated and optimised for use on lobster tissues. The colorimetric hexokinase based assay was identified as the preferred method and was used for measurement of GLUC. The GLUC concentrations were then converted to oyster glycogen equivalents using a similarly digested oyster glycogen reference curve.

These data revealed significant variations in stored glycogen at different stages of the moult cycle. The predominant trend observed was a significant decrease in the post- moult category with tissue specific variations observed in the inter-moult and pre- moult categories. A significant increase during the LPM suggests that the role of glycogen in the lobster could be related to chitin synthesis rather than as a primary energy source. Further examination of the effect of year on glycogen reserves is required as inconsistent differences in glycogen concentrations were seen between the

2009 and 2010 samples.

55 4.2. Introduction

Glycogen is the main storage polysaccharide in animal and microbial cells (Mathews et

al., 2000). A molecule of glycogen is composed of long chains of D-glucose units. The

D-glucose units are connected by a-1-4 linkages to form chains about 10-18 units long.

Other chains are connected to these via a-1-6 linkages to create a highly branched

molecule (Matsui et al., 1996). In vertebrates, glycogen is generally found in the liver

for storage and in muscle where it is a readily available energy source (Mathews et al.,

2000). The highly branched glycogen molecules are metabolised into GLUC and allow

for the maintenance of hemolymph GLUC levels, which are integral to the proper

functioning of organs (Verri et al., 2001).

As a potentially important energy reserve, knowledge of glycogen levels is integral in

the assessment of nutritional condition. In crustaceans, glycolysis and the pentose

phosphate metabolic pathways for GLUC metabolism have been identified (Chang and

O'Connor, 1983). The importance of glycogen has been investigated and identified as a

significant energy reserve in many crustacean species including: P. marginatus (Parrish

and Martinelli-Liedtke, 1999), Marsupenaeus japonicas (Yu et al., 2009), Orconectes

limosus (Gade, 1984), Aristeus antennatus, Parapenaeus longirostris, and Nephrops norvegicus (Rosa and Nunes, 2003b).

Glucose can play an essential role in structural physiology on a molecular level. Its

structural role is evident in the cellulose walls of woody plants, bacterial cell walls, and the chitinous exoskeleton of , which are all composed of a GLUC backbone

56 (Mathews et al., 2000). It has also been suggested that in crustaceans glycogen may supply the main GLUC pool for chitin synthesis (Travis, 1955).

Chitin is a linear homopolysaccharide that consists of long chains of modified GLUC units in which the hydroxyl group on carbon two (C-2) is replaced by an acetylated amino group. Several of these modified GLUC molecules are then connected via 0-1-4 linkages to create long chains (Figure 4.1) (Lehninger et al., 1993). Chitin is considered to be the second most abundant polysaccharide found in nature (Urich, 1994). Its widespread abundance is due in part to its presence in the hard exoskeleton of arthropods. The lobster shell is composed of long chains of protein-wrapped chitin molecules (Candy and Kilby, 1962). Evidence of the role of glycogen as a precursor to chitin has been observed in several crustacean species including:

(Salaenoi et al., 2006), Metapenaeus sp. (Dall, 1964), Orconectes obscures (Gwinn and

Stevenson, 1973) and Emerita asiatica (Parvathy, 1971).

CH, CH3 C- O C—O H NH CIL.OH H NH r ° "v£T \H »A\ r^/V^« 4< 1 H --o - sijy*OH nA -\HH > — ' A' o-'\c-/\OH iiny\ H^—o\H - n NH CH,OH H NH CH2OH I K " ! c-o c -o CH:j CH,

Figure 4.1: Diagram of a chitin molecule. Chitin molecule with 0-1-4 linked acetylated (GLUC) molecules (Lehninger et al., 1993).

57 In other lobster species, such as P. argus, glycogen is important for the deposition of calcium phosphate (calcospherites) in the HP during the pre-moult stages, as it provides phosphoric esters, a substrate of the phosphatase enzymes found in the HP

(Travis, 1955). The phosphatase releases free phosphate ions, which bind calcium to form the calcospherites (Travis, 1955). The calcium and phosphate for these deposits in the hepatopancreas originate in the exoskeleton. The mineral deposits are then remobilised after ecdysis to mineralize the new shell (Travis, 1955). The demineralisation of the shell in crustaceans during the moult preparatory stages allows for an easier escape at ecdysis and the conservation of minerals for new shell formation (Travis, 1960). In H. americanus, the demineralisation of the shell is associated with storage of minerals in the subcuticular layer of the stomach known as gastroliths (Waddy et al., 1995).

Research on the spiny lobster Panulirus argus (Travis, 1955) and the crab P. crassipes

(Baumberger and Olmsted, 1928) suggested the indirect role of glycogen stores in osmotic pressure changes that take place during the moult. More recently, Waddy et al. (1995) stated that changes in osmotic pressure allow for the uptake of water and the expansion of the new shell to increase in size. These changes are dictated by particulate concentrations in the hemolymph.

Tissue glycogen can be determined by chemical reduction methods or enzymatically.

Enzymatic methods have been selected because they are considered to be more specific than the reduction methods (Huijing, 1970; Carr and Neff, 1984). Indirect

58 enzymatic methods are carried out through the digestion of glycogen into its GLUC

subunits with an enzyme, such as amyloglucosidase (AMG). The AMG enzyme

hydrolyzes a-D-(l-4) and ct-D-(l-6) branched linkages between GLUC residues in

glycogen starting from the non-reducing end of a linear chain (Pazur and Ando, 1959).

The resulting GLUC concentrations are then measured. Results can be converted to

glycogen equivalents using a standard curve generated from similarly digested purified

glycogen (Burton et al., 1997).

Glucose concentrations are often determined enzymatically. Two common enzymatic

methods for GLUC determination are glucose oxidase (GOx) and hexokinase (HK). The

GOx enzymatic pathway works through the oxidation of GLUC, which generates

peroxide molecules (Caraway and Watts, 1986). The peroxide molecules then react

with a chromogenic oxygen acceptor that generates a colour change when hydrolyzed

as depicted below:

Glucose0> dase Glucose + 02 < " > D-Glucono-6-lactone + H202

Peroxidase o-Dianisidine (Colourless) + H202 < > Oxidized o-Dianisidine (Coloured) + H20

The colour change in the solution can then be read at a wavelength of 570 nm

(Caraway and Watts, 1986). This method has been validated for use in the mussel,

Mytilus edulis, another marine invertebrate (Burton et al., 1997).

59 The hexokinase enzyme phosphorylates GLUC molecules into glucose-6-phosphate (G-

6-P). A dehydrogenase enzyme then dehydrogenates the G-6-P and results in the production of hydrogen ions, as depicted below:

Glucose + ATP Glucose-6-phosphate + ADP

GIUC0Se-6-ph0Sphate + NAD(P) 6.phosphogluCOnate + NAD(P)H + H+

The absorbance of NADH generated in this reaction can be measured at a wavelength of 340 nm on the ultra violet (UV) index to determine GLUC concentrations (Caraway and Watts, 1986).

Validation of diagnostic procedures allows for reliable and diagnostically relevant data to be obtained (Murray et al., 1993). Validation requires the assessment of assay stability and linearity, precision and the standardisation of recovered values.

Glycogen is considered important for maintaining normal physiological function and growth. An adequate means of assessing glycogen reserves is necessary to obtain a full understanding of its role in crustacean nutrition. Potential implications of reserve distributions are discussed to reveal their physiological role in the lobster and potential confounding factors (such as season and year) that must be considered when examining these reserves.

In this chapter, protocols for the digestion of glycogen were optimised for the four lobster tissues and oyster glycogen standards. The HK and GOx assays for the determination of glucose, following glycogen digestion, were examined. The HK method was selected for use on lobster tissues. Subsequently, this method was

60 validated through examination of precision, linearity and stability of the samples and standards. The optimised AMG-HK protocol was then used to identify natural fluctuations in glycogen distribution in the four H. americanus tissues (HP, PCM, CCM and TM) at different stages of the moult cycle.

4.3. Materials and Methods

4.3.1. Optimisation of Tissue and Oyster Glycogen Standard Digestion

Lyophilised tissue samples were prepared as per section 3.3. A 2 mg/mL oyster glycogen (OG) stock standard solution in 100 mM sodium citrate buffer pH 5 (CB)

(Appendix F) was used unless otherwise stated. Approximately 10 to 20 mg of lyophilised tissue or 1 mL of OG stock was aliquoted into a 2 mL microfuge tube

(Axygen, Union City, CA, USA). Tissue samples were reconstituted in 1 mL of CB. Each sample was then vortexed vigorously until homogenous. A heat inactivation (HI) step

(10 min, 95°C) (VWR digital heat block, Radnor, PA) was followed by digestion in a shaking incubator (MaxQ 4000 E Class, Barnstead| Lab-Line, Melrose Park, IL) with

100 u.1 of 0.5% amyloglucosidase (AMG) (2h, 50°C, @150 rpm). Samples were centrifuged (30 min, 10,000xg) (Microlite RF Thermo, IEC, Needham Heights, MA) to separate the cellular debris and lipid from the rest of the solution. The supernatants were carefully removed and transferred into a pre-labeled 1.5 mL microfuge tube. The supernatants were used for GLUC determinations.

The completeness of the digestion was investigated through evaluation of a HP tissue digested in triplicate for 2.5 and 4 hour incubation periods and comparing the

61 standardised glycogen values obtained for both. Five HP tissue samples, ranging from

5.9 to 42 mg, were also digested as described above and values were standardised to

u.mol/mg of tissue.

4.3.2. Evaluation of the Glucose Oxidase Method for Glucose Determination in Lobster Tissues

The GOx assays were carried out in flat bottom 96 well plates. Samples on each plate were as depicted in Table 4.1. A total of 18 tissues in triplicate could be assayed on a single plate. Glucose concentrations in the supernatant were determined in triplicate for each sample according to the protocol for the colourimetric assay with the

BioVision glucose assay kit (Catalog #K606-100). A 5 u.1 aliquot of each digested sample was added to the appropriate wells containing 45 u.1 of buffer and 50 u.1 of reaction mix.

A standard GLUC curve was also run in duplicate in each plate. The standard curves run over time, were compared to assess the inter-assay precision and stability of the standards. A detailed description of the protocol can be found in Appendix D.

Table 4.1: Summary of solutions used in the glycogen assays. Summary of solutions prepared for one glucose oxidase assay. Each column represents a sample type and rows indicate amounts of various components added to each during the digestion procedures (Section 4.3.).

Digested Undigested Digested Undigested Enzyme Buffer Tissue Tissue OG OG Blank Blank CB1 lmL lmL lmL lmL lmL lmL AMG2 100 u.1 - 100 u.1 - 100 u.1 Tissue 10-20 mg 10-20 mg - - - - OG3 - - lmg lmg - - Replicates 3 1 3 3 3 3__ titrate buffer, 2amyloglucosidase and 3oyster glycogen

62 4.3.3. Evaluation of the Hexokinase Method for Glucose Determination in Lobster Tissues

The Cobas c501 biochemistry analyzer (Roche Diagnostics Corporation, Indianapolis,

IN) was used to perform a gluc3 enzyme based (HK) assay for determination of GLUC.

Digested tissue and oyster glycogen (OG) samples, as prepared in Section 4.3.1, and blanks (Table 4.1) were generated. A set of OG standards and blanks were run at three points during the assay, beginning, middle and end, with digested tissue samples assayed between. Each set included three AMG digested standard curves (2, 1, 0.5,

0.25, 0.125, 0.063, and 0.031 mg/mL), one non-digested curve, one enzyme blank and one buffer blank (Table 4.1).

Approximately 500 u.1 of supernatant, prepared as per Section 4.3.1, was pipetted into pre-labeled plastic vials provided by Diagnostic Services2 and sent for GLUC determination. Glucose recovery of a standard stock solution was examined over time to assess inter-assay variability and stability from data provided by the Diagnostic

Services quality control protocols.

4.3.4. Calculation of Oyster Glycogen Equivalents

Glucose concentrations were converted to OG equivalents using the regression equation generated with the mean values of the nine standard OG reference curves performed with each assay.

2 Diagnostic Services Laboratory at the Atlantic Veterinary College of the University of Prince Edward Island, Charlottetown, PE, Canada

63 4.3.5. Validation of Glucose Determination Using the Hexokinase Assay

4.3.5.1. Precision of Hexokinase Method for Glucose Determination in Oyster Glycogen and Tissue Samples

Two 20 mL OG stock solutions, one high (2 mg/mL) and one low (0.06 mg/mL) were prepared in CB. The solutions were heat inactivated (10 min, 95°C) before digestion with 4 mL of 0.5% AMG (2.5h, 50°C, @150 rpm). Twenty ~1 mL aliquots of each supernatant were submitted to Diagnostic Services for determination of GLUC with the

HK method (Section 4.3.3). These procedures were repeated on HP (17.02 mg) and a pooled pincher and crusher claw muscle (PCM and CCM respectively) (16.3 mg) tissue sample.

4.3.5.2. Evaluation of Tissue Stability Following Storage at -80°C

The HP of six Animal Care Committee (ACC) approved non-study lobsters were collected, pooled and separated into seven aliquots. Six of these were frozen immediately on dry ice and one, which was homogenised prior to freezing, was prepared for lyophilisation at week 0. Five of the aliquots were stored at -80°C and the sixth was allowed to thaw at room temperature (RT) before homogenisation and preparation for lyophilisation at week 0. Two aliquots of ~20 mg from each of the lyophilised 0 week samples were digested with AMG (See Section 4.3.1). One ~20 mg aliquot from each sample received the same treatments without the addition AMG.

Glucose concentrations of all the samples were measured with the HK assay (See

Section 4.3.3).

64 The tissue homogenisation, lyophilisation, digestion and GLUC determination steps were repeated on each of the frozen tissue samples in replicates of six at 4, 8, 16, 32, and 52 weeks. Mean GLUC concentrations were calculated and the medians compared for statistical significance (Mann-Whitney, Minitab® statistical software).

In addition, homogenised and lyophilised tail muscle (TM) and HP were digested and

GLUC concentrations measured before and after 9 months of storage at -80°C.

4.3.5.3. Linearity of Oyster Glycogen Derived Glucose Curve

A 2 mg/mL OG stock solution was prepared fresh in CB and serially diluted to generate concentrations of 2,1, 0.5, 0.25, 0.125, 0.063 and 0.031 mg/mL, in triplicate. The samples were digested, the GLUC concentrations were determined (See Section 4.3.3) and a standard curve was generated. Standard curves were prepared for each of the four days assays performed on the different tissue samples.

4.3.6. Assessment of Background Glucose in Lobster Tissues

Five study lobsters, representing each moult stage and each tissue type were chosen, based on tissue availability, for evaluation of glycogen levels resulting from autodigestion. All samples were subject to two different treatments when processing: heat inactivation (HI) and room temperature (RT). All the tissues were removed from storage at -80°C, minced and placed into a 15 mL plastic screw cap vial. The HI treatment tissues were thawed immediately by heat inactivation (10 min, 95°C) and the RT treatment tissue samples were allowed to thaw at RT (~60 min). Thawed samples were then homogenised (See Section 3.3.1) and lyophilised (See Section

65 3.3.2). Approximately 20 mg of lyophilised HI tissue was aliquoted into 2 mL microfuge tubes in triplicate. Similar aliquots of the RT samples were weighed out in replicates of four. All the tissue samples were then reconstituted in 1 mL of CB and vortexed vigorously to obtain a homogenous suspension. Three of the RT treated sample aliquots from each tissue were digested (See Section 4.3.1, excluding the HI step) and the fourth received the same treatment without the addition of AMG. Glucose concentrations were determined with the HK method (See Section 4.3.3).

4.3.7. Relative Distribution of Tissue Glycogen at Different Stages of the Moult Cycle

Tissue glycogen concentrations in the HP, PCM, CCM, and TMs representing the five moult categories (Table 4.2) were determined in triplicate with the HK assay (See

Section 4.3.3, excluding HI), which was determined to be the better method of glycogen determination in the lobster tissues (See discussion).

Table 4.2: Summary of glycogen assay sample sizes. The sample sizes for each of the four tissue types in the five different moult categories collected.

n Hepatopancreas (HP) Pincher (PCM) Crusher (CCM) Tail (TM) LIM1 20 20 20 18 EPM2 19 19 18 19 MPM3 18 17 17 18 LPM4 11 11 11 11 PoM5 20 18 16 19 :late inter-moult, 2early pre-moult, mid pre-moult, late pre-moult and 5post moult.

Glycogen concentrations were then standardised to make them comparable between lobsters. As the original tissue mass was not available for the muscle samples, an index was devised to take into account the internal changes in physiology (water content)

66 and variations in lobster size (carapace length). The equation for the standardisation of data that takes these factors into consideration is expressed as:

Glycogen Index (Gin) = ((%W *G)/CL) * lxlO4

Where %W represents percent water content of the tissue, G is mg of glycogen per mg of wet tissue, and CL is the carapace length of the lobster and the units are mg glycogen (mg tissue)_1 mm"1.

4.3.8. Statistical Analysis of Glycogen Data

Nonparametric analysis was performed on all of the glycogen data as sample sizes (n) were less than 20 in all groups (Table 4.2) and normal distributions were not observed.

Kruskal-Wallis analyses were initially performed to identify changes within groups.

Specific differences between the individual categories were determined with Mann-

Whitney analysis. All statistical analyses were performed with Minitab® statistical software version 15.1, 2006 and significance is reported at p < 0.05.

4.4. Results

4.4.1. Optimisation of Tissue and Standard Glycogen Digestion

Digestion incubation times of 2.5 and 4 hours showed no significant changes in GLUC concentrations with coefficients of variation being below 5% for both incubation periods (Table 4.3). The various amounts of tissue digested showed little variation with a CV of 2.96% (Table 4.4).

67 Table 4.3: The effect of incubation time on glycogen digestion. Mean glucose (GLUC) concentrations (HK) of digested hepatopancreas from one study lobster after 2.5 and 4 hour digestion incubations. The GLUC values are expressed as mean ± standard deviation and the coefficient of variation (CV) is displayed.

Median Glucose Time (h) (nmol/mg) (umol/mg) %cv n 4h 0.29a 0.29 ±0.01 3.8 3 2.5h 0.29a 0.28 ±0.00 1.2 3

Table 4.4: The effect of tissue amount on digestion of glycogen. Different amounts of hepatopancreas tissue from one lobster were digested with 100 u.1 of 0.5% amyloglucosidase. Glucose values were standardised to u.mol/mg for comparison and mean, standard deviation (SD) and coefficient of variation (CV) were calculated.

Mean Glucose Tissue (mg) (umol/mg) 42.0 0.28 29.9 0.28 21.8 0.29 9.9 0.29 5.9 0.30 Mean 0.29 SD 0.01 % CV 2.96

4.4.2. Evaluation of the Glucose Oxidase Method for Glucose Determination in Lobster Tissues

The GOx method showed a gradual decrease over time in optical density (OD) observed for the GLUC standard curve. The first standard curve generated was about

0.2 to 0.6 fold higher than the last, which was run one month later (Figure 4.2). The slopes of the curves also differed as observed by the regression lines crossing in many of the samples (Figure 4.2). Coefficient of variation for each GLUC concentration was calculated as 17.79,18.08, 14.81, 12.87 and 12.85% for the 2, 4, 6, 8 and 10 nmol/well solutions, respectively.

68 2

1.8 • Feb. 22, 2010 Feb. 24, 2010 1.6 A Feb. XFeb. *> 1.4 XMar '•& fD M rS 1.2 C •2 1

Q O 0.8

0.6

0.4

0.2 4 6 10

Glucose (nmol/well)

Figure 4.2: Glucose standard curves generated with the glucose oxidase method over time. The optical density (OD) of standard glucose curves with the glucose oxidase method at a wavelength of 570 nm. The curves were generated on six different days over a one month period.

4.4.3. Evaluation of the Hexokinase Method for Glucose Determination in Lobster Tissues

The mean absorbance values obtained for a 10.8 mmol/L GLUC standard run as part of the Diagnostic Services quality control protocol over a three month period was 7183.3

± 43.8, with a CV of 0.70% (Figure 4.3).

69 7500 •v 1 7400 1 1 !i 7300 4 • • • 1 7200 • * • • • •• i 2 7100 • • < i • 7000

6900 [ i 6800 t

**/L °^0 ***** °^011 °**'°H Date

Figure 4.3: Glucose standards run with the hexokinase method over time. The absorbance values of a 10.8 mmol/L standard glucose solution over a three month period using the hexokinase method for glucose determination.

4.4.4. Validation of Glucose Determination Using the Hexokinase Assay

4.4.4.1. Precision of Hexokinase Assay for Glucose Recovery in Lobster Tissues and Oyster Glycogen

The HK assay showed good precision with %CVs of 12.82, 0.95, 2.04 and 0.00 for the

low OG, high OG, HP and muscle samples, respectively, with greater variation observed in the standard with the low GLUC concentration (Table 4.5).

70 Table 4.5: Precision of hexokinase assay on tissue samples and oyster glycogen standards. Glucose concentrations in high (2 mg/ml) and low (0.06 mg/ml) oyster glycogen solutions, hepatopancreas, and muscle tissue for twenty replicates following digestion with 0.5% amyloglucosidase. The median, mean, standard deviation (SD), and coefficient of variation (CV) for glucose are shown.

Low OG Glucose High OG Glucose Hepatopancreas Muscle (mmol/L) (mmol/L) (mmol/L) (mmol/L) Median 0.3 8.7 1.5 0.5 Mean 0.3 9.0 1.5 0.5 SD 0.04 0.09 0.03 0.00 %CV 12.82 0.95 2.04 0.00

4.4.4.2. Evaluation of Tissue Stability Following Storage at -80°C

The flash frozen and immediately homogenised 0 week HP samples showed no difference in GLUC concentration between each other and were pooled for analysis.

After 16 and then 32 weeks in storage, the GLUC concentrations showed significant changes from the original concentrations but in both cases returned to the original

levels at the next sampling (Table 4.6). The long term stability study revealed that the wet tissue samples were stable up to 52 weeks when held at -80°C (Table 4.6). The

GLUC concentrations in the TM and HP samples, stored as lyophilised tissue over a 9 month period showed no significant changes (Table 4.7).

71 Table 4.6: Stability of tissue glycogen following long term storage. Long term stability study pooled hepatopancreas samples stored at -80°C for 0, 4, 8, 16, 32, and 52 weeks. Glucose values were converted to u.mol/mg dry tissue. Glucose values are expressed as median, mean ± standard deviation of n sampless. Different letters represent significance at p < 0.05.

Median Glucose Mean Glucose Week (nmol/mg) (umol/mg) %CV n ab 0 0.096 0.094 ±0.017 18.12 4 ab 4 0.098 0.09610.009 9.05 6 a 8 0.088 0.088 ± 0.003 2.86 6 b 16 0.097 0.09610.001 1.20 6 c 32 0.119 0.11810.006 5.13 6 b 52 0.098 0.09810.003 2.60 6

Table 4.7: Stability of lyophilised tissue samples following long term storage. Glucose concentrations in hepatopancreas and tail muscle run nine months apart. Tissues were stored as homogenised and lyophilised samples between assays. Glucose values are expressed as median, mean 1 standard deviation. Different letters indicate significant differences between samplings at p < 0.05.

Median Glucose Mean Glucose Lobster Tissue Date (umol/mg) (p-mol/mg) %CV n a NSa91 TM2 Jun-10 0.043 0.043 1 0.002 4.554 3 a NS91 TM Feb-11 0.050 0.05010.001 1.427 3 b NS30 TM Jun-10 0.018 0.019 1 0.001 5.836 3 b NS30 TM Feb-11 0.023 0.02410.002 7.805 3 c NS100 HP3 Apr-10 0.288 0.30010.01 3.65 5 c NS100 HP Dec-10 0.300 0.29010.01 2.96 3 Nutritional status, 2tail muscle and hepatopancreas

4.4.4.3. Linearity of Oyster Glycogen Derived Glucose Curves

The standard curves generated with the HK method showed excellent linearity. The regression line generated gives an R2 value of 0.9958 (Figure 4.4).

72 12.0 i

0 0.5 1 1.5 2 Oyster Glycogen (mg/mL)

Figure 4.4: Linearity of oyster glycogen standard curves. Glucose concentrations of glycogen standard curves generated from a 2 mg/mL oyster glycogen stock solution digested with 0.5% amyloglucosidase. Each concentration has 12 data points plotted representing the standards run in triplicate with each of the four assays. Linear regression fit shown. The hexokinase method was used for glucose determination.

4.4.5. Assessment of Background Glucose in Lobster Tissues

Background GLUC concentrations were higher in the RT auto-digested tissue samples than in the HI auto-digested ones (Table 4.8). The RT AMG digested tissue samples had higher GLUC concentrations than both of the autodigested tissues (Table 4.8). Similar changes in GLUC concentrations when subject to HI or RT thawing during processing were observed in all HP, PCM, CCM, and TMs.

73 Table 4.8: Evaluation of baseline glucose concentrations in tissue samples. Standardised glucose (umol/mg dry weight) concentrations with the hexokinase method in tissues digested with AMG, autodigestion with heat inactivation (HI) and thawed at room temperature (RT) (autodigestion) prior to homogenisation without HI. Four tissue samples are represented and each GLUC value for the HI autodigestion and AMG digested samples is a mean of three. The RT autodigestion samples represent only one value. Number of tissues and moult category used were dictated by tissue availability.

Lobster Moult HI1 RT2 AMG3 ID Tissue Stage Autodigestion Autodigestion Digested NS13 PCM4 LIM8 0.01 0.06 0.06 NS23 PCM EPM9 0.01 0.06 0.06 NS55 PCM MPM10 0.01 0.09 0.10 NS98 PCM LPM11 0.00 0.03 0.03 NS83 PCM PoM12 0.00 0.00 0.00 NS13 CCM5 LIM 0.03 0.08 0.12 NS57 CCM EPM 0.02 0.12 0.38 NS38 CCM MPM 0.01 0.13 0.19 NS98 CCM LPM 0.01 0.05 0.05 NS83 CCM PoM 0.01 0.01 0.01 NS57 TM6 EPM 0.01 0.03 0.03 NS64 TM EPM 0.01 0.03 0.03 NS98 TM LPM 0.01 0.03 0.03 NS87 TM PoM 0.00 0.00 0.00 NS94 HP7 LIM 0.01 0.00 0.00 NS27 HP EPM 0.01 0.03 0.03 NS75 HP MPM 0.02 0.11 0.11 NS100 HP LPM 0.04 0.24 0.30 NS69 HP PoM 0.01 0.01 0.01 1heat inactivation, 2room temperature, 3amyloglucosidase, "pincher claw muscle, 5crusher claw muscle, tail muscle, hepatopancreas, late inter-moult, early pre-moult, mid pre-moult, late pre-moult and post-moult.

4.4.6. Relative Distribution of Tissue Glycogen at Different Stages of the Moult Cycle

Glycogen concentrations of all tissue samples were determined by digestion with 0.5%

AMG followed by GLUC determination with the HK method, excluding the HI step. The

GLUC values obtained were converted to OG equivalents with the standard curves

74 generated on the same day (Table 4.9). Glucose values were standardised for both water content and lobster size using the standardisation equation (Gin) before statistical analyses were performed using the 2009 primary cohort data. Comparison of trends in the total HP glycogen concentrations and the standardised index values were carried out to ensure that trends in total glycogen were conserved during conversion (Figures 4.5 and 4.6, respectively).

The HP, PCM, and CCM (Figures 4.6, 4.7, and 4.8, respectively) all showed very similar trends in the Gin values with no significant changes in the LIM, EPM, and MPM categories followed by a significant decrease in the PoM category. The CCM had the highest glycogen concentrations with the average glycogen index being above 0.4 in the LIM, EPM and MPM categories while the average index values for all other tissues in those categories were below 0.4. The TM (Figure 4.9) had a small yet significant increase in the glycogen index from the LIM and EPM to the MPM category followed by a significant decrease, similar to what was observed in all other tissues.

Table 4.9: Regression equations for oyster glycogen curves used for the calculation of tissue glycogen. Linear regression equations and regression statistics (R2) for the standard oyster glycogen (OG) curves generated with each tissue. Lines were generated with the average of three reference curves run throughout the day with each point run in triplicate.

Linear Regression Tissue Date Equation for OG R2 Hepatopancreas 13-Dec-10 y = 5.2147X - 0.006 0.9999 Pincher 31-Jan-ll y = 4.7477X - 0.0078 0.9998 Crusher 07-Feb-ll y = 5.4094x - 0.0078 0.9997 Tail 28-Feb-ll y = 5.4094x- 0.0465 0.9997

75 160 a a n=18 n=ll g 140- | I 120- 8 .&• 100- a b (9 n=19 n=19 in 80- (B ' * £ u c 60- * ro a. o is 8" 40- i o 20--

i LIM EPM MPM LPM PoM Moult Category

Figure 4.5: Total hepatopancreas glycogen in lobsters from the 2009 primary moult cohort. Average total hepatopancreas (HP) glycogen (mg/HP) content of lobsters in different moult categories: late inter-moult (LIM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM). Statistically significant differences were observed in groups with different letters (p < 0.5, Mann-Whitney). Asterisks represent outliers within groups and were included in statistical analysis.

1.8

1.6

1.4 *—> c 3 12 I I.O c c 0.8 w a o> a n=ll n= b 8 0.6 n=19 =18 .£> n=19 u> *

0.4

0.2 1

0.0 QM EPM MPM LPM PoM Moult Category

Figure 4.6: Standardised hepatopancreas glycogen in lobsters from the 2009 primary moult cohort. Average glycogen index values of the hepatopancreas of lobsters in different moult categories: late inter-moult (LIM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM). Statistically significant differences were observed in groups with different letters (p < 0.5, Mann-Whitney). Asterisk represents an outlier within groups and was included in statistical analysis.

76 1.8- * 1.6-

1.4-

0 "- •5 1.0 H

* a a n==1 0 a n=18 P p b > o Glycoge n n=lS * 0.4- i I b 0.2- n=17 1 1 1 1 1 1 0.0- LIM EPM MPM LPM PoM Moult Category

Figure 4.7: Standardised pincher claw muscle glycogen in lobsters from the 2009 primary moult cohort. Average glycogen index values of the pincher claw muscle of lobsters in different moult categories: late inter-moult (LIM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM). Statistically significant differences were observed in groups with different letters (p < 0.5, Mann-Whitney). Asterisks represent outliers within groups and were included in statistical analysis.

l.O- a 1.6- n= 17 * a 1.4- n=19 a 8 i-2- n=10 •a 5c 10- -0.8V -

W 0.6- b 0.4- n=15 0.2- 1 * 1 ' 1 n n. LIM EPM MPM LPM PoM Moult Category

Figure 4.8: Standardised crusher claw muscle glycogen in lobsters from the 2009 primary moult cohort. Average glycogen index values of the crusher claw muscle of lobsters in different moult categories: late inter-moult (LIM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM). Statistically significant differences were observed in groups with different letters (p < 0.5, Mann-Whitney). Asterisks represent outliers within groups and were included in statistical analysis.

77 J..O-

1.6-

1.4-

•o £ 1.0- c -0.8- a n=18 » 0.6- a n=17 * b 0.4- n=10 c 1 n-18 0.2- * •* 1 ^ 1 ' 1 i UM EPM MPM LPM PoM Moult Category

Figure 4.9: Standardised tail muscle glycogen in lobsters from the 2009 primary moult cohort. Average glycogen index values of the tail muscle of lobsters in different moult categories: late inter-moult (UM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM). Statistically significant differences were observed in groups with different letters (p < 0.5, Mann-Whitney). Asterisks represent outliers within groups and were included in statistical analysis.

Figures 4.10 to 4.15 represent the changes in Gin values when the 2010 data and both of the 2009 moult cohorts are included. The significant decrease in the PoM category was consistently observed in the glycogen indices (Reported as median, mean ± standard deviation) of the HP (0.08, 0.12 ± 0.11), PCM (0.03, 0.06 ± 0.06), CCM (0.05,

0.10 ± 0.12), and TM (0.03, 0.06 ± 0.05) (Figures 4.10, 4.11, 4.12 and 4.13, respectively).

The standardised glycogen values in the HP showed a significant peak (0.81, 0.82 ±

0.26) in the mean glycogen concentrations during the LPM category. There were no significant changes in mean glycogen concentrations in the LIM, EPM, and MPM (0.25,

0.24 ± 0.11; 0.21, 0.26 ± 0.15; and 0.27, 0.41 ± 0.33, respectively) moult categories

78 (Figure 4.10). The PCM muscle showed a peak (0.44, 0.48 ± 0.34) in the LPM stage with statistically significant differences from the EPM and PoM categories. There were no significant changes between the LIM (0.25, 0.31 ± 0.36), EPM (0.20, 0.22 ± 0.10), and

MPM (0.24, 0.34 ± 0.35) categories (Figure 4.11).

The CCM showed a very different trend in glycogen reserves with a significant decrease from the MPM (0.72, 0.81 ± 0.37) to LPM (0.62, 0.52 ± 0.24) and then PoM (0.05, 0.10 ±

0.12) categories. No significant changes were observed between the LIM (0.48, 0.63 ±

0.38), EPM (0.54, 0.60 ± 0.37), and MPM (Figure 4.12). The glycogen concentrations in the LIM, EPM and MPM categories of the CCM were higher than those of any of the other tissues in those categories. The mean Gin was between 0.6 and 0.8, while the average Gin for the other tissues in these categories were below 0.5.

In the TM there was a marked peak in glycogen reserves in the MPM and LPM stages

(0.18, 0.18 ± 0.08 and 0.28, 0.31 ± 0.22 respectively), although these were not statistically different from one another. No significant changes were observed between the LIM (0.11, 0.13 ±0.08) and EPM (0.12, 0.14 ± 0.09) categories (Figure

4.13).

When comparing collection years, there was a significant difference in Gin observed between the 2009 and 2010 samples in the MPM category of the HP and the LPM category of the TM (Figures 4.14 and 4.15, respectively). No significant differences were observed between sampling years in any moult stages for the PCM and CCM.

79 1.8

1.6

b 1.4 a N=ll N=18 S 1-2-1 •o H 1.0 c OJ a g 0.8-1 a c N=20 N=19 N=20 W 0.6 *

0.4

0.2 T LIM EPM MPM LPM PoM 0.0 Moult Category

Figure 4.10: Standardised hepatopancreas glycogen in lobsters from all moult cohorts collected in 2009 and 2010. Changes in the glycogen index of the hepatopancreas of lobsters in different moult categories: late inter-moult (LIM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM). Statistically significant differences were observed in groups with different letters (p < 0.5, Mann-Whitney). Asterisks represent outliers within groups and were included in statistical analysis.

1.8- * 1.6- * 1.4- b 12 S " N=ll •o •5 i.o- * a ab ab N=19 N==1 7 o e n b o

Glycog e N=20 c 0.4- N=18 0.2-

0.0- 1 1 1 LIM EPM MPM LPM PoM Moult Category

Figure 4.11: Standardised pincher claw muscle glycogen in lobsters from all moult cohorts collected in 2009 and 2010. Changes in the glycogen index of the pincher claw muscle of lobsters in different moult categories: late inter-moult (LIM), early pre-moult (EPM), mid pre- moult (MPM), late pre-moult (LPM) and post-moult (PoM). Statistically significant differences were observed in groups with different letters (p < 0.5, Mann-Whitney). Asterisks represent outliers within groups and were included in statistical analysis.

80 1.8 ab a 1.6 N=18 N=17 ab 1.4-1 N=20

8 ^ b 5 i.o N=ll c V g1 0.8 H u U 0.6 * 0.4-1 c N=16 0.2

0.0 LIM EPM MPM LPM PoM Moult Category

Figure 4.12: Standardised crusher claw muscle glycogen in lobsters from all moult cohorts collected in 2009 and 2010. Changes in the glycogen index of the crusher claw muscle of lobsters in different moult categories: late inter-moult (LIM), early pre-moult (EPM), mid pre- moult (MPM), late pre-moult (LPM) and post-moult (PoM). Statistically significant differences were observed in groups with different letters (p < 0.5, Mann-Whitney). The asterisk represents outliers within groups and was included in statistical analysis.

1.8

1.6

1.4

S 1-2 •a H 1.0 b c N=ll V O.0.8 .£• a N=19 13 0.6 b N=18 N=18 c 0.4 N=19

0.2

LIM EPM MPM LPM PoM 0.0 Moult Category

Figure 4.13: Standardised tail muscle glycogen in lobsters from all moult cohorts collected in 2009 and 2010. Changes in the glycogen index of the tail muscle of lobsters in different moult categories: late inter-moult (LIM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM). Statistically significant differences were observed in groups with different letters (p < 0.5, Mann-Whitney). Asterisks represent outliers within groups and were included in statistical analysis.

81 1.2- o • • o 1.0- o o A o o 0.8- o o a • •a 0.6- • o en • ! 8 • i • • .a- 0.4- • • o • • • • 0.2- • i i 1 1 i 0.0- • 1 Year 2009 2010 2009 2010 2009 2010 2009 2010 2009 2010 Moult Category LIM EPM MPM LPM PoM

Figure 4.14: The effects of year on glycogen concentrations in the hepatopancreas. Individual value plot of glycogen index values in the hepatopancreas separated by moult category; late inter-moult (LIM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM), for 2009 (•) and 2010 (o). The "A" indicates a significant difference between years.

0.9-

0.8- 0

0.7-

x 0.6- (U •a A 0 S 0.5- • c 0 0) 0.4- o> o • 0 0.3- • 0 • • 0 0 • 0 0.2- • e • • s • 0.1- • 1 ! •! 1 • 0.0- • i Year 2009 2010 2009 2010 2009 2010 2009 2010 2009 2010 Moult Category LIM EPM MPM LPM PoM

Figure 4.15: The effects of year on glycogen concentrations in the tail muscle. Individual value plot of glycogen index values in the tail muscle separated by moult category; late inter-moult (LIM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM), for 2009 (•) and 2010 (o). The "A" indicates a significant difference between years.

82 4.5. Discussion

Trends in tissue glycogen concentrations during the moult were successfully established in all four lobster tissues examined. Glycogen digestion with AMG followed by GLUC determination was optimised. The hexokinase assay was established as the preferred method for the determination of glycogen equivalents in lobster tissues.

Fluctuations observed during the moult identified glycogen as a subtle yet significant metabolic reserve in the lobster, which can be influenced by numerous environmental and physiological factors.

The GOx method was initially assessed because it is often used for GLUC determination in tissues (Carr and Neff, 1984; Huijing, 1970; Murat and Serfaty, 1974; Roehrig and

Allred, 1974) and has been validated for use on marine invertebrate tissue (Burton et al., 1997). It is a manual assay, which required that the GLUC concentrations be determined over several days to weeks due to processing times. The downward shifts and variable slopes in O.D. readings of the standard curves suggested that some unidentified aspects of the reaction were unstable and attempts at determining the source of variation were unsuccessful. These differences in GLUC concentrations over time could have caused significant variations in the dataset, making the comparison of tissue samples assayed on different days difficult. Another commonly used enzymatic assay, the HK, was assessed as an alternative means of GLUC determination

(Giampietro et al., 1982; Neeley, 1972).

83 It was possible to run the HK assay on an automated biochemistry analyzer (Cobas c501), which allowed for all samples of the same tissue type to be assayed on the same day and converted to glycogen equivalents using the same reference curve. This eliminated any within tissue day to day variation. The within-run precision study with

HK on two OG concentrations, HP and pooled muscle (CCM and PCM) showed it to be very precise with % CVs of 0.95 (OG high), 12.8 (OG low), 2.04 and 0.00, respectively.

The high coefficient of variation (12.8%) observed in the low OG sample is the result the very low GLUC concentrations. These values may still be considered reliable because the standard deviation is low and variation observed in the GLUC concentrations was ± 0.1 mmol/L However, the large percentage change that a 0.1 mmol/L variation in the data can exert on low GLUC values should be taken into consideration when interpreting the %CV results.

Oyster glycogen was used as a standard as purified lobster glycogen was not commercially available. The consistent results obtained through digestion and HK glucose evaluation of OG suggests that it is a reliable standard that can be used to generate the glycogen equivalents for the lobster tissue samples. Conversion of glucose to oyster glycogen equivalents allows for the relative assessment of stored glycogen in the lobster and the potential for comparison with reserves reported in other species.

The good linearity and precision of the standard curves generated from digested glycogen indicated that digestion was consistent. Furthermore, the CV below 1%

84 demonstrates the HK method as precise means of GLUC determination. This method has also been shown to be much less susceptible to outside interference than other enzymatic procedures (Neeley, 1972). Digestion of glycogen in the tissue and OG samples could then be assessed using this method to ensure that complete digestion was achieved.

Complete digestion of glycogen, shown by the digestion studies, allows for accurate recovery of GLUC for glycogen determination. The consistent GLUC values obtained under the digestion conditions used in this study indicate that complete digestion of glycogen can be obtained after a 2.5 hour incubation for up to 42 mg of HP tissue per mL. As no more than 30 mg of tissue was ever digested, complete digestion of samples was assumed.

The long term stability of glycogen reserves in the stored HP samples was investigated

(See Section 4.3.5.2) to ensure that accurate glycogen values were being obtained as the NS study samples were stored for up to one year. This study indicated that the HP samples were stable in storage at -80°C for up to 52 weeks. The decrease at 8 weeks and increase at 32 weeks is most likely due to variation in the sample or assay for that day, as the concentrations at 16 and 52 weeks return to the values observed originally at 0 and 4 weeks. Similar observations were made by Burton et al. (1997) with a significant decrease in glycogen concentrations in mussel tissues stored

(-29°C) for one day while those tested at one month showed no change from the original concentrations.

85 The HPs used for the LTS study were divided into seven parts with one part from each pooled to generate one of the HP samples. Thus, the variation observed at weeks 8 and 32 could be due to unequal distribution of glycogen in the HP itself or within the pooled samples following homogenisation and lyophilisation. Further examination of the HK assay standards run near week 32 revealed no significant alterations in GLUC concentrations recovered during Diagnostic Services regular quality control protocols.

Data for the 8 week samples were not available. This suggests either unequal distribution of glycogen in the samples or unidentified errors occurring during the assay/sampling procedures for the 8 and 32 week samples

The data obtained for the homogenised and lyophilised TM and HP shows no significant changes in GLUC concentrations over a 36 week period of storage as dry tissue. This further supports the long term stability of glycogen in stored tissues. As all tissues in this study were processed in under 52 weeks, the data collected can be considered accurate and reliable. However, the possible unequal distribution of tissue glycogen must be kept in mind and should be elucidated to ensure a more accurate representation of tissue glycogen concentrations in the future.

Background GLUC in the four tissues was examined and subjected to heat inactivation studies to determine the impact of autodigestion. Stored glycogen in is closely associated with the enzymes responsible for its synthesis and catabolism (Urich, 1994).

The close association between the reserves and enzymes could allow for the autodigestion of glycogen stores in the tissues. Autodigestion of stored glycogen has

86 been observed in other tissue samples thawed at room temperature (Carr and Neff,

1984) identifying it as a potential source of the background GLUC observed in this

study.

The HI step in the glycogen assays was meant to denature the tissue associated

enzymes and stop autodigestion from occurring. Stopping autodigestion would

eliminate any background GLUC observed in the non AMG digested tissues if it had

originated from glycogen stores. However, this step was not carried out until just

before digestion leaving the period during tissue extraction and thawing prior to

homogenisation as potential time points for autodigestion to occur. The ability to

lower background GLUC with HI, prior to homogenisation and lyophilisation, of the

samples suggests that the background GLUC observed is due to autodigestion of

glycogen stores and can be included in the calculation of total tissue glycogen.

The minimal GLUC concentrations still measured in the samples that received the HI treatment before homogenisation and lyophilisation were assumed to be from

autodigestion of the glycogen reserves that took place during the interim between

excision of tissue from the body and freezing of the samples for storage. These

background values could also be attributed to hemolymph GLUC that was trapped in the tissue. These data led to the elimination of the HI steps in the original preparation

protocols for the tissue samples as the concentrations were minimal and most likely came from glycogen stores.

87 The glycogen concentrations obtained using AMG digestion and the HK assay were measured in mg of glycogen per mg of dry tissue. This measure does not accurately represent the fluctuations in stored energy reserves throughout the moult cycle as it does not take into account internal changes in physiology (i.e., water content) and size of the lobsters. Muscle degradation, especially in the claw muscles, occurs in the preparatory stages of the moult cycle (Mykles and Skinner, 1990; Mykles, 1992). The change in metabolic reserves expressed as milligrams of glycogen per milligram of dry tissue could therefore be affected by the decrease in muscle protein mass rather than an overall change in concentration. Changes in the composition and size of the hepatopancreas during the moult have also been observed (Heath and Barnes, 1970).

These physical changes taking place in combination with the different sized lobsters examined could cause discrepancies when interpreting the amount of stored metabolic reserves measured in the sample tissues.

A method of standardisation was devised to take into account the internal changes in physiology and the external physical changes in size. The standardisation of the glycogen concentrations allowed for a more accurate depiction of changes in glycogen reserves during the moult while justifying the comparison of different sized lobsters.

To ensure that the index was an accurate representation of changes in glycogen reserves, the trends in absolute HP glycogen concentrations were compared to those of the HP Gin values. Comparison of the absolute glycogen concentrations and the index values ensured that the trends in glycogen concentrations were being conserved during conversion to index values.

88 The conservation of significant trends between the index values and the absolute concentrations identified the Gin as an accurate representation of trends in glycogen reserves. This index was then used for all tissue samples assuming that all trends in absolute glycogen concentrations would still be reflected in the index values. The calculations of absolute glycogen concentrations in the HP were possible because the total HP weights were recorded. The total tissue weight of the muscle samples was not recorded, thus absolute glycogen concentrations for each tissue could not be used to standardise the muscle data.

Sample sizes for glycogen assessment in the four tissues examined varied because all tissues could not be run, as there was insufficient tissue available. Furthermore, there was no evidence available to identify the outliers observed in the glycogen data as true outliers. They are most likely reflections of the natural variations that can occur in wild lobster and were included in the statistical analysis of the data.

The data from the 2009 primary moult cohort discussed in Chapter Two were examined for the most accurate representation of glycogen fluctuations during the moult cycle as physiological differences between year and cohort are poorly understood. The significant decrease in the PoM category for all tissues suggests that these stores are being used or redistributed during or shortly after ecdysis. Under stress, the mobilisation of these reserves as an energy source has been observed in many crustaceans including: N. norvegicus (Baden et al., 1994), Penaeus japonicas

(Cuzon et al., 1980), Chasmagnathus granulate (Vinagre and Silva, 1992), Procambarus

89 clarkia (Schirf et al., 1987) and L. vannamei (Zhou et al., 2011). The stress of the moult,

due to an extended period of inanition and high energy demand, is the most likely

reason for the rapid depletion or redistribution of what little glycogen reserves are

present in the lobster tissues as observed in this study.

The lack of data available for LPM category of the primary cohort in 2009 makes

accurate interpretation of moult related changes over the entire cycle difficult. When

including the 2010 data and lobsters believed to be from a secondary moult cohort in

2009 (See Chapter 2), there was a significant increase in stored glycogen observed in

the LPM. The examination of the data including these lobsters may help with the

interpretation of the significant increase in the MPM of the tail muscle and the higher

glycogen values observed in the CCM. These data could be suggestive of the natural

fluctuations in glycogen reserves during the moult since increases in HP glycogen

preceding ecdysis have been observed in other crustaceans (Travis, 1955; Passano,

1960). More information on the effects of cohort and year on glycogen reserves are

necessary to fully support whether these trends are common to moult category and

not an artifact of cohort or year variability.

The significant trends observed in all tissues at different moult categories when including the full dataset suggest that glycogen is an important resource in the lobster during the moult. These data corroborate with that of other studies, which have shown muscle and HP tissues to house important glycogen reserves (Yu et al., 2009;

Rosa and Nunes, 2003b; Vinagre and Silva, 1992). Trends in glycogen concentrations

90 observed were very similar in the HP, PCM, and TM with a marked increase around the

LPM followed by a near depletion in the PoM. The increase in reserves in the LPM is

interesting because it is during this stage of the moult cycle that the lobster slowly

stops feeding (Dall, 1990). This suggests that the reserves are not coming from a food

source but are being reallocated from body stores. One explanation for this is the

conversion of chitin to GLUC for transport through the hemolymph to tissues for

storage as glycogen, followed by the remobilisation after the moult for chitin re-

synthesis as suggested by Travis (1955). Glucose is a necessary precursor to chitin

synthesis as observed in spiny lobsters Panulirus sp. (Scheer and Scheer, 1951), the

crab E. asiatica (Parvathy, 1971) and even some insect species (Candy and Kilby, 1962;

Lipkeetal., 1965).

Degradation of the exoskeleton, as a means of mineral conservation (Travis, 1960), is

observed in decapod crustaceans during the moult (Gwinn and Stevenson, 1973;

Wheatly, 1999). The minerals removed are stored for use in the re-mineralisation of

the shell after moulting (Waddy et al., 1995). Storage sites can vary among species but

in H. americanus these minerals are stored in the gastroliths (Waddy et al., 1995). The gastroliths are highly mineralized deposits that form in a sac between the epidermis

and cuticular lining of the cardiac stomach (Travis, 1960).

Glycogen has been shown to be important in the deposition of calcium and phosphate in the HP of the lobster P. argus (Travis, 1955) and could also play an important role in the deposition of minerals in the gastroliths. Although its role in gastrolith formation

91 has not been investigated in H. americanus, glycogen was detected in the gastroliths of the Orconectes virilis (Travis, 1960). The breakdown of chitin from the exoskeleton for storage in the muscle and HP would explain the trends in glycogen storage observed in H. americanus.

The conversion of lipid to glycogen in the HP during the pre-moult stages has also been suggested as a cause of the LPM increase in glycogen concentrations (Renaud, 1949 as translated in Passano, 1960). The increased glycogen reserves could then be mobilised for chitin synthesis. The conversion of lipid to glycogen more rapidly than the mobilisation of glycogen for chitin synthesis would cause the significant increase in glycogen reserves during this stage (Renaud, 1949 as translated in Passano, 1960).

Definitive data is required to determine the efficiency of glycogen conversion to chitin and the conversion of stored lipid to glycogen in the HP.

Trends observed in the CCM were very different from those of the other tissues with high glycogen concentrations in the pre-moult that gradually decreased through to the

PoM. This suggests that glycogen in the CCM could be a significant energy reserve being utilised when feeding slows and eventually ceases in the LPM. The difference could be due to the high energy requirements of this muscle to facilitate the pronounced degradation or atrophy of the muscle taking place in the pre-moult

(Mykles, 1992). Muscle atrophy allows for easier escape from the old shell at ecdysis and could be more pronounced in the CCM to allow for easier withdrawal through the knuckle, as was observed in the fiddler crab, Uca pugnax (Ismail and Mykles, 1992).

92 The pronounced atrophy in the CCM could also create the illusion of increased glycogen. As muscle mass is decreasing due to muscle atrophy, glycogen concentrations would appear to increase per mg of tissue even if they remain constant.

The differential atrophy of the CCM versus the PCM (Ismail and Mykles, 1992) would also explain why this is not also observed in the PCM. The decrease in muscle mass would be replaced with water, so the index correction should eliminate this illusion as it corrects for water content in the tissue. Therefore, this suggests that the changes observed are real.

On average, the HP had a higher concentration of glycogen than the muscle tissue, similar to what was observed in A. antennatus, P. longirostris, and N. norvegicus (Rosa and Nunes, 2003a). The ratio of muscle to HP is large, which could mean that although there is more glycogen per mg of tissue in the HP, the muscle will comprise a more significant portion of the absolute glycogen content for the lobster as a whole.

Glycogen has been shown to act as a significant energy reserve during starvation and hypoxia in the Norwegian lobster, N. norvegicus (Hagerman et al., 1990; Baden et al.,

1994) suggesting it is a significant energy source. These earlier studies examined the effects of stress on energy reserves whereas the present study investigated natural fluctuations. Thus, glycogen reserves may naturally be associated more with chitin synthesis but in cases of extreme stress, these reserves can also be utilised as an energy source (Baden et al., 1994; Cuzon et al., 1980; Vinagre and Silva, 1992; Schirf et al., 1987).

93 Glycogen depletion in the PoM category has also been suggested to be due to an increased mobilisation of GLUC into the hemolymph to aid in changing the osmotic pressure (Travis, 1955; Baumberger and Olmsted, 1928). In H. americanus, the role of glycogen in osmotic regulation is unclear because the concentrations of free GLUC in the hemolymph are very low. They rarely exceed 2 mmol/L, as was observed in

Chapter 6 and elsewhere (Urich, 1994). Previous studies have also shown that in juvenile H. americanus, water absorption occurs with little to no measurable changes in osmotic gradients (Mykles, 1980). Further investigation into the role of glycogen in the regulation of osmotic pressure is necessary for a better understanding of its role in osmotic regulation in crustaceans.

The temporal effect on glycogen reserves was examined since the samples were collected in two separate years. Glycogen concentrations from the 2009 and 2010 samplings showed significant differences between the MPM of the HP and the LPM of the TM with no differences observed in the PCM and CCM muscle. Since the differences are not consistent within moult categories and between the tissues, the trends observed in the tissues may still be valid. The temporal differences could be due to seasonal variations in food availability, which would ultimately affect the amount of glycogen stored in the tissues prior to the moult. The changing food abundance would dictate reserve distribution and ultimately be the determining factor in which reserves are available as energy. The differences could also be the result of variations in the overall glycogen reserves in the different moult cohorts discussed in

Chapter 2. Thus, although trends observed here suggest a role of glycogen in chitin

94 synthesis, it could also play a role as an energy reserve seasonally depending on diet.

Diet has been shown to dictate glycogen reserves in the penaeid shrimp L. vannamei

(Rosas et al., 2001). A closer look at seasonal changes in glycogen concentrations during the moult is required to confirm its role in H. americanus.

Glycogen reserves in the HP, PCM, CCM, and TM of H. americanus are integral to its moult. Its role in chitin synthesis is yet to be thoroughly assessed but is a likely explanation for the trends observed at the different moult stages. Overall, glycogen has potential as a significant reserve that could be used for the monitoring of changes in chitin production and shell formation during the moult cycle.

Although glycogen in the TM of the spiny lobster, P. marginatus, was found to be a good indicator of nutritional condition during the inter-moult (Parrish and Martinelli-

Liedtke, 1999), it does not appear to be true during the moult cycle in H. americanus.

Glycogen concentrations throughout the inter-moult stages should also be assessed as they may be utilised then as an energy reserve. However, the very low concentrations observed will most likely render it insignificant in comparison to other energy sources such as lipid. The temporal changes in glycogen concentrations noted could also affect the role of these reserves in the lobster. When examining these changes, spatial, temporal and environmental factors should be kept in mind as they can also cause fluctuations in glycogen storage (Winget et al., 1977; Vinagre and Silva, 1992; Nery and

Santos, 1993; Rosa and Nunes, 2003a and 2003b).

95 In conclusion, the data here offer no definitive role for tissue glycogen as a primary energy reserve in H. americanus during the moult cycle. There is strong evidence to suggest its significance in chitin synthesis, but a better understanding of the temporal changes in reserves is required to confirm this. The role of glycogen could change temporally due to environmental manipulation, such as changes in food type and abundance. To ensure that the trends observed here are accurate, a new dataset must be obtained that includes lobsters from each moult category in one year and from the same moult cohort. This will help to determine if the significant peaks in glycogen observed here were factors of month or year collected or if there is a significant increase in glycogen storage in the LPM category.

A better understanding of the natural fluctuations in reserves and their environmental constraints will aid in identifying their purpose and significance in the lobster. Such knowledge will contribute to a complete understanding of lobster physiology and elucidate the role of glycogen in nutritional condition. Should these reserves be found to be an indicator of nutritional condition, the data could have applications in methods for the proper monitoring, management and conservation of wild and cultured populations.

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101 5. Fluctuations in Stored Lipid of the Hepatopancreas and Muscle Tissues of the American Lobster Homarus americanus during the Moult Cycle

5.1. Abstract

Three methods for lipid extraction; Folch centrifugation (FoC), Folch filtration (FoF) and

Bligh and Dyer (B&D), were examined as a means of monitoring fluctuations in levels of lipid storage in lobster tissues. The Folch centrifugation method was established as the optimal method for lipid extraction as it showed the least amount of variation. The lipid concentrations demonstrated significant variations among the five moult categories examined: late inter-moult (LIM), early pre-moult (EPM), mid pre-moult

(MPM), late pre-moult (LPM) and post-moult (PoM). The hepatopancreas (HP) and tail muscle (TM) lipid concentrations showed a significant decrease in the PoM category, suggesting its use as an energy reserve during the moult. Lipid in the claw muscles showed very different trends with a significant increase in the LPM followed by a decrease to the LIM and pre-moult values in the PoM. The LPM increase in lipid concentrations suggests re-allocation of internal reserves as lobsters are not feeding during this period. This increase could be interpreted as an additional means of conserving metabolites derived from chitin degradation prior to ecdysis.

5.2. Introduction

Lipids represent a significant source of energy in many species (Jeffs et al., 1999; Rosa and Nunes, 2003; Schirf et al., 1987; Moore et al., 2000; Stuck et al., 1996). One gram of stored lipid will supply approximately 39 kJ of energy in comparison with glycogen,

102 another common form of energy storage, which will supply about 17 kJ/g. Unlike

glycogen, lipid stores are not hydrated, making them more compact, allowing for

increased storage efficiency (Urich, 1994). The energetic efficiency of lipids makes

them especially important in species that require large reserves to cope with certain

aspects of their life history. Hibernation, migration, and long-distance flight are three

important life history characteristics resulting in a heavy dependence on lipid reserves

(Urich, 1994). In each of these instances the organism experiences an extended period

of inanition, which in some cases corresponds to an increased energy requirement as

in migration and long distance flight.

Crustaceans have a cyclical life history punctuated with periods of physiological change

including moulting and reproduction. During the moult cycle crustaceans experience a

period of inanition, similar to that observed during hibernation, migration and long

distance flight. This period is followed by pronounced physiological changes, which

incur a high energy requirement. The high energy demand suggests that an

accumulation of metabolic reserves is integral to a successful moult. The sequential

accumulation and depletion of metabolic reserves during the moult cycle has already

been identified in many decapod crustaceans including: Penaeus monodon (Suneetha

et al., 2009), Pleoticus Muelleri (Jeckel et al., 1990), Penaeus esculentus (Barclay et al.,

1983) and P. japonicas (Ando et al., 1977).

Determination of lipid concentrations in tissues is generally carried out through the exploitation of their solubility properties. Lipid molecules will typically only dissolve in

103 organic solvents such as chloroform, hydrocarbons or alcohol (Gurr et al., 2002). It is generally agreed that the most efficient method of extracting total lipid from tissue samples is with chloroform and methanol in a 2:1 ratio (Christie, 1973). A combination of organic and inorganic solvents is then used to generate a biphasic solution in which lipid constituents are dissolved in the organic phase (Bligh and Dyer, 1959).

Gravimetric determination of total lipid can then be carried out following the separation of the two phases and evaporation of the organic solvent.

The Folch methods are commonly used means of lipid extraction in tissue samples.

They employ 8:4:3 ratios of chloroform, methanol and water to generate a biphasic solution (Folch et al., 1957). There are two different protocols for lipid extraction with the Folch method in which cellular debris is removed either by filtration or centrifugation of the crude extract (Folch et al., 1957). Another commonly used method of lipid extraction is the Bligh and Dyer (B&D) method. The B&D method was devised for use on tissues with high water content (Bligh and Dyer, 1959) such as those of aquatic species. This method employs an initial chloroform, methanol and water ratio of 1:2:0.8 for the crude extraction of lipid followed by a final ratio of 2:2:1.8 to generate the biphasic solution (Bligh and Dyer, 1959).

These three methods of lipid extraction were assessed for use on lobster tissues and to identify the optimal method. Fluctuations in lipid reserves in the four tissue samples collected were examined at the different moult categories to elucidate trends across

104 the moult. These data were analyzed to enhance the understanding of natural

fluctuations in stored lipids and their importance during the moult cycle.

5.3. Materials and Methods

5.3.1.Comparison of Different Extraction Methods for the Determination of Lipid Concentrations in Lobster Tissues

5.3.1.1. Evaluation of the Folch Centrifugation and Filtration Methods of Lipid Extraction from Lobster Tissues

For the Folch centrifugation (FoC) method a 100 mg aliquot of lyophilised

hepatopancreas (HP) was diluted in a 3.75 mL mixture of chloroform:methanol (2:1,

Appendix B) in triplicate. An aliquot of methanol was added (0.75 mL or 0.02 %v) then

the sample was homogenised (OMNI International automated homogeniser with a

stainless steel generator probe EZ coupling, G5-75 5mm diameter and 75 mm length,

Kennesaw, GA), vortexed (1 min) and incubated in a fume hood (15 min, RT). The

sample tubes were then centrifuged (20 min @ 1000xg, Model TJ-6 Centrifuge,

Beckman, Mississauga, ON) and the supernatant was decanted into a 15mL glass

centrifuge tube. The samples were vortexed (1 min) and centrifuged again (20 min @

lOOOxg) following the addition of 1.5 mL each of chloroform and distilled water. Using

a Pasteur pipette, the lower organic phase was transferred to a pre-labeled and

weighed 10 mL beaker, which was then stored at 50°C (~3 days, Precision incubator

3520, Mandel Scientific Company Inc., Guelph, Ontario). The weights of the lipid samples were measured every day until all solvent had evaporated and no changes in

105 weight were observed. The lipid weights were used for the calculation of percent lipid in the tissue samples.

Lipid extraction in the muscle tissues was carried out in the same manner with several modifications (Appendix C). Approximately 150-200 mg aliquots of muscle tissue were extracted in duplicate and all solution volumes were doubled. The Folch filtration (FoF) method was carried out in the same manner described above with the initial centrifugation step replaced by filtration (Buchner funnel, Whatman Qualitative 1 filter paper) and the addition of methanol (0.02 %v) eliminated.

5.3.1.2. Evaluation of the Bligh and Dyer Method of Lipid Extraction from Lobster Tissues

Approximately 70-90 mg aliquots of lyophilised tissue were weighed out into glass piston homogenisers (GPH) in triplicate. Two mL of chloroform:methanol (2:1) solution was added and then the sample was homogenised until the tissue was uniformly dispersed. The solution was then transferred to a 15 mL glass centrifuge tube. The

GPH was rinsed with 1.75 mL of chloroform: methanol, which was then transferred to

the same centrifuge tube. A final rinse was performed with 1 mL distilled H20. Each tube was vortexed vigorously (1 min) then incubated (15 min, RT). Following the incubation, 1.25 mL each of chloroform and distilled water were added and each sample was vortexed vigorously (1 min). The samples were centrifuged (20 min, lOOOxg) and the lower organic layers were pipetted into pre-labeled and weighed 10

106 mL beakers. Beakers were incubated (~3 days, 50°C) until no weight change was

observed.

5.3.1.3. Evaluation of the Manual and Electronic Homogenisation Methods

The FoC method was carried out on excess HP tissue from another UPEI Animal Care

Committee (ACC) approved study. Tissues were homogenised either manually with the

GPH (n=6) or electronically with the electronic homogeniser (n=6 replicates). Manual

homogenisation was carried out as described for the B&D method (Section 5.3.1.2)

using the solvent volumes described for the Folch methods and electronic

homogenisation as per the FoC method (Section 5.3.1.1).

5.3.2. Precision of the Folch Centrifugation Method for Lipid Extraction

Lipid was extracted from lyophilised nutritional status (NS) HP, PCM, CCM and TM

tissue (n=6/tissue) with the FoC method (Section 5.3.1.1). The lipid concentrations

were evaluated to determine the precision of the extraction methods on the four

tissues examined.

5.3.3. Long Term Stability of Hepatopancreas Tissue Samples Stored at -80°C

The FoC methods for lipid extraction (Section 5.3.1.1) were carried out on LTS HP samples (n=6) at 0, 4, 8, 16, 32, and 52 weeks following homogenisation (Section 3.3.1) and lyophilisation (Section 3.3.2) procedures.

107 5.3.4. Assessment of Lipid Concentrations in Lobster Tissues Representing Different Moult Categories

Lipid concentrations in the NS lobsters were determined in triplicate for the HP and duplicate for the PCM, CCM and TM using the FoC method (Section 5.3.1.1).

Concentrations were then standardised as described in section 4.3.7 to take into account the physical and physiological differences among the lobsters. The equation for the standardisation of the lipid data is expressed as:

Lipid Index (Lin) = ((%W * L) / CL) * lxlO4

Where %W represents percent water content of the tissue, L is mg of lipid per mg of wet tissue, CL is the carapace length of the lobster and the units are mg lipid (mg tissue)"1 mm"1.

5.3.5. Statistical Analysis of Lipid Data

Kruskal-Wallis and Mann-Whitney nonparametric analyses were used for determination of statistically significant differences in lipid concentrations within and between different moult categories. All statistical analyses were performed with

Minitab® statistical software version 15.1, 2006 and significance is reported at p < 0.05.

108 5.4. Results

5.4.1.Comparison of Different Extraction Methods for the Determination of Lipid Concentrations in Lobster Tissues

There were no significant differences in lipid yield among the three methods.

However, the FoC method showed less variation when carried out on the same tissue samples (Figure 5.1).

65

60

•o QJ N = 55 -C w Q. (0 o QJ c x g 40

Figure 5.1: Comparison of lipid extraction procedures. Percent lipid in aliquots of hepatopancreas from a single, non-study, lobster after extraction with the Bligh and Dyer (B&D), Folch centrifugation (FoC) and Folch filtration (FoF) methods. Error bars show standard error of sample mean (n=3).

5.4.1.1. Evaluation of the Manual and Electronic Homogenisation Methods for the Tissue Samples

Recovered lipid concentrations in the tissue samples following extraction with the electronic method were significantly greater than that of the manual homogenisation.

109 There was also less variation observed in the standard deviation (SD) and coefficient of variation (CV) when using electronic homogenisation (Table 5.1).

Table 5.1: Comparison of manual vs. electronic homogenisation of tissue samples. Percent lipid extracted from HP tissues (n=6) with the FoC method using manual and electronic homogenisation (MH and EH, respectively). The median, mean, standard deviation (SD) and coefficient of variation (CV) are displayed for each. Significance of means is observed at p <0.05 between the two methods.

Tissue Lipid (%) Tissue Lipid (%) Manual Homogenisation Electronic Homogenisation Median 57.4a 58.5b Mean 57.3 58.5 SD 1.0 0.2 % CV 1.7 0.4

5.4.2. Precision of the Folch Centrifugation Method for Lipid Extraction

Good precision was observed in all four tissue types. Variation was higher in the muscle tissues with %CV ranging from 4.20% to 5.25% compared to the 0.35% observed in the HP (Table 5.2).

Table 5.2: Precision of lipid extractions in four lobster tissues. Lipid concentrations in the four tissue types examined (n=6) as percent dry weight. Median, mean, standard deviation (SD) and coefficient of variation (% CV) are shown.

Total Lipid (%) Total Lipid (%) Total Lipid (%) Total Lipid (%) Hepatopancreas Pincher Claw Crusher Claw Tail Median 58.48 2.50 2.86 3.37 Mean 58.47 2.51 2.85 3.32 SD 0.21 0.11 0.15 0.14 %CV 0.35 4.20 5.25 4.24

110 5.4.3. Long Term Stability of Hepatopancreas Tissue Samples Stored at -80°C

Lipid concentrations in the long term stability (LTS) HP samples showed no significant changes until week 16 when a significant decrease from week 0 (3.08%) was observed.

There was then a 6.00% decrease in the 32 week sample but after 52 weeks in storage the value rose back to the original concentrations (Table 5.3).

Table 5.3: Stability of tissue samples after long term storage at -80°C. Total lipid concentrations (percent dry weight) in the long term stability (LTS) hepatopancreas (HP) tissue samples stored at -80°C for 0, 4, 8, 16, 32 and 52 weeks. Lipid concentrations were determined using the FoC method. Median, mean, standard deviation (SD) and coefficient of variation (CV) are shown. Different letters represent significance at p < 0.05 (Mann-Whitney).

% Lipid % Lipid % Lipid % Lipid % Lipid % Lipid WeekO Week 4 Week 8 Week 16 Week 32 Week 52 Median 57.14ac 57.70a 54.40ac 54.37b 54.51b 56.46c Mean 57.71 57.95 57.34 55.93 54.25 56.62 SD 2.76 0.77 1.00 0.52 1.76 0.50 %CV 4.78 1.33 1.75 0.92 3.25 0.88

5.4.4. Assessment of Lipid Concentrations in Lobster Tissues Representing Different Moult Categories

There were no significant differences observed in between the total lipid concentrations of the HP (g/HP) during the LIM, EPM, and MPM for the 2009 primary moult cohort (3.23, 3.68 ± 1.68; 4.53, 4.95 ± 2.07 and 4.05, 4.25 ± 1.01 g, respectively), the data are expressed as median, mean ± standard deviation. This concentration dropped significantly to 1.76, 1.84 ± 0.75 g in the PoM (Figure 5.2). This same trend was observed in the Lin values (standardised lipid concentrations) with the LIM, EPM and MPM Lin of 15.51, 15.72 ± 4.28; 17.22, 17.46 ± 2.74 and 17.89, 17.33 ± 3.03 respectively and the PoM Un dropped to 10.48, 10.43 ± 2.90 (Figure 5.3).

Ill Subsequently, all lipid concentrations will refer to index values. The LPM lobster were

all caught in 2010 or as part of the secondary moult cohort, so they are not present in

the primary 2009 moult cohort data.

12- * 10- a 8- a n=18 n= 19 a n=10 Q. i b _l n=19 4- * | 1 I 2- 1 1 0- LIM EPM MPM LPM PoM Moult Category

Figure 5.2: Total lipid concentrations in the hepatopancreas for the 2009 primary moult cohort. Mean total lipid content in the hepatopancreas of lobsters from the five different moult categories: late inter-moult (LIM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM). Different letters designate significant differences at p < 0.05 (Mann-Whitney). Asterisks represent outliers.

112 25 a a n=18 a n=19 n=10 20

b 3 n=19 •o c 15 3 n. 3 10-

5-

UM EPM MPM LPM POM Moult Category

Figure 5.3: Standardised lipid concentrations in the hepatopancreas for the 2009 primary moult cohort. Mean lipid index (Lin) values in the hepatopancreas of lobsters from the five different moult categories: late inter-moult (LIM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM). Different letters designate significant differences at p < 0.05 (Mann-Whitney).

Initial analyses of the 2009 primary moult cohort revealed similar trends in the PCM and TM as those seen in the HP. The Lin values were much lower in the muscle tissues than the HP; with the PCM showing Un values of 0.520, 0.509 ± 0.131 (LIM); 0.540,

0.515 ± 0.101 (EPM) and 0.498, 0.512 ± 0.145 (MPM) and a significant decrease from the EPM and MPM to 0.480, 0.482 ± 0.071 in the PoM (Figure 5.4). The CCM showed no significant changes in the lipid concentration in any of the moult categories with mean Un values of 0.574, 0.574 ± 0.124; 0.603, 0.584 ± 0.126; 0.561, 0.574 ± 0.108 and

0.559, 0.555 ± 0.69 in the LIM, EPM, MPM and PoM respectively (Figure 5.5). The Un values of the TM were 0.699, 0.703 ± 0.139 (LIM); 0.708, 0.703 ± 0.108 (EPM) and 0.727, 0.739 ± 0.121 (MPM) followed by a decrease to 0.541, 0.533 ± 0.102 in the PoM

(Figure 5.6).

2.0

1.5-

x 4) •a a •S 1.0 n=10 •o a '5. a n=19 b n=18 n=19

0.5

0.0 LIM EPM MPM LPM PoM Moult Category

Figure 5.4: Standardised lipid concentrations in the pincher claw muscle for the 2009 primary moult cohort. Mean lipid index values in the pincher claw muscle of lobsters from the five different moult categories: late inter-moult (LIM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM). Different letters designate significant differences at p < 0.05 (Mann-Whitney). The asterisk represents an outlier.

114 2.0

1.5

s •a a a •5 i.o a n=18 n=10 IS n=19 n=19

0.5 ^

0.0 UM EPM MPM LPM PoM Moult Category

Figure 5.5: Standardised lipid concentrations in the crusher claw muscle for the 2009 primary moult cohort. Mean lipid index values in the crusher claw muscle of lobsters from the five different moult categories: late inter-moult (UM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM). Different letters designate significant differences at p < 0.05 (Mann-Whitney).

2.0

1.5

a a •a a £ i.o n=19 n=10 n=18 b IB 1 1 Eb n=19 0.5- 1 * 1

0.0 UM EPM MPM LPM PoM Moult Category

Figure 5.6: Standardised lipid concentrations in the tail muscle for the 2009 primary moult cohort. Mean lipid index values in the tail muscle of lobsters from the five different moult categories: late inter-moult (LIM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM). Different letters designate significant differences at p < 0.05 (Mann-Whitney). The asterisk represents an outlier.

115 When including the secondary moult cohort and the 2010 dataset the LPM category is

represented. In the HP, lipid concentrations dropped significantly in the PoM (10.82,

10.89 ± 3.49) with no statistically significant changes observed in the LIM, EPM, MPM

and LPM categories (15.28, 15.43 ± 4.37; 17.12, 17.44 ± 2.66; 18.21, 17.35 ± 3.08 and

15.51,16.13 ± 2.70, respectively) (Figure 5.7). The same trend was observed in the TM

with an average PoM Un value of 0.54, 0.54 ± 0.10 and LIM, EPM, MPM and LPM

values of 0.69, 0.70 ± 0.14; 0.71, 0.70 ± 0.10; 0.76, 0.77 ± 0.13 and 0.78, 0.74 ± 0.14,

respectively (Figure 5.8). A significant increase of lipid in the LPM prior to depletion in

the PoM of the PCM (0.69, 0.70 ± 0.23 and 0.48, 0.48 ± 0.02 respectively) and CCM

(0.70, 0.78 ± 0.23 and 0.56, 0.56 ± 0.07 respectively) was observed (Figures 5.9 and

5.10, respectively). The LIM, EPM and MPM categories of the PCM (0.49, 0.48 ± 0.17;

0.55, 0.52 ± 0.02 and 0.52, 0.56 ± 0.04 respectively) and CCM (0.57, 0.57 ± 0.12; 0.61,

0.59 ± 0.13 and 0.63, 0.62 ± 0.15, respectively) showed no significant changes in lipid

concentrations.

When examining the distribution of Lin values for each moult category (cohorts

combined) with respect to year, there was a significant difference in lipid

concentrations observed in the LPM of the PCM (Figure 5.11). There were no

significant differences between years in any of the moult stages for any of the other tissues (Data not shown).

116 25 a a a n=19 n=18 a n=20 b n=ll n=20 20

8 5 is Q.

10

5-

LIM EPM MPM LPM PoM Moult Category

Figure 5.7: Standardised lipid concentrations in the hepatopancreas for all moult cohorts from the 2009 and 2010 samplings. Mean lipid index in the hepatopancreas of lobsters from the five different moult categories: late inter-moult (LIM), early pre-moult (EPM), mid pre- moult (MPM), late pre-moult (LPM) and post-moult (PoM). Different letters designate significant differences at p < 0.05 (Mann-Whitney).

2.0

1.5

8 a a n=18 a •o n=20 a n=ll 5 i.o n=19 b n=20

0.5

0.0 LIM EPM MPM LPM PoM Moult Category

Figure 5.8: Standardised lipid concentrations in the tail muscle for all moult cohorts from the 2009 and 2010 samplings. Mean lipid index in the tail muscle of lobsters from the five different moult categories: late inter-moult (LIM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM). Different letters designate significant differences at p < 0.05 (Mann-Whitney). The asterisk represents an outlier.

117 2.0-,

1.5-

ab x b a n=18 •o n=ll S 1.0 i •a a 'a. ab * a n=20 n=19 n=20 i 1 0.5- 1 1 1

0.0 i i LIM EPM MPM LPM PoM Moult Category

Figure 5.9: Standardised lipid concentrations in the pincher claw muscle for all moult cohorts from the 2009 and 2010 samplings. Mean lipid index in the pincher claw muscle of lobsters from the five different moult categories: late inter-moult (LIM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM). Different letters designate significant differences at p < 0.05 (Mann-Whitney). Asterisks represent outliers.

2.0

1.5 b ab n=ll S n=17 •o a a •5 I.O n=20 n=19 a Q. n=20

0.5-

0.0 LIM EPM MPM LPM PoM Moult Category

Figure 5.10: Standardised lipid concentrations in the crusher claw muscle for all moult cohorts from the 2009 and 2010 samplings. Mean lipid index in the crusher claw muscle of lobsters from the five different moult categories: late inter-moult (LIM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM). Different letters designate significant differences at p < 0.05 (Mann-Whitney). The asterisk represents an outlier.

118 1.4-

1.2-

1.0- o g «X •a • o S 0.8- a 0.6- I ! i : i 0.4- • S • o • | 0.2- • • • Year 2009 2010 2009 2010 2009 2010 2009 2010 2009 2010 Category UM EPM MPM LPM PoM

Figure 5.11: Evaluation of year effect on lipid concentrations in the pincher claw muscle. Lipid index values for each lobster from the five moult categories: late inter-moult (LIM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM). The 2009 (•) and 2010 (o) lobsters are shown with significant differences between years designated by "A". Significance is at a p < 0.05 level, Mann-Whitney.

5.5. Discussion

Gravimetric analysis of total tissue lipids following extraction with the FoC method was

identified as the optimal means of lipid extraction for the lyophilised lobster tissues.

The assessment of lipids in the various lobster tissues revealed significant fluctuations

in concentrations during the moult cycle. These changes identified lipids as a

substantial metabolic reserve in the lobster during the moult with the HP constituting

the largest reservoir.

The three extraction methods were examined to determine which procedure was the

most reliable and efficient for use on lyophilised lobster tissues. The two Folch

methods were evaluated as they represent a very commonly used extraction

119 procedure. The Bligh and Dyer (B&D) method, although modified for use on marine or

aquatic tissues, (Bligh and Dyer, 1959) proved to be more variable than the FoC

method when used on the lobster tissues. The most likely reason for the variation and

lower yield is that the lobster tissues used were lyophilised, eliminating the need to

account for tissue water content in the solvent ratios. The lack of water in the tissues

most likely resulted in an altered ratio of chloroform, methanol and water. Slight

alterations in this ratio could alter the efficiency of lipid extraction (Bligh and Dyer,

1959). The FoF method proved difficult as the small amount of solvent evaporated

quickly under the vacuum pressure used during filtration. This method was also time

consuming as the filtration apparatus had to be cleaned after each use. Few technical

difficulties were encountered with the FoC and B&D methods. The low variation and

relatively rapid procedures of the FoC method identified it as the optimal method for

lipid extraction from the lobster tissues.

The electronic homogenisation of tissue samples was assessed to increase efficiency of

the assay protocol. The electric homogenisation decreased the time required to

homogenise the tissue samples and the number of transfers between different vials

and centrifuge tubes. This most likely decreased residual loss from the transfer

between vials and resulted in the higher yields and lower variation observed between the two methods.

The method of lipid extraction for the muscle tissues had to be modified to account for the very low lipid concentrations present. Doubling the amount of tissue extracted

120 resulted in an overall increase in weighed lipid, and eliminated some of the potential

instrument variation that weighing small quantities may have caused. Doubling the

tissue amount required the doubling of the solvent volumes to ensure complete

dissolution of lipid from the tissue samples. Lipid extractions were reduced to two

replicates per tissue due to limited tissue availability.

The very low coefficients of variation (CV) observed through the precision studies

ensure that precise results could be obtained using the FoC method. The higher % CV

values observed in the muscle tissues are most likely a reflection of the low lipid

concentrations in the PCM, CCM and TM as compared to the HP. The low lipid

concentrations should be considered when examining the muscle lipid data as slight

changes in concentrations can result in large alterations during mathematical

conversion for the standardisation of the data.

The LTS studies of the HP tissue samples revealed some variation in stability at 16 and

32 weeks. The decrease in lipid concentrations observed in these two weeks was less than 6% of the average initial lipid concentration. However, the increase back to

original concentrations in the 52 week samples suggests that the sample was most

likely stable for up to 52 weeks in storage. The concentration decrease at 16 and 32 weeks could be due to inconsistencies in the homogenised tissue samples or unequal distribution of lipid in the tissues as each of the LTS samples represented 6 different HP that had been pooled to generate a sample large enough to be assayed several times over the course of one year.

121 Initial analyses of the lipid concentrations were carried out on the 2009 primary moult cohort to eliminate any potential discrepancies that may have been caused by year or cohort variations in physiology. The significant decrease in the Lin of PoM lobsters suggests that the lipid in these tissues is being used during or shortly after ecdysis. The most likely use of the lipid stores would be as an energy reserve, with the HP being the most significant source due to its high lipid content. This corresponds well with what has been reported in the literature where the HP is the primary organ involved in lipid storage with very low muscle lipid concentrations (Chapelle, 1977; Dall, 1981). The lack of significant differences in lipid concentrations of the CCM suggests that there are tissue specific variations in metabolic use and further examination of these variations are warranted.

Analysis of lipid concentrations by superimposing all cohorts from both 2009 and 2010 was performed in attempt to further elucidate the role of lipid during the moult cycle.

The additional data supplied information on potential changes in lipid concentrations during the LPM category. Significant changes are expected during this period due to its proximity to the moult and the significant changes in glycogen concentrations observed during this period (See Chapter 4).

Following the addition of the 2009 secondary cohort and 2010 data there were no changes in the trends observed in the HP and TM Lin values. Similar index values were measured between the LIM, EPM, MPM and LPM categories followed by a significant decrease in the PoM. Although there was no significant difference observed, the mean

122 Lin values in the HP and TM show a gradual increase from the LIM to MPM followed by

a slight decrease in the LPM. These trends support what has been reported in other

crustacean species where there is a buildup of metabolic reserves during the inter-

moult and pre-moult categories in preparation for the moult (Barclay et al., 1983; Ando

et al., 1977; Kanazawa et al., 1976; O'Connor and Gilbert, 1968). The natural variations

of wild populations are suspected to be hiding the actual changes in metabolic reserves

during these categories and hindering the ability to detect statistical significance. A

larger sample size may help to elucidate these changes and show significances

between the different moult categories.

These data verified the role of lipid stores in the HP and TM as significant energy

reserves in the lobster during the moult cycle. Although the low lipid concentrations in

the TM seem to be negligible in comparison to the HP, the size of the TM could result

in a large input of energy to the system. The average wet weight of a H. americanus

TM is 107.92 ± 28.34 g in a ~625 g lobster (Cawthorn, unpublished data), whereas the

average HP weight is approximately 18.97 ± 5.67 g in a ~370 g lobster. Consequently,

in a LIM lobster the TM contributes approximately 0.73 g of lipid to the system. The

approximate lipid concentrations in the whole TM were calculated using the mean lipid

concentration observed in the LIM NS lobsters. Late inter-moult lobsters were chosen

as they best represent the "typical or normal" state as lobsters spend the majority of their lives in the inter-moult stage. However, even with its smaller size the high lipid content of the HP will contribute ~3.63 g of lipid (~5x more than the TM) in the smaller

123 LIM lobsters. This further supports the fact that the HP is the primary site of lipid storage despite its size in comparison to the TM.

The PCM and CCM showed a significant increase in Lin during the LPM with values dropping back to the original concentrations in the PoM. This suggests that the role of lipid reserves in the claw muscles differ from that of the HP and TM. The increased stores could represent a buildup of energy reserves to be utilised by the claws when foraging begins again in the PoM.

During the LPM category feeding slows and eventually stops (Dall, 1990), suggesting that the significant increase in lipid content of the PCM and CCM during this time are a reallocation of internal reserves. The conversion of lipid to glycogen for use in chitin synthesis has been suggested by Renaud (1949) as translated in Travis (1955).

Mechanisms necessary for fatty acid synthesis from carbohydrate in mammals, including glycolysis and the pentose phosphate pathway, have also been identified in crustaceans (Chang and O'Connor, 1983). Reduced nicotinamide adenine dinucleotide

(NADPH) is required for fatty acid synthesis and supplied mainly through the pentose phosphate pathway (Lehninger et al., 1993). Acetyl-CoA is another key component of fatty acid synthesis, which is typically formed by pyruvate oxidation during glycolysis

(Lehninger et al., 1993). Thus, lipids in the claw muscles may be synthesised from chitin derived metabolites, similar to what was suggested for the glycogen reserves.

The lipid being stored during the LPM could also be indicative of the conservation of structural lipids from the shell for use in its re-synthesis following ecdysis. Small

124 quantities of lipids have been observed in the shell of the prawn P. japonicas

(Kanazawa et alv 1976). A more in depth analysis of the sequential mobilisation of lipid

stores is necessary to confirm the suggested functions.

In an attempt to conserve the most chitin for use following ecdysis, the lobster may

need to resort to a more efficient means of storage. The hydrophobic nature of lipids

makes them more compact than glycogen in the storage form, as glycogen molecules

are hydrated (Urich, 1994). This would also help the lobster during the moult by

allowing the maximum amount of energy stored without hindering its ability to exude

the claw muscle through the knuckle at ecdysis.

The effects of variable environmental conditions must be kept in mind when examining

the fluctuations in lipid stores as the present data were collected in different seasons

and years. Variations in lipid reserves in response to different temperatures were

observed in the , Paralithodes camtschaticus (Stoner et al., 2010). Depth

affects the lipid concentrations in the lobster N. norvegicus (Rosa and Nunes, 2003a).

Food availability will also play an fundamental role in lipid reserves as observed in

many crustaceans: E. sinensis (Wen et al., 2006), P. esculentus (Barclay et al., 1983), P. japonicas (Cuzon et al., 1980), P. semisulcatus, P. esculentus, P. monodon (Moore et al.,

2000) and L vannamei (Sanchez-Paz et al., 2007)

While the PoM decrease in Lin was found in both the 2009 and 2010 groups, the significant changes observed in the PCM during the LPM category could be due to a

125 year or cohort effect and not based on any particular physiological alterations in the lobsters.

The HP has been identified as the primary site of lipid storage with other tissues making a smaller, yet significant, contribution to the overall supply. Lipid reserves play an integral role in the moult cycle of the lobster. They appear to rely heavily on HP stores to supply the energy necessary to cope with the extended periods of inanition.

Lipids could potentially be acting as a more efficient means of metabolite conservation and storage for chitin synthesis in the claws. Closer examination of the mechanism of chitin synthesis, degradation and the sequential utilisation of metabolites is necessary to determine the role of lipids in chitin synthesis following the moult. A closer look at the specific year and cohort affects on physiology will also aid in confirming the role of lipid stores during the moult.

126 5.6. References

Ando, T., Kanazawa, A., Teshima, S., 1977. Variation in lipids of tissues during the molting cycle of prawn. Bull. Jap. Soc. Fish. Res. 43(12), 1445-1449.

Barclay, M.C., Dall, W., Smith, D.M., 1983. Changes in lipid and protein during starvation and the moulting cycle in the tiger prawn, Penaeus esculentus Haswell. J. Exp. Mar. Biol. Ecol. 68(3), 229-244.

Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37(8), 911-917.

Chang, E.S., O'Connor, J.D., 1983. Metabolism and Transport of Carbohydrates and Lipids, in: Bliss, Dorothy H. and Mantel, Linda H. (Ed.), The Biology of Crustacea Vol. 5: Physiological regulation. Academic Press, Inc., pp. 263-287.

Chapelle, S., 1977. Lipid composition of tissues of marine crustaceans. Biochem. Syst. Ecol. 5, 241-248.

Christie, W.W., 1973. The Isolation of Lipids from Tissues, in: Anonymous , Lipid Analysis, isolation, separation, identification and structural analysis of lipids. Pergamon Press, Headington Hill Hall, Oxford, pp. 30-41.

Cuzon, G., Cahu, C, Aldrin, J.F., Messager, J.L., Stephan, G., Mevel, M., 1980. Starvation effect on metabolism of Penaeus japonicus. Proc. World Maricult. Soc. 11, 410- 423.

Dall, W., 1981. Lipid absorption and utilization in the Norwegian lobster, Nephrops norvegicus (L.). J. Exp. Mar. Biol. Ecol. 50, 33-45.

Dall, W., 1990. Moulting and Growth, in: Russell, F.S. (Ed.), Advances in Marine Biology. London, New York, Academic Press, pp. 213-250.

Folch, J., Lees, M., Stanley, S.G.H., 1957. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226(1), 497-509.

Gurr, M.I., Harwood, J.L., Frayn, K.N., 2002. Lipid Biochemistry, 5th ed. Blackwell Science Ltd., Maiden, MA, USA.

Jeckel, W.H., Aizpun de Moreno, Julia Elena, Moreno, V.J., 1990. Changes in biochemical composition and lipids of the digestive gland in females of the shrimp Pleoticus muelleri during the moult cycle. Comp. Biochem. Physiol. B 96(3), 521-525.

127 Jeffs, A.G., Wilmott, M.E., Wells, R.M.G., 1999. The use of energy stores in puerulus of the spiny lobster Jasus edwardsii across the continental shelf of New Zealand. Comp. Biochem. Physiol. A 123, 351-357.

Kanazawa, A., Teshima, S., Sakamoto, Y., Guary, J.B., 1976. The variation of lipids and cholesterol contents in the tissues of prawn, Penaeusjaponicus, during the molting cycle. Bull. Jap. Soc. Sci. Fish. 42(9), 1003-1007.

Lehninger, A.L., Nelson, D.L., Cox, M.M., 1993. Lipid Biosynthesis, in: Anonymous , Principles of Biochemistry, 2nd ed. Worth Publishers, Inc., New York, NY, pp. 642-687.

Moore, L.E., Smith, D.M., Loneragan, N.R., 2000. Blood refractive index and whole- body lipid content as indicators of nutritional condition for penaeid prawns (Decapoda: Penaeidae). J. Exp. Mar. Biol. Ecol. 244(1), 131-143.

O'Connor, J.D., Gilbert, L.I., 1968. Aspects of lipid metabolism in crustaceans. Am. Zool. 8(3), 529-539.

Renaud, L, 1949. Le cycle des reserves organiques chez les Crustaces Decapodes. Ann. inst. Oceanog. (Paris) [N. S.] 24, 259-357.

Rosa, R., Nunes, M.L., 2003. Biochemical composition of deep-sea decapod crustaceans with two different benthic life strategies off the Portuguese south coast. Deep- Sea Res. Pt. 1 50,119-130.

Sanchez-Paz, A., Garcfa-Carreno, F., Hernandez-Lopez, J., Muhlia-Almazan, A., Yepiz- Plascencia, G., 2007. Effect of short-term starvation on hepatopancreas and plasma energy reserves of the Pacific white shrimp (Litopenaeus vannamei). J. Exp. Mar. Biol. Ecol. 340(2), 184-193.

Schirf, V.R., Turner, P., Selby, L., Hannapel, C, DeLa Cruz, P., Dehn, P.F., 1987. Nutritional status and energy metabolism of crayfish (Procambarus clarkii, Girard) muscle and hepatopancreas. Comp. Biochem. Physiol. A 88(3), 383-386.

Stoner, A.W., Ottmar, M.L., Copeman, L.A., 2010. Temperature effects on the molting, growth, and lipid composition of newly-settled red king crab. J. Exp. Mar. Biol. Ecol. 393,138-147.

Stuck, K.C., Watts, S.A., Wang, S.Y., 1996. Biochemical Responses during starvation and subsequent recovery in postlarval Pacific white shrimp, Penaeus vannamei. Mar. Biol. 125, 33-45.

128 Suneetha, Y., Sreenivasula Reddy, P., Jyothi, N., Srinivasulu Reddy, M., 2009. Studies on the analysis of proximal changes during molting process in the Penaeid prawn, Penaeus monodon. World J. Zool. 4(4), 286-290.

Travis, D.F., 1955. The moulting cycle of the spiny lobster, Panulirus argus Latreille. II. Pre-ecdysial histological and histochemical changes in the hepatopancreas and integumental tissues. Biol. Bull. Vol. 108(No. 1), 88-112.

Urich, K., 1994. Lipids, in: Anonymous, Comparative Animal Biochemistry. Springer- Verlag, New York, NY, pp. 562-623.

Wen, X., Chen, L., Ku, Y., Zhou, K., 2006. Effect of feeding and lack of food on the growth, gross biochemical and fatty acid composition of juvenile crab, Eriocheir sinensis. Aquaculture 252, 598-607.

129 6. Biochemical Analysis of Hemolymph Metabolites during the Moult Cycle in the American Lobster, Homarus americanus

6.1. Abstract

Hemolymph plasma samples were obtained from recently caught, wild lobsters for biochemical analysis. Concentrations of triglyceride (TG), cholesterol (CHOL), total protein (TP), glucose (GLUC), uric acid (UA) and hemolymph osmolality (OSM) were measured and evaluated in relation to the moult cycle. Examination of the dataset revealed significant changes among moult categories for all parameters measured. The

TP and GLUC concentrations increased as the lobsters approached the moult followed by a pronounced decrease in the post-moult (PoM). Hemolymph OSM showed a gradual decrease from the late inter-moult (LIM) through to the PoM. The only significant change observed in the UA concentrations was a significant decrease in the late pre-moult (LPM). Hemolymph TG and CHOL concentrations demonstrated significant changes during the moult, but the changes were highly associated with gender. Many factors influence hemolymph biochemistry and not all of them can be regulated when examining wild populations. A year effect on the data was observed in the hemolymph OSM and TP concentrations in the LPM male lobsters. The trends observed in hemolymph metabolite concentrations are closely associated with changing water content. The data also alluded to the potential mobilisation of metabolic reserves and shell constituents. Variations in physiology between years and cohorts due to changing environmental factors as well as sex may also influence hemolymph biochemistry.

130 6.2. Introduction

Examination of blood chemistry plays an integral role in human health assessment

(Marks, 1978). Alterations in specific blood constituents can be indicative of various changes in internal physiology and are very useful for the diagnosis of physiological well-being (Dufour, 2003). These techniques rely heavily on a general understanding of

"normal" or natural blood composition to identify abnormalities, which correspond to changes in condition (Farver, 1997).

The cyclical patterns of incremental growth and reproduction in crustaceans cause pronounced annual changes in metabolic physiology to occur (Passano, 1960;

McWhinnie et al., 1972). This complicates matters of nutritional assessment as reference or baseline values must be obtained for the various life history stages. In vertebrates, changes in physiology and nutrition are generally coupled with external alterations in physical appearance (i.e. muscle mass and fat deposits). Physical signs in crustaceans are masked by the hard calcified exoskeleton (Cockcroft, 1997).

Shedding of the hard shell and the formation of a new one occurs regularly with the moult and results in pronounced changes in physiology, metabolism and tissue structure. Even though these dramatic changes occur, very few external manifestations are observed (Chang, 1995). The lack of physical manifestations associated with changing physiological and metabolic state warrants the development of effective non-lethal methods for the assessment of nutritional physiology and health in crustaceans.

131 The hemolymph constitutes the largest tissue in crustaceans and is readily accessible

by non-lethal means (Noga, 2000). This offers the unique opportunity to optimise

methods of blood analysis, currently used on vertebrates, for use on crustacean

hemolymph. Advances in this field would allow for the development of more efficient

methods of nutrition and health assessment in crustaceans.

Marked alterations in the biochemical composition of the hemolymph and pronounced

changes in tissue composition (See Chapters 4 and 5) have been documented during

the moult cycle (Chang, 1995; Mercaldo-Allen, 1991). The rigorous metabolic

requirements of the moult cycle result in pronounced changes in hemolymph

metabolite composition, which may include carbohydrates, lipids and proteins. Moult-

induced changes in hemolymph glucose (GLUC) concentrations were observed in the

isopod Ligia exotica (Parvathy, 1971), the shrimp L. vannamei (Galindo et al., 2009),

and the lobster H. americanus (Mercaldo-Allen, 1991; Telford, 1968b). Fluctuations in

hemolymph lipid concentrations, such as triglycerides and cholesterol, have been

noted in the Southern rock lobster Josus edwardsii (Musgrove and Babidge, 2003) and the crab Carcinus maenas (Spindler-Barth, 1976) during the moult. Total hemolymph

protein (discussed in Chapter 2) also demonstrates significant changes throughout the

moult (Mercaldo-Allen, 1991; Glynn, 1968).

While methods and reference values for the evaluation of biochemical composition of the blood are well documented for many vertebrate species, the information available for crustaceans is limited and inconsistent. Furthermore, the wide range of aquatic

132 environments that crustaceans inhabit makes comparisons difficult among species

(Noga, 2000). Biochemical composition can also be highly influenced by environmental conditions including salinity, temperature, food availability and season (Noga, 2000;

Dove et al., 2005). This makes comparison among organisms even within a species problematic, as they can demonstrate a wide range of "normal" or reference values

(Noga, 2000).

The variability of biochemistry observed in crustaceans demonstrates the importance of generating "normal" or reference values for individual species, life stages and populations. In addition to the natural fluctuations in biochemistry discussed, stress can also play a significant role in the biochemical composition of the hemolymph (See

Chapter 2). Acute stress is generally associated with changes in GLUC concentrations

(Noga, 2000). A better understanding of the natural changes in hemolymph biochemistry and the variables affecting it could allow for hemolymph metabolite concentrations to act as potential indicators of nutritional state, which could also be indicative of overall health. Such metabolites may include GLUC, TP, UA, TG and CHOL.

Glucose is considered to be the main circulating carbohydrate in crustacean hemolymph (Sanchez-Paz et al., 2007). It plays an important role in lobster physiology as suggested by the significant fluctuations observed during the active phases of the moult cycle (Mercaldo-Allen, 1991; Galindo et al., 2009). Circulating GLUC is also an important remedial factor in coping with stressors such as emersion and hypoxia

(Paterson et al., 1997; Harris and Andrews, 2005; Lorenzon et al., 2007).

133 Circulating proteins in the hemolymph consist primarily of hemocyanin. Its primary function in crustaceans is as a copper-based respiratory protein (Goodwin, 1960).

Other circulating proteins can include moult specific proteins and female specific proteins (Ferrero et al., 1983). The various hemolymph proteins have been suggested to be involved in the synthesis of the new shell following ecdysis and the generation of energy (Uglow, 1969; Oliver and MacDiarmid, 2001).

Uric acid (UA) is another hemolymph metabolite often measured in human clinical chemistry (First, 2003). Uric acid is a waste product of purine degradation and catabolism. Purines are heterocyclic nitrogen compounds containing nucleotides that are integral to RNA and DNA synthesis (Urich, 1994). The measurement of UA in the hemolymph is important for the assessment of DNA or protein turnover and high concentrations can be indicative of renal failure in vertebrates (First, 2003).

Lipid concentrations in the hemolymph have been examined as a means of assessing health and growth in crustaceans (Floreto et al., 2000; Musgrove and Babidge, 2003).

Triglycerides (TG) and cholesterol (CHOL) represent two circulating lipid classes in crustacean hemolymph (Kanazawa, 2001; Musgrove and Babidge, 2003). Triglycerides are typically considered to be the primary metabolic reserve (Gurr et al., 2002) and are integral for the generation of energy. In contrast, cholesterol plays more of a structural role as a major component of cellular membranes (Gurr et al., 2002). It is also used for the synthesis of steroids or hormones (Kanazawa and Koshio, 1993),

134 which control many aspects of crustacean physiology especially during the moult cycle

(Chang etal., 1993).

Variations in hemolymph biochemistry have also received much attention on account of their potential role in altering the osmotic characteristics of the hemolymph (Travis,

1955; Pratoomchat et alv 2002). Changes in osmotic pressure have been implicated as an important aspect of water absorption during the moult (Galindo et al., 2009).

However, active regulation of osmotic pressure and its role in moulting varies among species. Water absorption in H. americanus occurs via a localised osmotic gradient across the gut into the hemolymph (Mykles, 1980) suggesting that changes in hemolymph osmolality may not be the driving force behind water absorption during the moult cycle (See Chapter 3).

The following chapter addresses changes in the composition of various metabolites in the hemolymph of lobsters representing different moult categories. These data will aid in the understanding of natural fluctuations in hemolymph biochemical composition and help to identify relationships between the various hemolymph constituents and the changes in physiology that occur during the moult cycle. Understanding fluctuations in hemolymph metabolite composition will also aid in the understanding of moult induced metabolic requirements. Osmolality was measured to better understand its relationship to water uptake during the moult cycle.

135 6.3. Materials and Methods

6.3.1. Biochemical Analysis of Hemolymph Metabolites and Osmolality During the Moult

Hemolymph plasma samples (n=88) were collected as per Section 2.3.2. Samples were stored at 4°C until brought to the AVC Diagnostic Services Laboratory for biochemical analysis within 24 hours of collection. The Cobas c501 biochemistry analyzer (Roche

Diagnostics Corporation, Indianapolis, IN) was used to measure TP, GLUC, TG, CHOL, and UA in the plasma samples. The TG, CHOL, UA and GLUC concentrations were determined by colourimetric analysis following enzymatic reactions with lipoprotein lipase, cholesterol esterase, uricase, and hexokinase, respectively (TRIGL, CHOL2, UA2, and GLUC3, Roche Diagnostics Corporation). The TP concentrations were determined colourimetrically through the generation a Cu-protein complex in an alkaline solution

(TP2, Roche Diagnostics Corporation). Measured osmolality (OSM) for each sample was determined by freezing point depression (Micro Osmette, Precision systems Inc.,

Natick, MA).

6.3.2. Statistical Analysis of Hemolymph Biochemistry and Osmolality Data

Kruskal-Wallis and Mann-Whitney non-parametric analyses were carried out on each parameter to identify significant changes between sex, moult category and year.

Pearson correlations and regression statistics were performed to identify significant correlations between the various plasma constituents. Statistical analyses were carried out using Minitab® statistical software version 15.1, 2006.

136 6.4. Results

6.4.1. Evaluation of Hemolymph Biochemical Parameters for the Primary 2009 Moult Cohort

Evaluation of the primary 2009 moult cohort revealed trends in hemolymph metabolite

concentrations and osmolality that mimicked those observed when both cohorts and

years were examined. Thus, these data are not shown and interpretation of

hemolymph biochemical composition will be carried out on the full dataset.

6.4.2. Evaluation of Hemolymph Biochemical Parameters for the 2009 and 2010 Moult Cohorts

Significant differences between sexes were observed in two (CHOL and TG) of the five

hemolymph parameters measured. Therefore, analyses were carried out on the

combined sexes for all parameters where significant differences between sexes were

not present (GLUC, TP, UA and OSM). The data are presented as median, mean ±

standard deviation.

A gradual increase in the TP concentrations was observed in the LIM (56.00, 57.85 ±

18.60 g/L), EPM (70.50, 69.22 ± 14.22 g/L) through the MPM (85.00, 83.33 ± 12.18 g/L).

The LPM (87.00, 87.18 ± 14.14 g/L) was significantly different from the LIM and EPM

but not the MPM. The PoM showed a significant decrease to 23.00, 23.89 ± 11.02 g/L

(Figure 6.1).

The mean UA concentrations were significantly lower in the LPM (46.0, 65.0 ± 57.3

u.mol/L) than in the LIM (86.0, 9.5 ± 49.1 u.mol/L), MPM (71.0, 84.2 ± 46.4 u.mol/L) and

137 PoM (77.0, 96.6 ± 61.2 u.mol/L) categories. The EPM (65.5, 84.2 ± 46.4 u.mol/L) was not statistically different from any of the other moult categories (Figure 6.2).

120 a c n=20 c n=ll n=18 100- b n=18 _1 o; 80 c '53 2 60 & T5 o H 40- d n=19 _L 20 T

LJM EPM MPM LPM PoM Moult Category

Figure 6.1: Variations in hemolymph total protein concentrations during the moult for all lobsters from 2009 and 2010. The mean total protein concentration in the hemolymph of lobsters in the different moult categories: late inter-moult (LIM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM). Different letters designate significant differences at p < 0.05 (Mann-Whitney). The asterisk represents an outlier.

138 250- a a =20 ab * n=] n=18 200- o E o i. 150- M E a 3 n=18 u 100- I b < ~H n=H .U i» 3 50-

0- LIM i?M MPM LPM PoM Moult Category

Figure 6.2: Variations in hemolymph uric acid concentrations during the moult for all lobsters from 2009 and 2010. The mean uric acid concentration in the hemolymph of lobsters in the different moult categories: late inter-moult (LIM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM). Different letters designate significant differences at p < 0.05 (Mann-Whitney). Asterisks indicate outliers in the data set.

Significant differences were observed in TG concentrations between sexes. In males there was an increase in the mean TG concentration from the EPM (0.24, 0.23 ± 0.08 mmol/L) to the MPM (0.30, 0.32 ± 0.07 mmol/L) and from the LIM (0.24, 0.24 ± 0.11 mmol/L) and EPM to the LPM (0.34, 0.35 ± 0.07 mmol/L) (Figure 6.3). The mean PoM

TG concentration (0.08, 0.08 ± 0.02 mmol/L) was significantly lower than all other moult categories. In the females, TG concentrations were always significantly higher than the males but showed no significant changes among the moult categories (Figure

6.3).

Hemolymph CHOL concentrations also demonstrated significant differences between the sexes. In males, there was a significant increase from the EPM (0.44, 0.46 ± 0.17

139 mmol/L) to the MPM (0.57, 0.63 ± 0.16 mmol/L). There was also a significant increase in the LPM (0.68, 0.74 ± 0.26 mmol/L) from the LIM (0.47, 0.50 ± 0.25 mmol/L) and

EPM categories (Figure 6.4). Concentrations in the PoM (0.19, 0.19 ± 0.04 mmol/L) were significantly lower than all other moult categories. In females, the changes in

CHOL concentrations between moult categories were less pronounced with a significant decrease observed only in the PoM (0.38, 0.44 ± 0.25 mmol/L) with respect to the EPM (0.66, 0.70 ± 0.17 mmol/L) and MPM (0.80, 0.73 ± 0.18 mmol/L) categories.

The LIM (0.56, 0.59 ± 0.20 mmol/L) was not significantly different from any of the other moult categories (Figure 6.4). The significant difference between sexes was only observed during the EPM and PoM with higher concentrations in the females.

A gradual increase in the GLUC concentrations in the LIM, EPM and MPM (1.10, 1.09 ±

0.30; 1.35, 1.30 ± 0.31 and 1.60, 1.74 ± 0.47 mmol/L, respectively) categories was observed. The LPM (2.00, 1.96 ± 0.24 mmol/L) GLUC concentration was not statistically different from the MPM (Figure 6.5). A significant decrease from all other moult categories was observed in the PoM (0.80, 0.85 ± 0.22 mmol/L).

Hemolymph OSM showed significant decreases from the LIM (927.50, 926.70 ± 30.06 mOsm/kg) and EPM (927.50, 921.00 ± 24.04 mOsm/kg) to the MPM (910.00, 901.78 ±

31.57 mOsm/kg) and LPM (885.00, 875.27 ± 20.67 mOsm/kg) with another significant decrease in the PoM (861.00, 860.26 ± 22.22 mOsm/kg) category (Figure 6.6).

140 LIM EPM MPM LPM PoM Female Male 1.4 n=10

1.2

1.0 E E 0.8

8 0.6 ab be n=10

n=i0 \n n=10 .SP n=10 i

fZ 0.4- & d n=10 0.2 k 0.0- LIM EPM MPM LPM PoM Moult Category

Figure 6.3: Variations in hemolymph triglyceride concentrations during the moult for all lobsters from 2009 and 2010. The mean triglyceride concentration in the hemolymph of lobsters in the different moult categories: late inter-moult (LIM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM). Different letters designate significant differences at p < 0.05 (Mann-Whitney) between moult categories. Diagonal bars indicate significant differences between sexes. Asterisks represent outliers.

LIM EPM MPM LPM PoM I 1_ Female Male

c 1.25 n=10

c ab ac n=9 n=10 IS- 1.00 n=10 a be o n=8 n=10 E n=10 E n=l I I" 0.75- X Vk v O 0.50- d T n=10 0.25 T I

0.00 LIM EPM MPM LPM PoM Moult Category

Figure 6.4: Variations in hemolymph cholesterol concentrations during the moult for all lobsters from 2009 and 2010. The mean cholesterol concentration in the hemolymph of lobsters in the different moult categories: late inter-moult (LIM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM). Different letters designate significant differences at p < 0.05 (Mann-Whitney) between moult categories. Diagonal bars indicate significant differences between sexes. The asterisk represents outlier that was included in analysis.

141 3.0 c n=18

2.5 • n-11 a b | 2.0 n=20 n=18 E, u d n=19 3 19

1.0-

0.5- QM EPM MPM LPM PoM Moult Category

Figure 6.5: Variations in hemolymph glucose concentrations during the moult in all lobsters from 2009 and 2010. The mean glucose concentration in the hemolymph of lobsters in the different moult categories: late inter-moult (LIM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM). Different letters designate significant differences at p < 0.05 (Mann-Whitney). The PoM outliers (*) were excluded from statistical analyses.

a 980 n=20 b 960 a n=18 940 n=18 2 d I 920 n=19 o c n=ll 900 £ •g 880 E V) O 860-

840-

820- UM EPM MPM LPM PoM Moult Category

Figure 6.6: Variations in hemolymph osmolality during the moult for all lobster from 2009 and 2010. The mean hemolymph osmolality of lobsters in the different moult categories: late inter-moult (LIM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM). Different letters designate significant differences at p < 0.05 (Mann- Whitney). Asterisks indicate outliers in the dataset that were included in analyses.

142 6.4.3. Evaluation of Year Effects on Hemolymph Biochemical Parameters

Significant differences in OSM and TP concentrations between years were observed in

males during the LPM (Figures 6.7 and 6.8). Lobsters representing both years were

only collected for the MPM and LPM categories (See Chapter 2). No significant

differences were observed between years in any other hemolymph analytes.

110- 8

100- • 0 • • _l o A° "-** 90- 0 • 9 • c • M • "5 80- • 4-2> 0 Q. • o o ra 70- • o o 1 H 60-

• 50- Year 2009 2010 2009 2010 2009 2010 2009 2010 Sex Male Female Male Female Category MPM LPM

Figure 6.7: Comparison of hemolymph total protein concentrations between collection years. Total protein concentrations in the hemolymph of lobsters representing the mid pre-moult (MPM) and late pre-moult (LPM) from 2009 (•) and 2010 (o) separated by sex. Each dot represents one lobster. "A" designates significant differences between the years at p < 0.05 (Mann-Whitney).

143 950- •

• 925- 1 0 Jt t (A A oE 900- 1 0

Year 2009 2010 2009 2010 2009 2010 2009 2010 Sex Male Female Male Female Category MPM LPM

Figure 6.8: Comparison of measured hemolymph osmolality between collection years. The osmolality of lobsters representing the mid pre-moult (MPM) and late pre-moult (LPM) from 2009 (•) and 2010 (o) separated sex. "A" designates significant differences between years at p <0.05 (Mann-Whitney).

6.4.4. Correlations between the Hemolymph Biochemical Parameters

The OSM in males showed significant correlations with TG and TP concentrations and

OSM in females only correlated with TP concentrations. Good correlations were observed between GLUC and all other parameters except UA in males, while GLUC in the females only showed significant correlations with TP and UA. The CHOL concentrations in the hemolymph correlated well with TG and TP concentrations in both males and females but the correlation coefficients were higher in the males.

Hemolymph TG concentrations also showed very significant correlations with TP in males. Few correlations were observed between the UA concentrations and the other

144 parameters measured. In females there were marginally significant correlations between UA concentrations and GLUC and TG. Table 6.1 summarises these findings.

Table 6.1: Pearson correlations between measured hemolymph biochemical parameters. Pearson regression analysis of hemolymph biochemistry parameters for male and female lobsters. The bottom boxes (in italics) are p values and the top boxes are R2 values. Bolded numbers represent significance at p < 0.05 and R2 values greater than 0.5.

OS M1 GLUC2 CHOL3 TG4 TP5 Male Female Male Female Male Female Male Female Male Female

GLUC -0.076 -t',i / O n.598 0 3G5 CHOL 0.223 0 091 0.562 0 270 0.120 0 596 0.000 0 112 TG 0 302 0 081 0.648 0 072 0.935 0.773 0.033 "'' 0.640 6.000~ 0 677 6.000 6.000 TP 0 419 0 421 0.669 0.600 0.795 , 0.567 0.909 | C 016 0.002 0.010 o.ooo 0.000 0.000 6.000 O.OOO] 0 92* 6 UA -0 106 [ 0 117 -3 042 0 334 is J. -j „ «• J^ i •0 159 0,144 0.465 I 0 495 3 770 0.047 0 1>5 0.179 0 364 0.031 0.26S 1 0 506 osmolality, glucose, cholesterol, triglycerides, total protein and uric acid

6.5. Discussion

This study examined changes in various hemolymph metabolic parameters including

TP, UA, TG, CHOL and GLUC to gauge the natural fluctuations occurring during the moult. Hemolymph OSM was examined to assess its potential association with water absorption. It has also been suggested that hemolymph metabolites (Such as protein and carbohydrates) may be partially responsible for changes in osmolality during the moult cycle (Pratoomchat et al., 2002).

Increased hemolymph TP concentrations during the pre-moult, including the LIM through LPM categories, have been observed in many crustaceans (See Chapter 2).

This increase could be due to an increase in protein synthesis, which has been

145 observed in crayfish during the pre-moult in response to increasing concentrations of

the moult hormone ecdysone (McWhinnie et alv 1972; Gorell and Gilbert, 1971). The removal of the chitin-protein complexes from the old shell has also been suggested as a cause of the increased protein concentrations (Passano, 1960). Thus, the changes seen in this study in hemolymph TP potentially represent the physiological response of the lobster to the moult cycle. Water loss in the lobster as a means of aiding in exuviation of the shell has also been implicated as a cause of the pre-moult increase in protein concentrations (Lavallee, 2006). The PoM decrease in TP likely reflects dilution of hemolymph with water after shell expansion. However, it could also be due to increased extraction of protein complexes from the hemolymph for formation of the new shell (Passano, 1960).

Uric acid is an end product of purine degradation (Urich, 1994), thus its presence in the hemolymph represents protein catabolism and degradation of DNA. The mean concentrations remain relatively stable over the moult cycle in this study, with the only changes occurring during the LPM. This suggests a baseline protein and DNA turnover in the lobsters with a disruption in the LPM category. The LPM decrease could result from the onset of the inanition period so there is a brief decrease in purine turnover from dietary sources (Battison, unpublished data). The slight increase back to the approximate baseline values in the PoM could represent compensation in protein turnover from internal sources or the end of the starvation period, which would occur in the early to mid PoM.

146 Increased water uptake is expected to cause a dilution effect of the UA concentrations during the PoM, as observed in most other hemolymph parameters. An increase in protein and/or DNA catabolism and increased UA production during the PoM category may be masking the expected dilution. An increase in protein catabolism during the

PoM would implicate protein as a potentially significant reserve utilised during the moult or that feeding has resumed. A more in-depth analysis of the mechanisms of UA generation and disposal in the lobster is necessary to fully explain the observed trends to elucidate the role of protein during the moult.

In the GLUC data there were two outliers with high glucose concentrations identified in two of the PoM lobsters collected in July of 2009. An increase in reserve mobilisation and metabolism is one explanation for the increased GLUC concentrations observed.

Warmer water temperatures in the summer months may induce an increase in metabolic rate, as metabolism is highly associated with temperature in poikilothermic organisms (Florkin, 1960; Dall, 1990). However, other lobsters caught at the same time did not show similarly high GLUC concentrations. Stress is another factor that could have caused hemolymph GLUC concentrations to increase. Increased mobilisation of

GLUC in the hemolymph has previously been noted during stress in crustaceans

(Paterson et al., 1997; Harris and Andrews, 2005; Lorenzon et al., 2007). These two lobsters could have unknowingly been sampled after extensive emersion or other stress. The data associated with them was eliminated from statistical analysis of the glucose data.

147 An increase in the hemolymph carbohydrate concentrations during the pre-moult of

the mud crab S. serrata was suggested to be due to dissolution of carbohydrates (i.e.,

acetylated glucose from chitin) from the cuticle into the hemolymph (Pratoomchat et

al., 2002). The mobilisation of metabolites from the old shell, as a means of

conservation for use in the reformation of the new shell, would explain the gradual

increase in hemolymph GLUC concentrations observed prior to ecdysis.

In the TG and CHOL data, gender played a significant role in hemolymph

concentrations, with higher concentrations in the females. Females also seemed to be

less susceptible to moult induced changes in these two analytes, which was reflected in

the lack of discernible trends during the moult cycle. In males there was a gradual

increase in hemolymph TG and CHOL concentrations observed through to the LPM

followed by a significant depletion in the PoM. This increase could be indicative of

increased mobilisation of lipid stores as an energy source during the impending moult.

It could also represent the mobilisation of chitin-bound lipids, which are present in

some crustaceans at low concentrations (Kanazawa et al., 1976). The less pronounced

trends observed in the lipid concentrations in females potentially represents the

differential effects of reproductive physiology between the sexes. The high TG

concentrations in females have been suggested to reflect ovary maturation (Castille

and Lawrence, 1989). Thus, female lobsters with high TG concentrations in the LIM

could be preparing to spawn (Battison et al., 2011). The more complex physiological state in females appear to hide moult related changes in physiology with respect to

148 lipid concentrations, as lipids are very important during the reproductive cycle in crustaceans (Ravid et al., 1999).

A significant decrease was observed in hemolymph OSM during the MPM and LPM creating a gradual decrease from the LIM through to the PoM. These data suggest that changing OSM is not the primary mechanism employed to generate a net influx of water at ecdysis, as OSM would have to increase to be the driving force behind the water influx. It is also interesting to note that as the OSM decreased there was a general increase in the other hemolymph metabolites measured. The increasing metabolite concentrations in the pre-moult should cause an increase in osmotic pressure unless the relative concentrations are small in comparison to other osmolytes. This suggests that active regulation of hemolymph osmolytes may control hemolymph composition and allow for alterations in specific constituents in response to the physiologically demanding life history stages as discussed in Chapter 3.

However, the role of metabolites in regulating osmotic pressure will depend on their overall concentrations in comparison to other circulating osmolytes, which warrants further evaluation. Active absorption of water through other mechanisms (Such as ingestion) may also contribute to the observed decrease in OSM.

Some of the changes in hemolymph biochemical composition measured in this study were affected by year. The two outliers in the LIM and EPM OSM data represent two of the 2010 lobsters. These changes most likely reflect environmental variations in salinity over time, as the lobsters are osmoconformers.

149 The effect of different moult cohorts on hemolymph biochemical parameters is unclear. However, the different cohorts essentially represent different seasons, which have been shown to influence biochemical composition (Dove et al., 2005). Therefore, the changes observed in the full dataset could potentially be due to these confounding factors; this warrants further evaluation. Biochemical analysis of lobsters from different cohorts and years with larger sample size will help to identify if there is a temporal effect on the data.

The significant positive correlations observed between hemolymph TP, TG, CHOL and

GLUC concentrations, which have been observed in other studies (Battison, unpublished data), likely reflects their related roles in metabolism. These correlations were more prominent in males, alluding to the differences in reproductive physiology between males and females. The significant correlations observed between hemolymph TP concentrations and OSM suggest that TP may play a role in the regulation of osmotic pressure as suggested in the mud crab S. serrata (Pratoomchat et al., 2002). However, concentrations of TP in extracellular fluid are typically considered to have minimal affects on osmolality (George, 1994), which could be reflected in the low R2 values.

Hemolymph TP and GLUC showed the most pronounced moult specific trends. These variations could be related to changes in feeding activity, the mobilisation of metabolic reserves, the conservation of shell constituents or a combination of these. Alterations in body water content will also play a role in hemolymph metabolite concentrations, as

150 demonstrated by the significant decreases in most hemolymph metabolites following expansion at ecdysis. The increase in hemolymph lipid concentrations during the pre- moult period could be indicative of a mobilisation of stored lipid as an energy source during the starvation period. A closer look at the year, cohort and sex variability in hemolymph biochemistry is warranted to elucidate actual changes in biochemical composition associated with the moult cycle. Correlations between tissue metabolic reserves and hemolymph biochemical composition will help to understand the changes occurring during the moult cycle as well as help identify lobsters in different stages

(See Chapter 7).

151 6.6. References

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Castille, F.L., Lawrence, A.L., 1989. Relationship between maturation and biochemical composition of the gonads and digestive glands of the shrimps Penaeus aztecus Ives and Penaeus setiferus (L). J. Crustacean Biol. 9(2), 202-211.

Chang, E.S., 1995. Physiological and biochemical changes during the molt cycle in decapod crustaceans: an overview. J. Exp. Mar. Biol. Ecol. 193, 1-14.

Chang, E.S., Bruce, M.J., andTamone, S.L., 1993. Regulation of crustacean molting: A multi-hormonal system. Am. Zool. 33(3), 324-329.

Cockcroft, A.C., 1997. Biochemical composition as a growth predictor in male west- coast rock lobster (). Mar. Freshw. Res. 48, 845-856.

Dall, W., 1990. Moulting and Growth, in: Russell, F.S. (Ed.), Advances in Marine Biology. London, New York, Academic Press, pp. 213-250.

Dove, A.D.M., Sokolowski, M.S., Bartlett, S.L., Bowser, P.R., 2005. Spatio-temporal variation in serum chemistry of the lobster, Homarus americanus Milne-Edwards. J. Fish Dis. 28, 663-675.

Dufour, R.D., 2003. Sources and Control of Preanalytical Variation, in: Kaplan, L.A., Pesce, Amadeo J. and Kazmierczak, Steven C. (Eds.), Clinical Chemistry: Theory, Analysis, Correlation, 4th ed. Mosby, St. Louis, Missouri, pp. 64-82.

Farver, T.B., 1997. Concepts of Normality in Clinical Biochemistry, in: Kaneko, J.J., Harvey, J.W., and Bruss, M.L. (Eds.), Clinical Biochemistry of Domestic Animals, 5th ed. Academic Press, Inc., San Diego, CA, pp. 1-19.

Ferrero, E.A., Graziosi, G., Marzari, R., Mosco, A., 1983. Protein pattern variability of the hemolymph of , L. (Crustacea, Stomatopoda). J. Exp. Zool. 225, 341-345.

First, M.R., 2003. Renal Function, in: Kaplan, L.A., Pesce, Amadeo J. and Kazmierczak, Steven C. (Eds.), Clinical Biochemistry: Theory, Analysis, Correlation, 4th ed. Mosby, St. Louis, Missouri, pp. 477-491.

152 Floreto, E., Prince, D.L., Brown, P.B., Bayer, R.C., 2000. The biochemical profiles of shell-diseased American lobsters, Homarus americanus Milne Edwards. Aquaculture 188, 247-262.

Florkin, M., 1960. Ecology and Metabolism, in: Waterman, T.H. (Ed.), The Physiology of Crustacea. Academic Press, Inc., New York, NY, pp. 395-410.

Galindo, C, Gaxiola, G., Cuzon, G., Chiappa-Carrara, X., 2009. Physiological and biochemical variations during the molt cycle in juvenile Litopenaeus vannamei under laboratory conditions. J. Crustacean Biol. 29(4), 544-549.

George, J.W., 1994. Water, Electrolytes, and Acid Base, in: Duncan, R.J., Prasse, K.W., Mahaffey, E.A. (Eds.), Veterinary Laboratory Medicine Clinical Pathology, 3rd ed. Iowa State University Press, Iowa, pp. 94-111.

Glynn, J.P., 1968. Studies on the ionic, protein and phosphate changes associated with the moult cycle of Homarus vulgaris. Comp. Biochem. Physiol. 26(3), 937-946.

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155 7. Correlations among the Changes in Physiology and Biochemistry of the Tissues and Hemolymph in the American Lobster, Homarus americanus, during the Moult Cycle

7.1. Introduction

In Chapters 3 through 6, fluctuations in various physiological and biochemical

components of lobster tissues and hemolymph were assessed in relation to the moult

cycle. The high metabolic requirement of the moult causes shifts in the metabolic

pathways exploited (McWhinnie et al., 1972), with proteins, lipids and carbohydrates

being the main metabolic resources. Water, glycogen and lipid concentrations in the

tissues, hemolymph metabolites and osmolality were measured. Fluctuations in these various parameters were assessed individually at different moult categories in an

attempt to elucidate their involvement in the moult cycle.

Previous studies have shown that metabolic requirements can vary greatly among species (Sanchez-Paz et al., 2006), warranting the assessment of metabolite functions

and importance for each species. Alterations in hemolymph metabolite composition

have also been correlated with changes in tissue reserves (Galindo et al., 2009),

marking them as potential non-lethal markers of internal changes in physiology. The relatively close relationship reported between tissue and hemolymph metabolites warrants comparative analysis of both. Enhanced knowledge of moult induced changes in metabolic physiology is integral to a complete understanding of nutrition and health.

156 In this chapter the correlations among tissue water, lipid and glycogen concentrations and circulating hemolymph metabolites will be examined with the various moult categories to further the understanding of the changes in metabolic physiology taking place during the lobster moult cycle. The evaluation of correlations between hemolymph metabolites and corresponding hepatopancreas (HP), pincher claw muscle

(PCM), crusher claw muscle (CCM) and tail muscle (TM) tissue reserves will also aid in the identification of potential biomarkers that can be used for non-lethal assessment of condition.

7.2. Materials and Methods

Water content was determined as described in Chapter 3. Tissue glycogen concentrations were measured with the methods discussed in Chapter 4. Lipid concentrations were evaluated as described in Chapter 5 and hemolymph biochemical analyses were carried out as reported in Chapter 6. Hemolymph protein concentrations were also measured with a Brix refractive index refractometer as described in Chapter 2. The glycogen and lipid concentrations are expressed as index values (Gin and Lin, respectively) calculated with the equations described in chapters 4 and 5. Pearson regression correlation statistics were carried out using Minitab® statistical software version 15.1, 2006.

157 7.3. Results

7.3.1. Relative Concentrations of Water, Lipid and Glycogen in the Hepatopancreas across the Five Moult Categories

The highest concentrations of lipid were observed in the HP with approximately 20% of the total HP being lipid in all moult categories. Glycogen constituted a very small portion of the HP with it being less than 1% in all moult categories except the LPM where it rose to a mean of ~1.4% of the total composition. The category entitled

"Other" made up approximately 17% of the HP in all moult categories (Figure 7.1).

100% 90% H 80% • •III c 70% o a Other '% 60% H Glycogen Lipid Water

LIM EPM MPM LPM PoM Moult Category

Figure 7.1: Relative composition of the hepatopancreas. The percent composition of water, lipid and glycogen in the hepatopancreas with all other tissue components represented in the "Other" category.

158 7.3.2. Relative Concentrations of Water, Lipid and Glycogen in the Muscle Tissues across the Five Moult Categories

The relative compositions of the three muscle tissues (PCM, CCM and TM) were similar.

All the tissues contained just below 80% water in the LIM, EPM, MPM and LPM categories with a slight increase to just above 80% in the PoM. Lipid and glycogen constituted less than 1% of all muscle tissues. Components of the "Other" category constituted about 20% of the muscle tissues during the LIM, EPM, MPM and LPM and showed a decrease to about 16-18% in the PoM (Figures 7.2, 7.3 and 7.4, respectively) as water content increased.

100% 90% 80% • •111 J 70% g 60% E 50% <•» 40% u 30% o. 20% 10% 0% LIM EPM MPM LPM PoM Moult Category

Figure 7.2: Relative composition of the pincher claw muscle. The percent composition of water, lipid and glycogen in the pincher claw muscle with all other tissue components represented in the "Other" category.

159 * Other • Glycogen Lipid • Water

L1M EPM MPM LPM PoM Moult Category

Figure 7.3: Relative composition of the crusher claw muscle. The percent composition of water, lipid and glycogen in the crusher claw muscle with all other tissue components represented in the "Other" category.

Figure 7.4: Relative composition of the tail muscle. The percent composition of water, lipid, and glycogen in the tail muscle with all other tissue components represented in the "Other" category.

160 7.3.3. Examination of the Correlations between Tissue Water Content and Biochemical Composition

Tissue water content and lipid index (Lin) values showed significant inverse correlations

in the HP and TM but not in the PCM and CCMs for both males and females. Tissue water content showed significant inverse correlations with glycogen index (Gin) values in the PCM, CCM and TM in males and CCM and TM in females. However, the

relationship between the TM in males and the CCM in males and females were slightly stronger than the other observed correlations, with Pearson correlations below -0.5

(Table 7.1).

Table 7.1: Correlations between tissue water content and metabolic reserves. Correlations between the tissue water content of each tissue and the respective lipid and glycogen index values for male and female lobsters. Pearson correlation values are shown on the top with the corresponding p value underneath in italics. Bolded values indicate significance at p <0.05 or R2values less than -0.5.

Female Lipid Index Glycogen Index Lipid Index Glycogen Index

Percent Water -0.903 -0.158 -0.799 -C /•? 'j Hepatopancreas 0.000 0.272 0.000 0.195 Percent Water -0.156 -0.537 -0 149 -0.521 Crusher Claw Muscle 0.256 0.000 0.394 0.003 i i Percent Water -u 2OD -U 31 '-J J..'C^ Pincher Claw Muscle 0.074 0.020 0.62/ 0.252 Percent Water -0.623 -0.690 -0.537 Tail Muscle 0.000 0.000 0.001 0.000

161 7.3.4. Examination of the Correlations between Tissue Energy Reserves and Metabolite Concentrations in the Hemolymph

Significant correlations between tissue lipid and glycogen concentrations and OSM

were observed sporadically among all tissues, between sample groups and between

the two sexes (Tables 7.2 and 7.3). The two sample groups represented all of the

moult cohorts from 2009 and 2010 (09-10) and the primary 2009 moult cohort only

(09).

The hemolymph GLUC concentrations showed significant correlations with the tissue

lipid concentrations for both groups in the males, while in females the PCM and CCM

only correlated well in the combined moult cohort group. Correlations between

hemolymph GLUC and tissue glycogen concentrations were less pronounced, being

observed primarily in the combined cohort group for males and females with the

exception of the CCM and TM Gin values in males, which showed significant correlations in both groups (Tables 7.2 and 7.3).

Hemolymph CHOL concentrations in males showed significant correlations with all tissue lipid and glycogen concentrations (Table 7.2), while in the females only weak correlations were observed with the HP and TM Lin and the HP Gin (Table 7.3).

Triglycerides (TG) in males showed significant correlations with the lipid and glycogen concentrations in all of the other tissues, with the exception of lipid concentrations in the PCM from the primary 2009 moult cohort (Table 7.2). In females TG did not correlate with any of the tissue metabolite reserves (Table 7.3).

162 The TP concentrations in the hemolymph demonstrated good correlations with all tissue metabolites except the lipid concentrations of the PCM in the primary moult cohort (09) in males and all PCM and CCM concentrations in females (Tables 7.2 and

7.3).

The Brix values showed the same patterns as the TP concentrations with the exception of the 2009 Primary cohort lipid concentrations in the CCM of males, which showed no significant correlations (Tables 7.2 and 7.3).

Hemolymph UA showed no significant correlations with any of the lipid and glycogen concentrations in any of the tissues for either males or females (Tables 7.2 and 7.3).

Figures 7.5 to 7.7 represent the correlations between hemolymph TP, TG, and CHOL and the HP lln values. The different moult categories are expressed and grouped together.

163 Table 7.2: Pearson correlations between tissue energy reserves and hemolymph biochemical parameters for males. Pearson correlation statistics for lipid and glycogen concentrations and hemolymph biochemical constituents. The top box shows the R2 value and the bottom box is the p value. Bolded values represent significance at p < 0.05 or R2 values above 0.5.

Male HP1 Lin5 PCM2 Lin CCM3 Lin TM4 Lin HP Gin6 PCM Gin CCM Gin TMGIn 09-10 i 09 09-10 09 09-10 i 09 09-10 ! 09 09-10 } 09 09-loJ~ 09 09-10 09 09-10 09

Q.4>9 ' 0.614 : "• > ":.3 0.574 0.24Q | 0.511_ 0 AM- 0.517 0.236 0.612 -- OSM7 0.001 0.000 0.011 0.000 o.tm 1 o.ooi 0.0011 0.001 0.102 0.000 0.446 > 0.543 C.,47 ; 0 183 0.537 1 0. ^87_ 0 374 0.543 0.333 | 0.195 0.352 1 0.646 0.537 0.629 GLUC8 0.002 0.001 0.001 0.019 0.000 0.018 0.007 0.019 0.000 „.uU> 0.018 \ 0,247 0.012 I 0.000 0.000 0.000 f 0.629 + 0.756 0.610 0.623 0.504 : 0.430 0.395 I 0.299 0.540 [ 0.697 0.563 0.645 CHOL9 0.002 0.000 0.005 0.049 0.000 0.024 0.000 0.000 0.000 I 0.008 0.004 0.072 0.000 0.000 0.000 0.000 1 0.639 0.751 0.513 '6 0.590 0.587 0.555 J 0.451 0A7& 0,345 0.573 0.720 0.658 0.682 ! TGio 0.000 0.000 0.002 0.000 0.036 0.000 0.000 0.000 0.005 0.001 0.037 0.000 0.000 0.000 0.000 t 0.655 0.723 0.4*;.• 1 i).;.36 0.608 0.604 0.548 0.566 0.449 0.375 0.615 0.760 0.695 0.766 TP11 0.000 0.000 0.002 0.000 0.042 0.000 0.000 0.000 0.000 0.001 0.022 0.000 0.000 0.000 0.000 - 93 -0.243 0.083 - v 12 • '- UA ; • .•- :': ' ,; • : S3 (1093 0.630 0.590 0.653 0.508 i: 0.569 i 0.565 0.587 0.575 0.491 1 0374 0.603 | 0.760 0.724 0.783 Brix 0.000 0.000 0.002 o.ooo c-yisi 0.000 0.000 0.000 0.000 0.000 | 0.023 0.000 1 0.000 0.000 0.000 hepatopancreas, pincher claw muscle, crusher claw muscle, tail muscle, 5lipid index, glycogen index, Osmolality, glucose, 9cholesterol,1 triglycerides, "total protein and 12uric acid

164 Table 7.3: Pearson correlations between tissue energy reserves and hemolymph biochemical parameters for females. Pearson correlation statistics for lipid and glycogen concentrations and hemolymph biochemical constituents. The top box shows the R2 value and the bottom box is the p value. Bolded values represent significance at p < 0.05 or R2 values above 0.5.

Female HP1 Lin5 PCM2 Lin CCM3 Lin TM4 Un HP Gin6 PCM Gin CCM Gin TMGIn 09-10 09 09-10 09 09-10 09 09-10 " 09 09-10 ! 09 09-10 09 09-10 09 09-10 09 - c q ] 0 388 r -43 0 359 0.523 r :G c^4 * C03C 1 /5S OSM7 0.005 / . i C "S, 0.020 ' 0.015 „ qgr~\ 0.021 0 063 "4?0 ?07S 0.579 4 0.550 " ' C r<'y ' J2C 0.518 0 ^0? •" /",' /l 0 404 GLUC8 0.000 0.010 0.009 t 0.004 1 0.001 , 0.013 0.006 0^? 0.000 .' 3? 0.003 0 "6? 0.009 0.045 i -2 i " r 32c. C 239 f 222 Z 291 0 295 OZ'7? 0 245 CHOL9 0.039 / y 0.041 ! •)- 0.032 ; f. J* / 0 18" 0 >^ a 7^ n r )3 9 2J1. f -- 0 of- 0 247 0 vi f 0 00f» TG10 f i 0 ?<\C 1 C "S," 0 9// 0.810 0.775 -> 0.693 0.742 "./ ! 0.522 ••JAl) ! 0.570 0.758 0.758 0.570 0.513 3 TP11 0.000 0.000 ^ '., 0.000 0.000 + O.O06To-0O3 0.000 0.000 0.000 0.009 0.002 i 0.005 d J c J s 1 " . ' ' ' if/" I ~ « 'i 1-,) i >?,(. I , ,R C20S UA12 hi • /•&/ ' v?^ J 4 "5 .0 326 0.774 0.743 ) lA 9 0.700 0.727 »41° 0.671^ 0.640 0.537 0 468 13 Brix 0.000 0.000 1 J w 0.000 0.000 0.002 1 0.0*8 0.018 0.037 0.000 0.001 0.001 0.018 hepatopancreas, pincher claw muscle, crusher claw muscle, 4tail muscle, 5hpid index, 6glycogen index, Osmolality, glucose, 9cholesterol, "triglycerides, total protein and uric acid

165 5 10 15 20 25 1 i Female Male • Y = -0.14 + 0.04x 1.0- • R2 = 0.629, p = 0.002 • *-> Category =? 0.8- •" • • UM + • •• o E • EPM E • MPM • / ~ 0.6- • •\^* V^\ " A LPM /+ 2 ^"^•- • PoM ^ •/ • t to • W m • •S 0-4- S .c • y\ • u • •

0.2- •• jf •• •• Y = 0.32 + 0.02x / • R2 = 0.345, p = 0.039 0.0- 5 10 15 20 25 Hepatopancreas Lipid Index (Lin)

Figure 7.5: Regression analysis of hemolymph cholesterol versus lipid concentrations in the hepatopancreas. The data represent the hemolymph cholesterol concentrations versus the Lin values of the hepatopancreas for the 2009 primary moult cohort with regression fits. The five moult categories: late inter-mouit (LIM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM) are designated by different colours and symbols.

10 15 20 25 Female Male 1.4 • Categ c ry • UM 1.2 • • EPM Y = -0.07 + 0.02x • • MPM R2 = 0.639, p = 0.000 Y = 0.58 + 0.01x A LPM o 1.0 • PoM E R = -0.155, p = 0.367 E 0.8 • u 0.6 • • 3 • —_^______^ • .&• 0.4 • * rvT~^ .• • > • •%i" 0.2 • * *£--* \ *>K • 0.0- 10 15 20 25 Hepatopancreas Lipid Index (Lin)

Figure 7.6: Regression analysis of hemolymph triglyceride versus lipid concentrations in the hepatopancreas. The data represent the hemolymph triglyceride concentrations versus the Lin values of the hepatopancreas for the 2009 primary moult cohort with regression fits. The five moult categories: late inter-moult (LIM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM) are designated by different colours/symbols.

166 10 15 20 25 Female Male

Y = -13.62 + 4.49x Category • 100 • UM 2 R = 0.810, p = 0.000 • EPM « MPM 80 A LPM y • PoM at / * y/\ « 60 u* • 2 / m a • / m/» 2 40 o

20 * • + • • / • Y = -9.66 + 4.51x R2 = 0.655, p = 0.000 5 10 15 20 25 Hepatopancreas Lipid Index (Lin)

Figure 7.7: Regression analysis of total hemolymph protein versus lipid concentrations in the hepatopancreas. The data represent the hemolymph total protein concentrations versus the Lin values of the hepatopancreas for the 2009 primary moult cohort with regression fits. The five moult categories: late inter-moult (LIM), early pre-moult (EPM), mid pre-moult (MPM), late pre-moult (LPM) and post-moult (PoM) are designated by different colours/symbols.

The correlations between the HP Lin values and hemolymph CHOL, TG, TP and Brix showed tighter correlations during the LIM than any other moult category in males. In the female population HP lipid concentrations did not demonstrate significant correlations with the CHO, TG, TP or Brix values in any of the moult categories (Table

7.4).

167 Table 7.4: Assessment of nutritional condition biomarkers by moult category and sex. The Pearson correlations (top box) for the hepatopancreas lipid index (HP Lin) versus hemolymph cholesterol, triglycerides total protein and Brix for the 2009 moult cohort. Significance at p < 0.05 is designated by bolded numbers in the bottom box. Bolded numbers in the top box designates R2 values above 0.5 or below -0.5.

Cholesterol Triglycerides Total Protein Brix Male LIM EPM MPM PoM LIM EPM MPM PoM LIM EPM MPM PoM LIM EPM MPM PoM 0.817 0.799 0.904 0.848 -c ;•* 0.816 0.703 0.649 C 23 -C '»"? 0 'b7 HPUn 0.004 0.010 0.000 0.004 0.004 0.035 o r, i 0.042 0 467 6 69 = Cholesterol Triglycerides Total Protein Brix Female LIM EPM MPM PoM LIM EPM MPM PoM LIM EPM MPM PoM LIM EPM MPM PoM -0.589 -0.627 0.658 0.522 N/A -0.535 0 439 0.534 N/A 0 246 HPUn 'V, 1 $ ^ (I 1 '3 0 r>

168 7.4. Discussion

The ability to assess metabolic reserves in crustaceans may aid in predicting growth

obtained through the moult, as metabolic reserves play an important role in tissue

formation and maintenance (Conklin, 1980; Johnston et al., 2003). In the West-coast

rock lobster Jasus lalandii lipids in the HP have been identified as a robust predictive

indicator of growth in adult males (Cockcroft, 1997). Phospholipids in the hemolymph

of the Southern rock lobster Jasus edwardsii have been implicated as a potential non-

lethal means of predicting moult increment (Musgrove and Babidge, 2003). Growth

prediction can be important in monitoring lobster populations to set the proper

management practices in place and to ensure the survival of a population.

Examination of the relative composition of the HP, PCM, CCM and TM confirmed that

the HP contains the highest concentrations of lipid and glycogen, which suggests that

this tissue is the main depot for energy storage. Glycogen comprised a very small

portion of all the tissues examined, suggesting that it is not acting as a significant

source of energy during the moult cycle. These data implicate HP lipid stores as the

primary metabolic energy reserve in the lobster, marking it as a potential indicator of

nutrition and condition.

The changes in standardised lipid concentrations in the HP are associated with inverse

changes in water content. The inverse relationship between water and tissue

metabolites can be observed in the HP through visual examination of the data and through statistical analyses, which revealed a significant inverse relationship between

169 lipid and water content in the HP and TMs. Baumberger and Olmsted (1928) suggested that the inverse relationship observed was due to the replacement of mobilised metabolic reserves with water. The significant correlations between water content and metabolic reserves observed in this study and in the shrimp P. muelleri (Jeckel et al., 1990) support the notion that the utilisation of metabolic reserves in some tissues results in the subsequent replacement with water.

The lack of significant correlations between water content and lipid reserves in the claw muscles reflects the tissue specific changes in physiology that take place during the moult. The process by which the claw muscles are exuded from the old shell is far more complex than in the other tissues. The claw muscles are large in comparison to the basi-ischial joint (knuckle) through which it must pass (Mykles and Skinner, 1990).

This means pronounced atrophy is required to facilitate removal at ecdysis (Mykles,

1992). A decrease in water content in the CCM could also aid in the exuviation of the old shell by decreasing the size of the muscle. This could explain the tissue specific differences in correlations observed between tissue water and metabolite concentrations.

Significant correlations between tissue glycogen concentrations and water content were not visible even in the LPM category when a significant increase in tissue glycogen was observed. This is most likely due to the fact that glycogen concentrations were so low that any reciprocal changes in water content were not detected. Another explanation could be that glycogen molecules are highly branched and closely

170 associated with water (Urich, 1994). The hydrated nature of the glycogen complicates the relationship between stored glycogen and water content.

Although the visual assessment of such trends is not obvious, statistical analyses of these data revealed significant inverse correlations between water content and glycogen concentrations in the CCM and TMs. These data further emphasise the tissue specific changes in physiology that occur during the moult. Similar changes in metabolic composition were observed in the crayfish P. clarkia during stress (Schirf et al., 1987). These variations in physiology highlight the importance of evaluating several individual tissues when examining physiological and biochemical changes in lobsters representing different life history stages.

The category entitled "Other" contains all non lipid, glycogen and water components of the tissues. This includes proteins, minerals, and heavy metals (Barrento et al., 2008).

Rosa and Nunes (2003) found that the protein content in the muscle of the Norwegian lobster N. norvegicus was approximately 19-21%. This corresponds well with the ~20%

"Other" category observed in H. americanus, suggesting that the majority of it may be protein. Changes in this category could represent actual utilisation of protein as an energy reserve or the changing tissue water content. More in-depth investigations of the components of the "other" category and the moult induced changes in protein storage and function are warranted to better understand the reasons for and mechanisms controlling the pronounced changes observed.

171 Comparison of the correlations among the various hemolymph metabolites and tissue reserves between the 2009 primary moult cohort and all cohorts from 2009 and 2010 were performed to determine if, by eliminating some potential sources of variation, the correlations between tissue energy stores and hemolymph biochemistry would improve. The two sexes were also examined separately because significant differences in hemolymph biochemistry with respect to the moult were identified previously in

Chapter 6.

Correlations between hemolymph OSM and GLUC concentrations differed between the two moult cohort groups indicating that there may have been a seasonal or year effect on the data. The inconsistencies in the OSM most likely reflect salinity changes in the natural environment over time, as lobsters are osmoconformers and alter internal osmolality to mimic that of the surrounding environment (Dall, 1970). The inconsistencies in the GLUC data are most likely due to the diverse nature of GLUC, which makes it susceptible to acute changes during periods of stress (Telford, 1968).

The variations observed in these two variables and the lack of any significant correlations between hemolymph UA and tissue metabolites make these three parameters poor candidates for monitoring condition in the lobster.

In males, the hemolymph CHOL, TG, TP and Brix index values showed significant correlations with the HP lipid concentrations with slight differences between the two groups assessed. The slight differences between the groups suggest that these parameters may be susceptible to year- and cohort-induced alterations, as data

172 including one year and a single cohort showed tighter correlation with the HP lipid

concentrations. However, this does not hold true in all the data with some correlations

being stronger when both years and cohorts are included. This demonstrates the need

for further evaluation of year and cohort affects on physiology.

In female lobsters the hemolymph TG and CHOL concentrations showed very poor

correlations with tissue metabolites. Although correlations between tissue

metabolites and TP concentrations in the hemolymph were still present they were

fewer than those observed in males. These data demonstrate the pronounced

differences in physiology that exist between genders most likely associated with

differences in reproductive physiology which have been observed in other crustaceans

(Castille and Lawrence, 1989). The high variations in TG concentrations in the LIM

lobsters have been suggested to be due to ovary maturation in preparation for

spawning (Battison et al., 2011).

The significant correlations observed between lipid concentrations in the HP and the

hemolymph TG, CHOL and TP concentrations and Brix indices identified them as

potentially effective biomarkers of nutritional condition in the lobster. As these

parameters were deemed potential markers of condition, they were further assessed

to evaluate potential sources of variation or error. The moult categories were

examined individually to identify moult induced changes within the data.

The highest correlations between hemolymph metabolites and HP lipid concentrations

(Lin) were observed in the LIM, with decreased significance in the other moult

173 categories. This suggests that these hemolymph parameters are not the most accurate

representation of metabolic reserves or condition during the active phases of the

moult cycle. The remobilisation of protein and lipid from the old shell could explain

the lack of correlation observed between hemolymph and tissue constituents during

the moult. Thus, the most accurate predictions of HP lipid concentrations as a measure

of lobster condition can be made during the inter-moult period when hemolymph

reserves are unaffected by the moult. The higher correlations observed between the

directly measured TP concentrations as opposed to the Brix indices, as a

representation of TP concentration, identify the direct measurement as a more

accurate assessment of condition.

It should also be noted that although the tissue metabolite concentrations have been standardised to account for variations in water content, the hemolymph metabolite

concentrations have not. Thus, the fluctuations in hemolymph metabolites could

reflect changing water content. This could mean that the variations in hemolymph

metabolite concentrations may not directly represent the changing tissue reserves.

Further analysis of absolute changes in hemolymph metabolites is required to confirm the observed correlations with tissue metabolic reserves.

These data have identified the HP as the primary site of lipid storage, which acts as a significant energy reserve in the lobster during the moult. These characteristics of HP lipid make it the most accurate representation of nutritional condition in the lobster.

Furthermore, CHOL, TG and TP have been shown to correlate well with HP lipid

174 concentrations in males, especially during the LIM category. This warrants a more comprehensive analysis of the relationship between the hemolymph TP, TG, CHOL concentrations and the HP lipid reserves that address all potential factors that could affect their concentrations in the hemolymph. The factors could include temperature, salinity, depth and disease state).

These biomarkers show promise for the development of non-lethal means of assessing nutrition or condition in male lobsters. The complex nature of the female reproductive cycle hinders the assessment of condition in females. A more in-depth analysis of female moult and physiology are warranted if accurate measures of condition are to be obtained. Further evaluation of the effects of spatial variations is also necessary before these biomarkers can be validated as an effective tool for determining nutritional condition. This is especially important among groups occupying different habitats with distinct differences in environmental characteristics. This will help to ensure that the methods are consistent and reliable for individual lobster populations.

175 7.5. References

Barrento, S., Marques, A., Teixeira, B., Vaz-Pires, P., Carvalho, M.L., Nunes, M.L., 2008. Essential elements and contaminants in edible tissues of European and American lobsters. Food Chem. Ill, 862-867.

Battison, A., Burton, M., Comeau, M., Silva, A., Summerfield, R., 2011. Hemolymph Plasma Triglycerides and Cholesterol Concentrations as Potential Aids to Determine Ovary Maturity in the American Lobster. 9th Annual International Conference and Workshop on Lobster Biology, Bergen, Norway.

Baumberger, P.J., Olmsted, J.M.D., 1928. Changes in the osmotic pressure and water content of crabs during the molt cycle. Physiol. Zool. Vol. l(No. 4), 531-544.

Castille, F.L., Lawrence, A.L., 1989. Relationship between maturation and biochemical composition of the gonads and digestive glands of the shrimps Penaeus aztecus Ives and Penaeus setiferus (L.). J. Crustacean Biol. 9(2), 202-211.

Cockcroft, A.C., 1997. Biochemical composition as a growth predictor in male west- coast rock lobster (Jasus lalandii). Mar. Freshw. Res. 48, 845-856.

Conklin, D.E., 1980. Nutrition, in: Cobb, Stanley J. and Phillips, Bruce F. (Ed.), The Biology and Management of Lobsters. Academic Press, Inc., New York, NY, pp. 277-300.

Dall, W., 1970. Osmoregulation in the lobster Homarus americanus. J. Fish. Res. Board Can. 27, 1123-1130.

Galindo, C, Gaxiola, G., Cuzon, G., Chiappa-Carrara, X., 2009. Physiological and biochemical variations during the molt cycle in juvenile Litopenaeus vannamei under laboratory conditions. J. Crustacean Biol. 29(4), 544-549.

Jeckel, W.H., Aizpun de Moreno, Julia Elena, Moreno, V.J., 1990. Changes in biochemical composition and lipids of the digestive gland in females of the shrimp Pleoticus muelleri during the moult cycle. Comp. Biochem. Physiol. B 96(3), 521-525.

Johnston, D.J., Calvert, K.A., Crear, B.J., Carter, C.G., 2003. Dietary carbohydrate/lipid ratios and nutritional condition in juvenile southern rock lobster, Jasus edwardsii. Aquaculture 220, 667-682.

McWhinnie, M.A., Kirchenberg, R.J., Urbanski, R.J., Schwarz, J.E., 1972. Crustecdysone mediated changes in crayfish. Am. Zool. 12, 357-372.

176 Musgrove, R.J.B., Babidge, P.J., 2003. The relationship between hemolymph chemistry and moult increment for the Southern Rock lobster, Jasus Edwardsii Hutton. J. Shellfish Res. 22(1), 235-239.

Mykles, D.L., 1992. Getting out of a Tight Squeeze: Enzymatic Regulation of Claw Muscle Atrophy in Molting. Am. Zool. 32(3), 485-494.

Mykles, D.L., Skinner, D.M., 1990. Atrophy of Crustacean Somatic Muscle and the Proteinases That Do the Job. A Review. J. Crustacean Biol. 10(4), 577-594.

Rosa, R., Nunes, M.L., 2003. Biochemical composition of deep-sea decapod crustaceans with two different benthic life strategies off the Portuguese south coast. Deep- Sea Res. Pt. 1 50,119-130.

Sanchez-Paz, A., Garcia-Carreno, F., Muhlia, A., Adriana, Peregrino-Uriarte, A.B., Hernandez-Lopez, J., Yepiz-Plascencia, G., 2006. Usage of energy reserves in crustaceans during starvation: Status and future directions. Insect Biochem. Mol. Biol. 36, 241-249.

Schirf, V.R., Turner, P., Selby, Lv Hannapel, C, DeLa Cruz, P., Dehn, P.F., 1987. Nutritional status and energy metabolism of crayfish (Procambarus clarkii, Girard) muscle and hepatopancreas. Comp. Biochem. Physiol. A 88(3), 383-386.

Telford, M., 1968. The effects of stress on blood sugar composition of the lobster, Homarus americanus. Can. J. Zool. 46, 819-826.

Urich, K., 1994. Lipids, in: Anonymous, Comparative Animal Biochemistry. Springer- Verlag, New York, NY, pp. 562-623.

177 8. Summary and Conclusions

This study evaluated several methods of lipid and glycogen determination in tissue to identify and optimise efficient methods of metabolic assessment in the lobster. The major purpose of this research was to investigate the natural fluctuations in tissue water content, tissue metabolic reserves, hemolymph biochemistry and the relationships that exist among them. Furthermore, methods of standardisation for the measured lipid and glycogen concentrations were devised. These methods allowed for the comparison of lobsters representing different moult categories even though physical and physiological differences existed between them. These data will help enhance the general understanding of H. americanus physiology and promote the development of effective management practices through a better understanding of lobster nutrition and health as well as the development of tools to evaluate health and moult stage more accurately.

The Folch centrifugation (FoC) method was identified as the preferred method of lipid extraction as it showed the least amount of variation. This method was also simpler and less time consuming than the other methods assessed. An automated HK assay was identified as the preferred method of glycogen determination over a bench top manual glucose oxidase based method due to the large sample capacity, which allowed all assays to be performed on the same day to eliminate between run variations.

178 Methods of glycogen determination with the HK assay were further validated through

the optimisation of glycogen digestion procedures, evaluation of precision and linearity

in the assay and standard oyster glycogen (OG) solutions, and the assessment of

stability in the tissues and OG standard. The validation procedures identified the assay

as a precise means of glycogen assessment in the four lobster tissues examined:

hepatopancreas (HP), pincher claw muscle (PCM), crusher claw muscle (CCM) and tail

muscle (TM).

The sampling procedure investigations identified sampling strategy as an integral

component to the assessment of condition and quality in lobsters, and the monitoring

of populations. Seasonal variations, stress and the potential effects of year may cause

alterations in physiology as observed previously in H. americanus (Telford, 1968; Dove

et al., 2005). Sampling procedures were devised to limit variation within the dataset

and to target moult-induced changes in physiology.

An increase in tissue water content was observed in the PoM, which corresponds well

to the water influx reported in H. americanus (Lowndes and Panikkar, 1941; Mykles,

1980) and other crustaceans (Lowndes and Panikkar, 1941; Baumberger and Olmsted,

1928; Romero et al., 2006; Suneetha et al., 2009). Changes in water content also

correlated inversely with the changing lipid and glycogen concentrations in the tissues.

This demonstrates the replacement of metabolic reserves with water and vice versa.

Tissue glycogen showed marked changes during the moult. However, the generally

low concentrations and significant increase during the period of inanition (LPM)

179 suggest that it is not being used as an energy reserve. These data reflect reported

observations in the adult spiny lobster P. argus (Travis, 1955), the crayfish O. virilis

(Jungreis, 1968) and larval spiny lobster J. edwardsii (Jeffs et al., 1999) where it was

shown that glycogen is not used as an energy source in these species. These

fluctuations may represent the storage of chitin-derived metabolites in tissues for use

in forming the new shell following ecdysis (Travis, 1955; Parvathy, 1971; Salaenoi et al.,

2006).

The HP had high lipid content in comparison to other tissues and demonstrated

significant changes in lipid concentrations during the moult cycle, with a prominent

decrease in the PoM following ecdysis. These characteristics marked HP lipid as a

potential energy reservoir and a good indicator of nutritional condition in the lobster.

Lipid has previously been implicated as the primary metabolic reserve in Homarus sp.

with the HP being the main site of storage (Factor, 1995).

Protein in the tissues represents another metabolic reserve not addressed here, but

the hemolymph TP and UA data suggests that protein plays an important role in the

moult cycle. A more in-depth analysis of the role of protein during the moult is

warranted as the protein rich muscle tissues can constitute as high as 40% of the

lobster total body weight (Cawthorn, unpublished data), depending on moult stage.

Significant fluctuations in various hemolymph analytes were observed between the

different moult categories examined. The common trend was a gradual increase in concentrations from the LIM through the LPM categories followed by a significant

180 decrease in the PoM, which most likely reflects dilution of the hemolymph by the influx of water at ecdysis (Retzlaff et al., 2007). This occurred in all the hemolymph metabolites except uric acid (UA) where the only change observed was a small but significant decrease in the LPM. The lack of a dilution effect on the UA concentrations in the PoM could indicate a significant increase in UA production at this time and warrants closer examination.

Hemolymph GLUC and UA concentrations were discounted as biomarkers of condition in H. americanus on account of their inconsistencies and lack of significant correlations between tissue metabolic stores. The effects of stress on hemolymph sugar have led to its being discounted as a measure of condition in the Western rock lobster Panulirus longpipes (Dall, 1974). Although the UA concentrations were discounted as a nutritional indicator, the stability of hemolymph concentrations could potentially be crucial in the assessment of other aspects of lobster health and nutrition. For example the stable levels could be used to generate ratios with other metabolites to standardise the values for changing hemolymph volume during the moult cycle. The use of stable body fluid analytes (Such as creatinine) has been used for the generation of ratios with other analytes for the assessment of physiological well being in human medicine (First,

2003). Further, examination of the mechanisms of UA generation and excretion are necessary to confirm its role and significance in lobster health.

Hemolymph TG, CHOL and TP concentrations demonstrated good correlation with lipid concentrations in the HP of male LIM lobsters. Hemolymph protein concentrations

181 determined through Brix refractive index values also demonstrated good correlations

with HP lipids. However, the directly measured TP concentrations showed higher

correlations, marking them as a more accurate parameter to measure.

Due to the suspected role of HP lipid stores in nutrition, these parameters represent

potential biomarkers of nutritional condition in the lobster that could be assessed non-

lethally. The higher correlations between tissue metabolic stores and hemolymph TG,

CHOL, and TP concentrations observed in the LIM males suggest that biochemical

analysis of the hemolymph may be more accurate during this category. It also suggests

that caution should be taken if evaluating them during the active phases of the moult

cycle such as the pre- and post-moult period and ecdysis.

Various lipid classes have been shown to be utilised differentially during the moult in

the crab C. maenas and the prawn P. japonicas (Heath and Barnes, 1970; Ando et al.,

1977). A more detailed analysis of specific lipid compounds in circulation may

elucidate nutritional indices that are less susceptible to moult induced changes than TG

and CHOL. In addition, a closer examination of specific protein or nucleic acid

concentrations, which were not examined in this study, could provide a superior

biomarker of condition. Nucleic acids as potential indicators of growth and nutritional

condition have been identified in the juvenile white shrimp P. vannamei (Moss, 1994) and the Norway lobster N. norvegicus (Parslow-Williams et al., 2001), respectively.

A better understanding of how dissolution and reformation of the shell affects

hemolymph protein and lipid concentrations would also be required to improve the

182 usefulness of hemolymph biochemical evaluation in the active pre- and post-moult

stages. Methods of monitoring nutrition that utilize multiple parameters will offer a

more reliable representation of condition that is less susceptible to variation caused by

environmental conditions, stress or disease (Noga, 2000). Although TP, CHOL, and TG

concentrations in the hemolymph show potential as markers of condition in males, the

results of this study indicate that a more in-depth analysis of female reproductive and

moult physiology is required if an accurate measure of condition is to be found in

females.

The results of this study offer insight into the natural fluctuations in nutritional

condition, which are expected to occur in Homarus americanus as it progresses

through its moult cycle. Understanding the natural fluctuations in tissue energy

resources is important in developing tools to assess foraging success, environmental

availability of resources and changes in metabolic physiology.

Insufficient diet can cause long-term susceptibility to disease (Tlusty et al., 2008) and

decreases in population growth (Tuck et al., 1997; Johnston et al., 2003; Conklin, 1980).

Diet will determine the amount and type of stored energy available to the organism

(Vinagre and Silva, 1992). Thus, biomarkers of nutrition could aid in the monitoring of food availability and habitat changes within a population. The ability to track such

changes could provide additional information to assist with management policy

decisions to maximize the sustainability of lobster populations in different fishing

areas.

183 Hemolymph biochemical evaluation may also offer a non-lethal tool for assessment of quality in the lobster. The significant decrease or dilution of biochemical constituents in the PoM corresponded to the significant increase in the tissue water content. As quality is inversely correlated with tissue water content (Dufour et al., 1997; Retzlaff et al., 2007) the low metabolite concentrations could be used to asses changes in meat quality.

A more extensive sample set that incorporates a larger sample size representing each moult category for males and females separately would be useful in eliminating some of the natural variation within the wild populations and allow for more accurate representations of physiological changes. Evaluation of year and cohort effects on the data is also important for a complete understanding of lobster physiology and the development of proper tools for nutritional and quality assessment.

Although these data offer insight into the complex nature and pronounced alterations in physiology that occur during the lobster moult cycle, it has brought about several questions that warrant investigations to obtain a full understanding of lobster physiology. The mechanisms and pathways for the synthesis and degradation of chitin, glycogen, lipid and protein must be assessed. The correlations observed between tissue and hemolymph metabolite concentrations may reflect changes in hemolymph water content as these measurements are expressed per unit volume. While relative concentrations may still be valuable as indicators of lobster condition and moult stage,

184 a more in-depth evaluation of absolute analyte concentrations and possible evaluation of various analytes in relation to UA may be more accurate tools.

Practical implications of this research include, but are not limited to, the generation of non-lethal methods for nutritional and quality assessment, predicting moult success in terms of frequency, increment or survival and the monitoring of lobster populations.

The relationship between tissue lipid concentrations and hemolymph biochemistry may be helpful for the development of non-lethal tools for population monitoring including general nutritional status and fluctuations in environmental constraints such as food availability, which plays an integral role in all aspects of crustacean life history.

185 8.1. References

Ando, T., Kanazawa, A., Teshima, S., 1977. Variation in lipids of tissues during the molting cycle of prawn. Bull. Jap. Soc. Fish. Res. 43(12), 1445-1449.

Baumberger, P.J., Olmsted, J.M.D., 1928. Changes in the osmotic pressure and water content of crabs during the molt cycle. Physiol. Zool. Vol. l(No. 4), 531-544.

Conklin, D.E., 1980. Nutrition, in: Cobb, Stanley J. and Phillips, Bruce F. (Ed.), The Biology and Management of Lobsters. Academic Press, Inc., New York, NY, pp. 277-300.

Dall, W., 1974. Indices of nutritional state in the western rock lobster, Panulirus longipes (Milne Edwards). I. Blood and tissue constituents and water content. J. Exp. Mar. Biol. Ecol. 16(2), 167-180.

Dove, A.D.M., Sokolowski, M.S., Bartlett, S.L, Bowser, P.R., 2005. Spatio-temporal variation in serum chemistry of the lobster, Homarus americanus Milne- Edwards. J. Fish Dis. 28, 663-675.

Dufour, R., Bernier, D., Brethes, J., 1997. Optimisation of meat yield and mortality during snow crab (Chionoecetes opilio O. Fabricius) fishing operations in Eastern Canada. Canadian Technical Report of Fishery and Aquatic Sciences 2152,1-31.

Factor, J.R., 1995. The Digestive System, in: Factor, J.R. (Ed.), Biology of the Lobster: Homarus americanus. Academic Press, Inc., San Diego, CA, pp. 395-440.

First, M.R., 2003. Renal Function, in: Kaplan, L.A., Pesce, Amadeo J. and Kazmierczak, Steven C. (Eds.), Clinical Biochemistry: Theory, Analysis, Correlation, 4th ed. Mosby, St. Louis, Missouri, pp. 477-491.

Heath, J.R., Barnes, H., 1970. Some changes in biochemical composition with season and during the moulting cycle of the common shore crab, Carcinus maenas (L.). J. Exp. Mar. Biol. Ecol. 5(3), 199-233.

Jeffs, A.G., Wilmott, M.E., Wells, R.M.G., 1999. The use of energy stores in puerulus of the spiny lobster Jasus edwardsii across the continental shelf of New Zealand. Comp. Biochem. Physiol. A 123, 351-357.

Johnston, D.J., Calvert, K.A., Crear, B.J., Carter, C.G., 2003. Dietary carbohydrate/lipid ratios and nutritional condition in juvenile southern rock lobster, Jasus edwardsii. Aquaculture 220, 667-682.

186 Jungreis, A.M., 1968. The role of stored glycogen during long-term temperature acclimation in the freshwater crayfish, Orconectes virilis. Comp. Biochem. Physiol. 24(1), 1-6.

Lowndes, A.G., Panikkar, N.K., 1941. A note on the changes in water content of the lobster {Homarus vulgaris M.-EDW.) during moult. J. Mar. Biol. Assoc. U. K. 25(1), 111-112.

Moss, S.M., 1994. Use of nucleic acids as indicators of growth in juvenile white shrimp, Penaeus vannamei. Mar. Biol. 120, 359-367.

Mykles, D.L., 1980. The mechanism of fluid absorption at ecdysis in the American lobster, Homarus americanus. J. Exp. Biol. 84, 89-101.

Noga, E.J., 2000. Hemolymph biomarkers of crustacean health, in: Fingerman, Milton and Nagabhushanam, Rachokonda (Ed.), Recent Advances in Marine Biotechnology: Immunobiology and pathology. Science Publishers, Inc., Enfield, NH, pp. 125-163.

Parslow-Williams, P.J., Atkinson, R.J.A., Taylor, A.C., 2001. Nucleic acids as indicators of nutritional condition in the Norway lobster Nephrops norvegicus. Mar. Ecol. Progr. 211, 235-243.

Parvathy, K., 1971. Glycogen storage in relation to the moult cycle in the two crustaceans Emerita asiatica and Ligia exotica. Mar. Biol. 10, 82-86.

Retzlaff, A., Claytor, R., Petrie, B., Frail, C, Tremblay, J., Pezzack, D., Lavallee, J., 2007. Variation in moult timing and market quality in the American lobster (Homarus americanus). Cat. No. Fsl01-3/2006E, 22-26.

Romero, CM., Lovrich, G.A., Tapella, F., 2006. Seasonal changes in dry mass and energetic content of Munida subrugosa (Crustacea: Decapoda) in the Beagle Channel. J. Shellfish Res. 25(1), 101-106.

Salaenoi, J., Bootpugdeethum, M., Mingkwan, Thongpan, A., 2006. Chitobiase, proteinase, glycogen and some trace elements during molting cycle of mud crab (Scylla serrata Forskal 1775). Kasetsart J.: Nat. Sci. 40, 517-528.

Suneetha, Y., Sreenivasula Reddy, P., Jyothi, N., Srinivasulu Reddy, M., 2009. Studies on the analysis of proximal changes during molting process in the Penaeid prawn, Penaeus monodon. World J. Zool. 4(4), 286-290.

187 Telford, M., 1968. The effects of stress on blood sugar composition of the lobster, Homarus americanus. Can. J. Zool. 46, 819-826.

Tlusty, M.F., Myers, A., Metzler, A., 2008. Short- and long-term dietary effects on disease and mortality in American lobster Homarus americanus. Dis. Aquat. Org. 78, 249-253.

Travis, D.F., 1955. The moulting cycle of the spiny lobster, Panulirus argus Latreille. II. Pre-ecdysial histological and histochemical changes in the hepatopancreas and integumental tissues. Biol. Bull. Vol. 108(No. 1), 88-112.

Tuck, I.D., Chapman, C.J., Atkinson, R.J.A., 1997. Population biology of the Norway lobster, Nephrops norvegicus (L.) in the Firth of Clyde, Scotland. I. Growth and density. ICES J. Mar. Sci. 54, 125-135.

Vinagre, A.S., Silva, R.S.M., 1992. Effects of starvation on the carbohydrate and lipid metabolism in crabs previously maintained on a high protein or carbohydrate- rich diet. Comp. Biochem. Physiol. A 102(3), 579-583.

188 9. Appendices

Appendix A Homogenisation and Lyophilisation of tissue samples

1. Using a scalpel blade mince enough tissue frozen at -80 °C to fill a 5 mL plastic screw cap vial (pre-weigh tube with cap). (Be sure to label both the tube and cap) **The more finely minced the sample is the easier it will be to homogenise. 2. Re-weigh the vial, with the cap and tissue then allow it to thaw at room temperature (~30-60 min). 3. Homogenise the thawed tissue using the designated stainless steel adaptor (OMNI G-5 generator probe) and electric homogeniser (OMNI International TH- 01) until it is a uniform consistency (~15-30 seconds). **Add distilled water as needed to ensure complete homogenisation. 4. Freeze the homogenate in vials in upright position at -80 °C then remove cap and cover the opening of the tube with a piece of gauze, held in place with an elastic band. **Approximately 40 tissue samples can be processed (steps 1-4) in an 8 hour work day.** 5. Place homogenates on the lyophiliser (Labconco lyophiliser Model 75035, Central Services), according to operating procedures on machine, for a minimum of 48 hours. After lyophilisation is complete the weight of the tube (+cap) with the dry tissue is recorded. **lfthe sample is to be used immediately place on ice, otherwise store at -80X.

189 Appendix B Folch centrifugation method of total lipid extraction from hepatopancreas

1. Pre-label 12 ~10 mL round bottom glass centrifuge tubes with the sample name and date for four hepatopancreas tissues in triplicate. Remove samples from storage and place on ice. 2. Zero the balance with a 10 mL round bottom glass centrifuge tube on it then weigh a ~100 mg aliquot of lyophilised hepatopancreas into it (four tissues in triplicate for a total of 12 tubes). Store on ice after weighing out. 3. Pre-label and weigh 12 10 ml glass beakers and write the mass on the label (place label on beaker before weighing). NOTE: Perform steps 4-15 in the fume hood.

4. To each of the hepatopancreas tissue aliquots, add 3.75 ml chloroform:methanol (2:1 v/v) and cap the tube. **Plastic sheath's found at central services can be used as caps. 5. Add 0.75 mL of 100% methanol to one of the samples, and then homogenise using the designated stainless steel adaptor and electric homogeniser (OMNI International) until a uniform consistency is achieved. Re-cap and store at room temperature. Repeat this for each sample. 6. Vortex each tube vigorously for 1 min, taking care not to spill the contents. 7. Let the homogenates stand in the fume hood at room temperature for 15 min. 8. Centrifuge each tube at 1000 x g for 20 min to pellet cell debris. 9. Decant supernatant into a 15 mL round bottom glass centrifuge tube. Transfer label from the 10 mL tube to the new 15 mL tube. (Discard pellet)

10. Add 1.5 mL chloroform and 1.5 ml dH20 to each supernatant. Cap all tubes when finished. 11. Vortex each tube vigorously for 1 min, taking care not to spill the contents. 12. Centrifuge samples at 1000 x g for 20 min. The supernatant will divide into an upper water-soluble layer and a lower organic layer, divided by a thin layer of tissue debris. 13. CAREFULLY remove the bottom organic layer using a Pasteur pipette and place into its respective pre-labelled and weighed beaker. If any debris or water droplets are transferred, try to remove them from the beaker with the pipette. Repeat for all tissue aliquots. 14. Dry each beaker in the incubator in a fume hood at 50 °C until only lipid residue remains (mass no longer decreases) or for 96 hours. **Keep samples in a capped container during transfer. 15. Weigh each beaker using the analytical balance and calculate the % total lipid per mg dry weight:

((Beaker with lipid residue (mg) - Beaker (mg))/Lyophilised tissue aliquot (mg) * 100

190 Appendix C Folch centrifugation method of total lipid extraction from muscle

1. Pre-Label 12 15 mL glass centrifuge tubes with the sample name and date for six muscle tissues in duplicate. Remove lyophilised samples from storage and place on ice. 2. Zero the balance with a 15 mL round bottom glass centrifuge tube on it then weigh a ~200 mg aliquot of lyophilised muscle into it (six tissues in duplicate for a total of 12 tubes). Store on ice after weighing. 3. Pre-label and weigh twelve 10 ml glass beakers and write the mass on the label (place label on beaker before weighing). NOTE: Perform steps 4-15 in the fume hood.

4. To each of the muscle tissue aliquots, add 7.5 ml chloroform:methanol (2:1 v/v) and cap the tube. **Plastic sheaths found at central services can be used as caps. 5. Add 1.5 mL of 100% methanol to one of the samples, and then homogenise using the designated stainless steel adaptor (OMNI G-5 generator probe) and electric homogeniser (OMNI International TH) until a uniform consistency is achieved. Re-cap and store at room temperature. Repeat this for each sample. 6. Vortex each tube vigorously for 1 min, taking care not to spill the contents. 7. Let the homogenates stand in the fume hood at room temperature for 15 min. 8. Centrifuge each tube at 1000 x g for 20 min to pellet cell debris. 9. Decant supernatant into a 30/50 mL round bottom glass centrifuge tube. Transfer label from the 15 mL tube to the new 30/50 mL tube. (Discard Pellet) **Cap 30 mL tubes with provided screwcap and 50 mL tubes with a rubber stopper which can be obtained from Central Services.

10. Add 3 mL chloroform and 3 ml dH20 to each supernatant. Cap all tubes when finished. 11. Vortex each tube vigorously for 1 min, taking care not to spill the contents. 12. Centrifuge samples at 1000 x g for 20 min. The supernatant will divide into an upper water-soluble layer and a lower organic layer, divided by a thin layer of tissue debris. 13. CAREFULLY remove the bottom organic layer using a Pasteur pipette and place into its respective pre-labelled and weighed beaker. If any debris or water droplets are transferred, try to remove them from the beaker with the pipette. Repeat for all tissue aliquots. 14. Dry each beaker in the incubator in a fume hood at 50 °C until only lipid residue remains (mass no longer decreases) or for 96 hours. **Keep samples in a capped container during transfer between labs. After one night at 50°C a container ofdesiccant should be kept in the closed container for transport to weigh samples. 15. Weigh each beaker using the analytical balance and calculate the % total lipid per mg dry weight:

(((Beaker with lipid residue (mg) - Beaker (mg))/Lyophilised tissue aliquot (mg))* 100

191 Appendix D Glucose oxidase method for glucose determination

BioVision Glucose Assay

1. Dilute glucose standard to 1 nm/u.1 by adding 10 u.1 of glucose standard to 990 u.1 of Glucose Assay Buffer, mix well. Add 0, 2, 4, 6, 8,10 u.1 to each well individually. Adjust volume to 50 uJ per well with Glucose Assay Buffer to generate 0, 2, 4, 6, 8,10 u.1 per well of glucose standard. 2. Add 5 u.1 of glucose sample to a well and then add 45 u.1 of Glucose Assay Buffer to make a 50 u.1 reaction volume. 3. Enough glucose reaction mix was prepared to add 50 u.1 to each well. Reaction mix is as follows: 46 u.1 Glucose Assay Buffer 2 u.1 Glucose Probe 2 ul Glucose Enzyme Mix 4. Mix reactions well. Add 50 u.1 of glucose reaction mix to each well for a total well volume of 100 u.1. 5. Incubate the reaction in the dark for 30 minutes at 37°C. 6. Measure the O.D. at 570 nm. 7. Calculate glucose concentrations by first subtracting the 0 glucose control from all readings. Then determine glucose content for each sample from the standard curve generated. Calculate sample glucose with the following equation:

C = Sa/Sv = u,mol/mL

Where Sa is the sample amount from the standard curve and Sv is the sample volumes added to the sample wells.

192 Appendix E Hexokinase method for glucose determination

1. Weigh out three aliquots, 10 - 20 mg each, of lyophilised tissue directly into a pre-labelled 2mL microfuge tube. **10 mg is the suggested minimum amount but closer to 20 mg would be ideal. May be prepared ahead and stored at -80°C. 2. Prepare a 2 mg/mL stock solution of oyster glycogen (OG) in a 100 mM sodium citrate buffer pH 5. 3. Prepare a serial dilution of OG stock with the concentrations 2 mg/mL, 1 mg/mL, 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL, 0.063 mg/mL and 0.031 mg/mL as described below.

a) Label five 2 mL microfuge tubes accordingly b) Add lmL of buffer to each of the 1, 0.5, 0.25, 0.125, 0.063, and 0.031 mg/mL tubes. c) Place one mL of 2 mg/mL stock solution into the 2 and 1 mg/mL tubes and vortex to mix. d) Pipette 1 mL from the 1 mg/mL tube into the 0.5 mg/mL tube and vortex to mix. e) Pipette 1 mL from the 0.5 mg/mL tube into the 0.25 mg/mL and vortex to mix. f) Pipette 1 mL from the 0.25 mg/mL tube into the 0.125 mg/mL tube and vortex to mix. g) Pipette 1 mL from the 0.125 mg/mL tube into the 0.063 mg/mL tube and vortex to mix. h) Pipette 1 mL from the 0.063 mg/mL tube into the 0.031 mg/mL tube and vortex to mix i) Pipette 1 mL from the 0.031 mg/mL tube and discard. ** The serial dilutions should be prepared in Replicates of four. Three will be digested to generate a standard curve and the third will not be digested and will act as blanks to ensure that there is no background glucose in the OG samples.

4. To each tissue aliquot add 1 ml 100 mM sodium citrate pH 5.0. 5. Also prepare three microfuge tubes with controls and blanks: Add 1 ml of Na Citrate buffer to a negative control (buffer + enzyme) and buffer blank (buffer only) and add 1 ml of OG (2 mg/mL working solution) to the positive control sample tube. 6. Vortex each tube prepared in steps 8-10 vigorously until a homogenous mixture is achieved. 7. Add 100 u.1 0.5% amyloglucosidase to each sample (except the buffer blank and the third serial dilution that will serve as the OG blanks and vortex gently to mix. 8. Incubate for 2.5 h at 50°C in the shaking incubator with shaker set at 150 rpm. 9. Centrifuge samples at 10,000x g for 30 min at room temperature. 10. Transfer liquid to a new 1.5 mL microfuge tube with a 1 mL pipette being careful not to transfer any of the upper lipid layer or the cell pellet. **Or transfer liquid to a pre-labelled sample vial provided by Diagnostic Services. 11. Submit samples to Diagnostic Services for glucose determination. 12. The equation of the regression line generated with the standard curves was then used to determine glycogen concentrations, which are then standardised as follows: Mass of Glycogen (mg) / Mass of Lyophilised Tissue (mg) = Glycogen / mg dry tissue

193 Appendix F Solution Preparations

Tryptic Soy Broth with salt (TSB*):

Supplied by diagnostic services, Atlantic Veterinary College, UPEI Charlottetown PE

Ciliate Media:

Ingredients per 5 litres of stock medium:

Proteose peptone (Difco 0120-01) 50 g Bacto-Tryptone (Difco 0123-01) 50 g Yeast RNA (Sigma R-6625) 5 g Distilled water 500 ml Filtered artificial seawater (Instant Ocean) 4000 ml

Ingredients to be added to 450 ml of above before use:

Heat inactivated Fetal Bovine Serum (Gibco 12483-020) 50 ml RPMI 1640 vitamins (Sigma R-7256) 1 ml Penicillin/Streptomycin solution 5 ml (Gibco 15140-148,10000 units/ml penicillin, 10000 u.g/ml streptomycin)

Procedure to prepare 5 litres of medium (stock stored at 4°C):

1. Dissolve the proteose peptone, bacto-tryptone and yeast RNA in artificial seawater at room temperature. 2. Place media on heated stir plate and heat to 80°C until all ingredients are dissolved. 3. Place media in 1 L media bottles (do not fill above 700 ml mark). Loosely cap bottles and autoclave for 25 min at 121°C. 4. Aseptically aliquot 450 ml of ciliate culture media to 500 ml media bottles and store at 4°C. 5. Before use, add to 450ml of medium: 50 ml of heat-inactivated fetal bovine serum 1 ml of RPMI vitamin solution 5 ml of penicillin/streptomycin.

194 Sodium Citrate Buffer 100 mM pH 5 (CB):

1. 7.35 g Na Citrate (S279-500 Fisher Scientific) weighed into a plastic beaker. 2. 200 mL distilled water was added to the beaker. 3. Solution was mixed and pH was adjusted to 5 with HCI acid (Accumet® Basic AB15 Fisher Scientific). 4. Solution was transferred to a graduated cylinder and distilled water was added to 250 mL 5. Solution was then transferred to a glass bottle and stored at 4°C.

Amvloglucosidase 0.5% (AMG)

1. 5 mg Amyloglucosidase. 2. 1 mL Citrate Buffer.

195