THESIS

Feeding Energetics and Carbohydrate Digestion in Juvenile edwardsii

ABSTRACT

To enhance the on-growing of Jasus edwardsii in culture, it is important to understand the feeding physiology of juveniles. In , there is a loss of energy and an increase in oxygen consumption (specific dynamic action (SDA)) associated with feeding. The present research measured the SDA of juvenile J. edwardsii fed either in the morning or at night held at 15°C. The present research also investigated important issues affecting the successful on­ growing of rock in culture, diet and nutrition. This was achieved by investigating carbohydrate digestion and metabolism. The present research also investigated the growth of juvenile J. edwardsii in response to three different algal carbohydrates; agar, carrageenans and alginate.

Closed box respirometry was used to measure juvenile lobsters oxygen consumption (M02) and ammonia excretion. Juveniles exhibited a nocturnal rhythm in both M02 and ammonia excretion. The factorial rise in M02 (1.58±0.03 times) for lobsters fed in the morning was significantly less than lobsters fed at night (1.80±0.01 times). Lobsters fed in the morning had a significantly shorter SDA (30±1.2 h) response compared to lobsters fed at night (36±1 h). Energy loss as a result of digestion was less for lobsters fed in the morning. Therefore, if juvenile J. edwardsii are fed in the morning, they could optimise the energy content of the meal and this could result in increased growth.

At 18 °C, over 80 days juvenile J. edwardsii that were fed squid in the morning had a specific growth rate (SGR) of0.92±0.07 %body weight d- 1 and grew faster than lobsters that were fed at 1 night (SGR = 0.76±0.01 %bw d- ). Morning fed lobsters had a percent weight gain (%WG) of

107.0±3.1%, which was significantly higher than lobsters fed at night (%WG = 78.4±3.1 %). Survival was also greater for morning fed lobsters (50%) than night fed lobsters (25 %).

The SDA was used as a tool to determine if juvenile J. edwardsii could utilise a range of general carbohydrates from simple monosaccharides (glucose, fructose), disaccharides (maltose, sucrose) and polysaccharides (glycogen) (general carbohydrates). Three algal binding agents (polysaccharides) were also tested; agar, carrageenans and alginate (algal carbohydrates). For Feeding Energetics and Carbohydrate Digestion in Juvenile Jasus edwardsii ii

lobsters fed the general carbohydrates, oxygen consumption increased to a peak followed by a slow decline back to pre-feeding levels. The more complex the carbohydrate, the greater the oxygen consumption profile. However, when lobsters were fed on algal carbohydrates they did not exhibit this trend and had similar oxygen consumption profiles to unfed lobsters. Oxygen consumption magnitudes were significantly higher in lobsters fed a meal of glycogen, sucrose, maltose and agar than unfed lobsters.

If juvenile J. edwardsii were able to digest and utilise carbohydrates, there should have been a rise in haemolymph glucose concentration following feeding. The results confirm that juvenile J. edwardsii can digest all the general carbohydrates, but require more energy to digest and metabolise glycogen and sucrose. Lobsters fed agar had a significantly higher haemolymph glucose concentration than unfed lobsters. This was the only algal carbohydrate to exhibit this response as alginate and carrageenans had similar response to unfed. These results suggest that the SDA can be used as a technique for determining carbohydrate digestion in juvenile J. edwardsii and that the magnitude of the response is the most sensitive SDA parameter.

Growth rates of juvenile J. edwardsii fed algal carbohydrates were measured at 18 oc, over 80 days. Growth of lobsters fed fresh blue mussels (Mytilus galloprovincialis) was higher (SGR = 1 1.64±0.02 %bw d" ) than any of those produced with the algal carbohydrate diets. Lobsters fed a diet of mussels also had higher survival rates (87 %). Growth rates were similar amongst the 1 1 algal carbohydrates (agar SGR = 0.91±0.10 %bw d" ; carrageenans SGR = 1.14±0.04 %bw d" ; 1 alginate SGR = 0.97±0.35 %bw d" ) but were superior to lobsters fed a diet of squid (SGR = 1 0.76±0.01 %bw d" ). The survival of lobsters fed an agar (50 %) diet was significantly higher than those fed carrageenans (37.5 %) and alginate (25 %) diets. Based on these results juvenile J. edwarsii can digest and utilise agar more efficiently than carrageenans and alginate. The growth experiment, oxygen consumption and haemolymph glucose results from the present research will aid in the development of a nutritional, cost effective diet for the successful aquaculture of J. edwardsii. Acknowledgements iii

ACKNOWLEDGEMENTS

Many thanks must go to my supervisor Associate Professor Islay D. Marsden for her support throughout this study. Your promptness in returning drafts and helpful criticism were much appreciated and made my life a lot less stressful. Thank you for always having your door open and allowing me to bounce ideas off you. Due thanks must also be given to my associate supervisors, Associate Professor Bill Davison and Associate Professor Harry H. Taylor, who also gave me constructive criticism on drafts and experimental procedures.

Huge thanks must be given to Dr Andrew Jeffs of NIWA who kindly supplied me with all my , diet ingredients and papers that were not available in the library. Thank you for your constructive criticisms on drafts. Without your help, this research would not have been possible. I look forward to undertaking my PhD under your supervision in the near future.

Thank you to the technical staff of the Zoology Department for their advice and willingness to find ways around problems. Special thanks must be given to ·Gavin Robinson for advice on holding facilities and experimental equipment. You provided unlimited help throughout this project while also meeting the needs of other research students and running laboratories for undergraduates.

A HUGE BIG THANK YOU to the following people:

Dr Steven Gieseg for the use of the Biochemistry laboratory and for the help with techniques and calculations of lipid and glycogen analysis.

Liam Cassidy for showing me the initial procedures involved.

Associate Professor Laurie Greenfield from PAMS for allowing numerous protein samples to be processed in his laboratory and Jackie Healy for freeze drying my samples.

John Pirker for much appreciated advice on the grim world of statistical analysis. Acknowledgements iv

Aeron Storey and Jo Alcock for feeding and taking care of my growth experiment over the Christmas break. With out your help my growth experiment would have been a disaster.

Jan McKenzie for your help with my poster, which was a great success.

Thanks to my office buddy Melanie Bressington for your intellectual conversions regarding the estuary and my lobsters.

A big thank you to Glen Thompson for providing with valuable break time during my writing and watch out for the Chiefs - they will take out the Super 12 next year!

Last but not least, a huge thank you to my wife (Hannah) who put in the hard yards with me. Without your love and support I would not have been able to do this. Thank you for providing helpful comments and proof reading all drafts.

Thanks must also be given to friends and family for their love and support. Table v

TABLE OF CONTENTS

PAGE ABSTRACT i ACKNOWLEDGMENTS iii LIST OF FIGURES ix LIST OF TABLES xiv

CHAPTER ONE: GENERAL INTRODUCTION 1.1. Spiny Lobsters 1 1.2. Status of the Rock Fishery 3 1.3. Aquaculture 4 1.4. Nutrition 7 1.5. Feed Energetics and Specific Dynamic Action 10 1.6. Aims 11

CHAPTER TWO: SPECIFIC DYNAMIC ACTION 2.1. Introduction 13 2.2. Methodology 16 Collection and Storage 16 Experimental Design 17 Determination of Oxygen Consumption 19 Determination of Ammonia Excretion 19 Diel Rhythms 20 SDA Determination 22 Parameters and Statistical Analysis 22 2.3. Results 23 2.3.1 Effect ofTime on Oxygen Consumption 23 2.3.2 Effect of Feeding Time on Oxygen Consumption 24 2.3.3 Effect of Time on Ammonia Excretion 27 Table vi

2.3.4 Effect of Feeding Time on Ammonia Excretion 28 2.3.5 Oxygen Nitrogen Ration 29 2.4. Discussion 32 2.4.1 Oxygen Consumption 32 2.4.2 SDA Coefficient 34 2.4.3 Ammonia Excretion and O:N Ratio 34

CHAPTER THREE: OXYGEN CONSUMPTION & AMMONIA EXCRETION IN TO FEEDING DIFFERENT CARBOHYDRATES 3 .1. Introduction 37 3.2. Methodology 40 Diet Formulation 40 Experimental Design 41 Experimental Procedure 41 Determination of Oxygen Consumption 41 Determination of Ammonia Excretion 42 Statistical Analysis 42 3.3. Results 42 3.3.1 General Carbohydrates 42 Oxygen Consumption 42 Ammonia Excretion 45 3.3.2 Algae Carbohydrates 52 Oxygen Consumption 52 Ammonia Excretion 53 3.3.3 General and Algal Carbohydrates 56 Oxygen Consumption 56 Ammonia Excretion 57 3.4. Discussion 63 3.4.1 Oxygen Consumption 63 3.4.2 Ammonia Excretion 65 Table of Contents vii

CHAPTER FOUR: HAEMOLYMPH GLUCOSE 4.1. Introduction 67 4.2. Methodology 68 Experimental Design 68 Experimental Procedure 69 Haemolymph Sampling 71 Glucose Determination 71 Statistical Analysis 72 4.3. Results 72 4.3.1 General Carbohydrates 72 4.3.2 Algae Carbohydrates 77 4.3 .3 General and Algal Carbohydrates 79 4.4. Discussion 84

CHAPTER FIVE: GROWTH EXPERIMENT 5 .1. Introduction 89 5.2. Methodology 90 Collection and Storage 90 Experimental System and Water Quality 90 Preparation of Diets 92 Experiment One 93 Experiment Two 93 Experimental Procedure 94 Leaching 95 Food Intake 95 Glycogen and Lipid Analysis 96 Total Protein Analysis 98 Calculations 98 Statistical Analysis 99 Table of Contents viii

5.3. Results 100 5.3.1 Diets 100 5.3.2 Experiment One 100 Lobster Growth and Feed Consumption 100 Moults and Survival 105 Biochemical Composition 107 5.3.3 Experiment Two 108 Lobster Growth and Feed Consumption 108 Moults and Survival 116 Biochemical Composition 118 5.4. Discussion 118 5.4.1 Experiment One 118 5.4.2 Experiment Two 123 5.4.3 Conclusion 126

CHAPTER SIX: GENERAL DISCUSSION 128

REFERENCES 134 List ofFigures ix

LIST OF FIGURES

PAGE Chapter Two 2.1. Photo of the respirometer used during the experiment A Reservoir 21

syringe; B = Sampling syringe; C = Incurrent tube; D = Excurrent tube; E Incurrent to water jacket.

2.2. Average oxygen consumption of undisturbed rock lobsters (n=6). 24 Shaded areas represent night time. 0 and 24 hrs = 0800 hrs.

2.3. Oxygen consumption of lobsters fed during the day (n=6). The star 26 represents when fed lobsters' oxygen consumption was not significantly (P>0.05) different from unfed lobsters = 'duration of SDA'. The shaded areas represent the night periods.

2.4. Oxygen consumption of lobsters fed at night (n=6). The star represents 26 when fed lobsters' oxygen consumption was not significantly (P>0.05) different from unfed lobsters = 'duration of SDA'. The shaded areas represent the night periods.

2.5. Average ammonia excretion of undisturbed rock lobsters (n=6). Shaded 27

areas represent night time. 0 and 24 hrs = 0800 hrs.

2.6. Ammonia excretion of lobsters fed during the day (n=6). The star 30 represents when fed lobsters' ammonia excretion was not significantly (P>0.05) different from unfed lobsters = 'duration of SDA'. The shaded areas represent the night periods. List X

2.7. Ammonia excretion of lobsters fed during the night (n=6). The star 30 represents when fed lobsters ammonia excretion was not significantly (P>O.OS) different from unfed lobsters = 'duration of SDA'. The shaded areas represent the night periods.

2.8. Oxygen nitrogen ratios of lobsters fed during the day (n=6). The star 31 represents when the fed lobsters O:N ratio was not significantly different (P>O.OS) from base level lobsters = 'duration of SDA'. The shaded areas represent the night periods.

2.9. Oxygen nitrogen ratios of lobsters fed during the night (n=6). The star 31 represents when the fed lobsters O:N ratio was not significantly different (P>O.OS) from base level lobsters = 'duration of SDA'. The shaded areas represent the night periods.

Chapter Three 3.1. Oxygen consumption rates over time of lobster fed with different general 44 carbohydrates. Means ± S.E., n=6. The control was unfed lobsters (n=6). The solid horizontal line represents base level metabolism.

3.2. Ammonia excretion rates over time of lobsters fed with different general 46 carbohydrates. Means ± S.E., n=6. The control was unfed lobsters (n=6). The solid horizontal bar represents base level metabolism.

3.3. Oxygen consumption rates over time of lobsters fed with different algae 52 carbohydrates. Means ± S.E., n=6. The control was unfed lobsters (n=6). The solid horizontal bar represents base level metabolism. List ofFigures xi

3.4. Ammonia excretion rates over time of lobsters fed with different algae 54 carbohydrates. Means ± S.E., n=6. The control was unfed lobsters (n=6). The solid horizontal bar represents base level metabolism.

3.5. Combined oxygen consumption rates over time of lobsters fed with both 58 algae and general carbohydrates. Means± S.E., n=6. The control was unfed lobsters (n=6). The solid horizontal bar represents base level metabolism.

3.6. Combined ammonia excretion rates over time of lobsters fed with both 59 algal and general carbohydrates. Means ± S.E., n=6. The control was unfed lobsters (n=6). The solid horizontal bar represents base level metabolism.

Chapter Four 4.1. Photo of the container used for holding juvenile J. edwardsii during the 70 haemolymph glucose experiments. A = Incurrent; B = Excurrent; C = Stones used for attachment points for the lobsters.

4.2. Haemolymph glucose concentrations of lobsters in response to feeding 74 different carbohydrate diets, glucose, glycogen, maltose, fructose, sucrose, gelatine and unfed. Means ± S.E., n=6. The solid horizontal line represents base level metabolism.

4.3. Haemolymph glucose concentrations of lobsters in response to feeding 79 different algal carbohydrate sources, agar, carrageenan, alginate and unfed. Means ± S.E., n=6. The solid horizontal bar represents base level metabolism. List ofFigures xii

4.4. Haemolymph glucose concentrations of lobsters in response to feeding 81 different carbohydrates glucose, glycogen, maltose, sucrose, fructose, gelatine, agar, carrageenan, alginate and unfed. Means± S.E., n=6. The solid horizontal line represents base level metabolism.

Chapter Five 5.1. Equipment used during the experiments: A, recirculating sea water 91 system; B, 2 x 1000 L sump tanks used to hold the lobsters for each experiment; C, containers used to house the lobsters during the experiments.

5.2. Mean growth (a: wet weight; b: carapace length) of juvenile J. edwardsii 103 fed either at night or during the day over 80 days (means ± S.E.). * denotes significantly different.

5.3. Mean (± S.E.) consumption and weight gain of juvenile lobsters fed 104 either in the morning (a) or at night (b) over 80 days.

5.4. a: percentage of moults of both morning and night fed treatments over 105 80 days; b: mean survival of lobsters of both morning and night fed treatments over 80 days.

5.5. Mean growth (a: wet weight; b: carapace length) of juvenile J. edwardsii 109 fed either squid, mussels, agar, carrageenans or alginate over 80 days (means± S.E.).

5.6. Mean (± S.E.) consumption and weight gain of juvenile lobsters fed 115 either squid (a), mussel (b), agar (c), carrageenan (d) or alginate (e) over 80 days. List xiii

5. 7. a: percentage of moults of squid, mussel, agar, carrageenan and alginate 117 treatments over 80 days; b: mean survival of lobsters fed squid, mussel, agar, carrageenan and alginate treatments over 80 days.

Chapter Six 6.1. Mean carapace length of lobsters measured over 70 days. Results of 131 interest to the present research were malt (maltose), glucose, fructose, sucrose, and glycogen. Graph obtained with the permission of Jeffs and Devey. List of Tables xiv LIST OF TABLES

PAGE Chapter Two 2.1. Summary of SDA parameters of lobsters fed during the day and at night. 25 * = values significantly different at P<0.05; ** = values significantly different at P<0.001; t =significantly different to fasting metabolic rates; t = significantly different from lobsters fed at night; a = ratio of peak

metabolic rate to fasting metabolic rate; b = total post-prandial oxygen

consumed; c = the percentage of the energy content of the meal used metabolically in the SDA response. Means expressed as means±S.E.

2.2. Summary of ammonia excretion parameters of lobsters fed during the day 28 and during the night. * = values significantly different at P<0.05; ** = values significantly different at P< 0.001 level; t = significantly different to fasting metabolic rates; t = significantly different from lobsters fed at

night; a = ratio pf peak metabolic rate to fasting metabolic rate; b = total post-prandial ammonia excreted. Means expressed as means±S.E.

2.3. Summary of data for oxygen nitrogen ratio for lobsters fed during the day 29 and at night. * = values significantly different at P<0.05; ** = values significantly different at P<0.001; t = significantly different to fasting metabolic rates; t = significantly different from lobsters fed at night. Means expressed as means±S.E.

Chapter Three 3.1. Summary of oxygen consumption results comparing lobsters fed general 47

carbohydrate diets. a = The ratio of peak oxygen consumption to base

level metabolism. b =total oxygen consumed up to 12 hrs after feeding. Means followed by the same letter are not significantly different (P>0.05). * = P<0.05; ** = P<0.01; *** = P

3.2. Summary of ammonia excretion results comparing lobsters fed general 48

carbohydrate diets. a = The ratio of peak ammonia excretion to base level

metabolism. b = total oxygen consumed up to 12 hrs after feeding. Means

followed by the same letter are not significantly different (P>0.05). * = P<0.05; ** = P

3.3. Oxygen consumption and ammoma excretion of juvenile lobsters fed 49 different carbohydrates. Summary of (a) repeated measures univariate analysis of variance model (F values) between diets through time and, (b) repeated measures multivariate analysis (F values) within subjects through time and interactions between time and treatments within subjects. % Variance = percentage of the variance explained. * = P

3.4. Tukeys HSD test comparing the effect of diet (carbohydrate type) (Table 50 3.3) on oxygen consumption of lobsters fed general carbohydrates.

3.5. Tukeys HSD test comparing the effect of diet (carbohydrate type) (Table 50 3.3) on ammonia excretion oflobsters fed general carbohydrates.

3.6. Tukeys HSD test comparing the effect of time (Table 3.3) on ammonia 50 excretion of lobsters fed general carbohydrates.

3.7. Tukeys HSD test comparing the effect of time (Table 3.3) on oxygen 51 consumption oflobsters fed general carbohydrates.

3.8. Summary of oxygen consumption results comparing lobsters fed algal 53 carbohydrate diets. Means followed by the same letter are not significantly different (P>0.05). * = P<0.05; ** = P

3.9. Summary of ammonia excretion results comparing lobsters fed algal 55 carbohydrate diets. Means followed by the same letter are not significantly different (P>0.05). * = P<0.05; ** = P

3.10.Tukeys HSD test comparing the effect of diet (carbohydrate type) (Table 55 3.3) on ammonia excretion oflobsters fed algae carbohydrates.

3.11.Tukeys HSD test comparing the effect of time (Table 3.3) on ammonia 56 excretion oflobsters fed algal carbohydrates.

3.12.Summary of oxygen consumption results comparing lobsters fed both 60 algal and general based carbohydrate diets. Means in each row followed by the same letter are not significantly different (P>0.05). * P<0.05; ** = P

3.13.Tukeys HSD test comparing the effect of diet (carbohydrate type) (Table 60 3.3) on oxygen consumption of lobsters fed both the general and algal carbohydrates.

3.14.Tukeys HSD test for the effect of time (Table 3.3) on oxygen consumption 61 of lobsters fed general and algal carbohydrates.

3.15.Summary of ammonia excretion results comparing lobsters fed both algal 61 and general carbohydrate diets. Means in each row followed by the same letter are not significantly different (P>0.05). * P<0.05; ** = P

3.16.Tukeys HSD test comparing the effect of diet (carbohydrate type) (Table 62 3.3) on ammonia excretion oflobsters fed general and algal carbohydrates. 3.17.Tukeys HSD test comparing the effect of time (Table 3.3) on ammonia 62 excretion of lobsters fed general and algal carbohydrates.

Chapter Four 4.1. Haemolymph glucose results. Summary of (a) repeated measures 75 univariate analysis of variance modal (F values) between treatments through time and, (b) repeated measures multivariate analysis (F values) within subjects through time and interactions between time and treatments within subjects. * P<0.05, ** = P

4.2. Summary of haemolymph glucose parameters (means ± S.E., n=6) of 76 lobsters fed either glucose, glycogen, maltose, sucrose, fructose, gelatine or unfed control. Means followed by the same letter are not significantly

different. a = peak haemolymph glucose divided by base level metabolism.

4.3. Tukeys HSD test comparing the effect of diet (carbohydrate type) (Table 77 4.1) on haemolymph glucose concentrations of lobsters fed different general carbohydrate sources.

4.4. Tukeys HSD test comparing the effect oftime (Table 4.1) on haemolymph 77 glucose concentrations of lobsters fed general carbohydrates combined.

4.5. Summary of haemolymph glucose parameters (means ± S.E., n=6) of 78 lobsters fed either agar, carrageenen, alginate or unfed control. Means

followed by the same letter are not significantly different. a = peak haemolymph glucose divided by base level metabolism. 4.6. Tukeys HSD test comparing the effect of diet (carbohydrate type) (Table 78 4.1) on haemolymph glucose concentrations oflobsters fed different algal carbohydrates.

4.7. Tukeys HSD test comparing the effect oftime (Table 4.1) on haemolymph 79 glucose concentrations of lobsters fed algal carbohydrates combined.

4.8. Tukeys HSD test comparing the effect of time (Table 4.1) on haemolymph 80 glucose concentrations of lobsters fed algal and general carbohydrates.

4.9. Summary of haemolymph glucose parameters (means ± S.E., n=6) of 82 lobsters fed either glucose, glycogen maltose, sucrose, fructose, gelatine, agar, carrageenen, alginate or unfed. Means followed by the same letter

are not significantly different. a = peak haemolymph glucose divided by base level metabolism.

4.10.Tukeys HSD test comparing the effect of diet (carbohydrate type) (Table 83 4.1) on haemolymph glucose concentration of lobsters fed algal and general carbohydrates.

Chapter Five 5.1. Summary of dry matter percentages for each of the diets used for growth 100 experiments.

5.2. Growth, feeding rates and survival (mean± S.E.) of juvenile J. edwardsii 101 fed either in the morning or at night for 80 days. List xix

5.3. Growth experiment results. Summary of repeated measures univariate 102 analysis of variance model (F values) between treatments through time (a) and, repeated measures multivariate analysis (F values) within subjects through time and interactions between time and treatments within subjects (b). * = P<0.05, ** = P

1 5.4. Summary of mean (± S.E.) consumption rates (g dai ) (a) and weight 104 1 gains (g dai ) (b) for lobsters fed in the morning or at night over 80 days. Values followed by the same letter are not significantly different.

5.5. Percent moult increment and intermoult period (mean ± S.E.) of juvenile 106 lobsters fed either in the morning or at night over 80 days.

5.6. Percent lipid and % glycogen of the hepatopancreas, % protein of the 107 abdomen muscle and the digestive gland index (DGI) of juvenile lobsters fed either in the morning or at night over 80 days. Means ± S.E. Means followed by the same letter are not significantly different (P>0.05). Control lobsters = lobsters that were sacrificed at the start of the 80 day growth experiment.

5.7. Growth, feeding rates and survival of juvenile J. edwardsii fed either 110 squid, mussels, agar, carrageenens or alginate over 80 days (mean± S.E.).

5.8. Growth experiment results. Summary of repeated measures univariate 111 analysis of variance model (F values) between treatments through time (a) and, repeated measures multivariate analysis (F values) within subjects through time and interactions between time and treatments within subjects (b). * = P

5.9. Tukey's post hoc test comparing the effect of diet (Table 5.8) final on wet 111 weight of juvenile lobsters fed either squid, mussel, agar, carrageenens or alginate.

5.10.Tukey's post hoc test comparing the effect of time (Table 5.8) on wet 112 weight of lobsters fed either squid, mussel, agar, carrageenens or alginate.

5.11.Tukey's post hoc test comparing the effect of time (Table 5.8) on carapace 112 length of lobsters fed either squid, mussel, agar, carrageenens or alginate.

5.12.Tukey's post hoc test companng the effect of diet (Table 5.8) on 112 consumption rates of juvenile lobsters fed either squid, mussel, agar, carrageenens or alginate.

5.13.Tukey's post hoc test comparmg the effect of time (Table 5.8) on 113 consumption rates of lobsters fed either squid, mussel, agar, carrageenens or alginate.

1 5.14.Summary of mean (± S.E.) consumption rates (g daf ) (a) and weight 114 1 gains (g daf ) (b) of lobsters fed either squid, mussel, agar, carrageenen or alginate over 80 days. Values followed by the same letter are not significantly different.

5.15.Percent moult increment and intermoult period (mean± S.E.) of juvenile 114 lobsters fed squid, mussel, agar, carrageenen or alginate over 80 days. Values followed by the same letter are not significantly different. List xxi

5.16.Percent lipid and% glycogen of the hepatopancreas, %protein of the tail 119 muscle and the digestive gland index (DGI) of juvenile lobsters fed either squid, mussel, agar, carrageenen, alginate over 80 days and a control group of lobsters. Means ± S.E. Values followed by the same letter are not significantly different. Control lobsters = lobsters that were sacrificed at the start of the 80 day growth experiment.

5.17.Comparisons among several studies of the growth, survival, feed intake of 121 0+ J. edwardsii in culture. Arranged from highest SGR to lowest SGR. Chapter One: General Introduction 1

CHAPTER ONE

GENERAL INTRODUCTION

1.1. Spiny Lobsters

Spiny (or rock) lobsters belong to the family Palinuridae. There are 49 and about 33 of these support some of the largest commercial fisheries worldwide (Kanciruk, 1980). The world market for spiny lobsters is around 123,000 tonnes per year and is supplied almost entirely by wild fisheries (Jeffs and Hooker, 2000). Spiny or rock lobsters are large, mobile and benthic crustaceans (MacDiarmid, 1991 ), which are ubiquitous inhabitants of tropical and temperate seas (Kanciruk, 1980; Lipcius and Eggleston, 2000). Their name is derived from the many spines on the carapace and the basal segments of the long second antennae (Phillips et al., 1980).

In New Zealand there are two species of Palinuridae, which inhabit the shallow coastal zone, red spiny rock lobster, Jasus edwardsii and Jasus verreauxi the packhorse or green lobster. J. edwardsii occurs from the Three Kings Islands in the north to the subantarctic in the south, to the Chatham Islands in the east as well as southern Australia (Booth and Breen, 1994; Booth, 2000).

Palinurid life history reflects the predominant developmental patterns of many marine crustaceans, with the release of a pelagic larva after embryonic development. The exhibits five major phases within its life cycle; egg, phyllosoma, puerulus, juvenile and adult (Lipcius and Cobb, 1994; Lipcius and Eggleston, 2000). In J. edwardsii mating occurs shortly after the female moults (Lipcius and Cobb, 1994). The female carries broods of eggs during the breeding season which is a few months long (Booth and Phillips, 1994). The eggs are released by the female and phyllosoma hatch in spring and summer in offshore reefs inhabited by the female (Lipcius and Cobb, 1994; Lipcius and Eggleston, 2000). One: General Introduction 2

Phyllosomas moult many times, are long lived (12 -18 months) and occur in giant oceanic gyres up to hundreds of kilometres offshore (Kittaka and Booth, 1994; Jeffs et al., 1999; 2001a; 2001c; 2002). Phyllosomas are adapted for planktonic life and adult distribution is affected by the planktonic stage of their life history. Phyllosomas are poor horizontal swimmers (Lefever, 1999), but are very good vertical swimmers (Booth and Phillips, 1994). The giant oceanic gyres transports the phyllosomas from the nearshore environment to deep oceanic environment then brings them back into the nearshore again. The phyllosomas complete their last moult on the way back to the nearshore from the deep ocean (Kanciruk, 1980; Booth and Phillips, 1994; Lipcius and Cobb, 1994; Lipcius and Eggleston, 2000). The pueruli are the post larvae stage, the transition between phyllosoma and juvenile and as strong swimmers completes the oceanic journey (Booth and Phillips, 1994; Lipcius and Cobb, 1994; Lipcius and Eggleston, 2000). Pueruli settle subtidally in shallow inshore areas (Booth and Phillips, 1994; Jeffs et al., 1999). The pueruli stage does not eat, they get their energy reserves from the lipids which are accumulated during the phyllosoma stage (Jeffs et al., 1999; 2001a; 2001c; 2002). The metabolic rate of post settlement puerulus is low compared with the pre-settlement puerulus. This conserves energy reserves during the moult to the juvenile stage (Lefever, 1999).

Two to four weeks after settlement the puerulus moults into the first moult post puerulus juvenile which develops camouflage colours (Kanciruk, 1980; Booth and Kittaka, 1994; Jeffs et al., 1999). Juvenile spiny lobsters undergo at least two ecologically distinct phases. Firstly there is an early benthic phase resident primarily in habitats similar or equivalent to settlement habitat and this is followed by a later benthic phase where typical lobsters inhabit such as crevices (Lipcius and Cobb, 1994).

The deep water adult habitats differ from the juveniles' shallow bay nurseries (Kanciruk, 1980). The most characteristic feature of the habitat for many spiny lobsters is the residence area or den. Spiny lobsters distribute randomly among many available den sites and these sites are defended against predator intrusion (Kanciruk, 1980; Lefever, 1999). Environmental cues, such as temperature and light control breeding, moulting and population movements (Kanciruk, 1980). Sexual maturity is reached three to twelve years after settlement depending on location (Booth and Breen, 1994). Chapter One: General Introduction 3

Spiny lobsters such as J. edwardsii and exhibit nocturnal rhythms, showing a general peak of activity and feeding just after dusk, correlating with the departure from the den (Fielder, 1965; Lipcius and Cobb, 1994; Crear, 1998; Crear and Forteath, 2000; 2001a). Activity continues at a somewhat lower level throughout the night and is generally depressed at times of greater nocturnal illumination (Phillips et al., 1980; Lipcius and Cobb, 1994; Lipcius and Eggleston, 2000). Daily metabolic losses can be replenished during the night when they are foraging and feeding (Lefever, 1999). Mating and mass migrations occur with substantial diurnal and nocturnal incidence (MacDiarmid et al., 1991; Lipcius and Cobb, 1994; Lipcius and Eggleston, 2000).

Where diel rhythms are present in crustaceans light is the main controlling factor (Dall, 1986; Williams and Dean, 1988). Photoperiod is a reliable and widely used cue regulating cyclic processes. In numerous organisms, these processes include migration, diapause, hibernation and reproductive activity (Lefever, 1999). In lobsters this includes the migration from their dens up current to release their eggs. The form of lobster activity varies as a function of metabolic requirements as indicated by activity levels, but the locomotory activity pattern differs from that of feeding (Lefever, 1999).

1.2. Status of the New Zealand Rock Lobster Fishery

J. edwardsii is the basis of the important commercial 'red' rock lobster fishery of New Zealand (Taylor and Waldron, 1997; Booth, 2000; Jeffs and Hooker, 2000; Speed et al., 2001; Ward et al., 2003) and the southern rock lobster fishery in Australia (Phillips et al., 2000; Thomas et al., 2000; Crear and Forteath, 2000; Crear, et al., 2002; Ward et al., 2003). The fishery for the red rock lobsters is valued at over $100 million annually in New Zealand ( NZ). Spiny lobster landings in the 1998/99 seasons were 2,700 tonnes, the bulk of which (90.7%) was destined for the Asian live lobster market. Despite being spread throughout NewZealand, J. verrauxi comprises less than 1% of the landings, as it is only caught commercially in the north of the North Island (Booth, 2000). Chapter One: General Introduction 4

Natural marine resources are threatened by pressure exerted by a rapidly growmg human population. This has pushed many of New Zealand's harvested species, including spiny lobsters, to the limits of sustainability. When demand is met by the uncontrollable removal of biomass over prolonged periods, the biomass levels of the targeted species may fall to, or below, a point from which they cannot recover naturally (Sheppard, 2001).

Spiny lobster populations in New Zealand have declined steadily since the 1960s, or possibly even earlier, although records are not comprehensive enough to prove this. If the rate of harvest had been maintained at their original level, over-harvesting of the stocks would have occurred (Booth, 1994). Fortunately there were changes to fisheries legislation in 1988/89 and the fishery became a limited entry fishery for commercial fishers (Booth, 2000). The next major legislation change occurred in 1990 with the introduction of 10 Quota Management Areas (QMAs) (Booth and Breen, 1994; Booth, 2000). Each of the QMAs was allocated a total allowable commercial catch (TACC) and each fisher received a transferable term quota based on their recent catch history (Booth, 2000). Fishery stocks must now be maintained near the biomass associated with the Maximum Sustainable Yield (BMsv). Breen and Kendrick (1998) predict that most harvested areas around New Zealand should reach BMsY over the next 10- 15 years, but for this to be attained, the TACC levels will almost certainly be lower than those needed to meet current or foreseeable demand.

1.3. Aquaculture

Spiny lobsters form the basis of major fisheries throughout the world, commanding high market prices due to their high demand. Increased pressure on wild fishery resources has fuelled international interest in their culture potential (Booth and Kittaka, 1994; Hooker et al., 1997; Ward, 1999; Jeffs and Hooker, 2000; Jeffs and James, 2001). There is considerable interest in the aquaculture potential of the southem rock lobster, Jasus edwardsii, in New Zealand and southern Australia (Crear et al., 2000; Crear and Forteath, 2000).

Aquaculture is recognised internationally as an alternative to wild fisheries for certain molluscs (e.g. abalone, oysters, mussels), fish (e.g. salmon, trout, kingfish), and crustaceans (e.g. , Chapter One: General Introduction 5 , lobsters). Booth (1988) has suggested that farmed lobsters could fetch a higher market price than wild stock. This is because of the potential for better consistent supply of the spiny lobsters of particular sizes, and greater uniformity of the product. Four criteria are considered important for the culture of species (Anon, 1994). These include:

1. Controllable spawning in laboratory conditions and simple larval development; 2. Fast growth rate and high efficiency of food conversion; 3. Hardy animals that are tolerant of high densities and resistant to disease; 4. High retail price.

Spiny lobsters score well in only the last two categories. There have been recently major advances in larval rearing and understanding aspects of their biology. Improvements in the development of more efficient pueruli collection indicate that with some more research, large scale commercial aquaculture of spiny lobsters is possible (Jeffs and Hooker, 2000).

There are further criteria for culturing New Zealand crustaceans, with particular reference to site and species management (Sheppard, 2001). These include:

1. Adequate land area for grow out; 2. Ability to regulate and control salinity; 3. Suitable year-round environmental temperatures; 4. Suitable broodstock in adequate and continuing supply; 5. Adequate supply of wild or hatchery seed; 6. Market potential and ready access to those markets.

J. edwardsii fulfils 1,2 and 6 of these criteria (Sheppard, 2001). Aquaculture techniques for spiny lobsters have been previously underdeveloped because of an inability to acquire adequate numbers of new recruits (pueruli, the transitional stage between pelagic larvae and benthic juveniles) to grow to marketable size (Booth and Phillips, 1994). Typical of most spiny lobsters, J. edwardsii has a long and complex early life history (Thomas et al., 2000). The planktonic phyllosoma stage is spent dispersed by oceanic currents for a period of 12-18 months before settling as pueruli in shallow waters around the coast (Jeffs et al., 1999; 2001a; 2001c; 2002). Chapter One: General Introduction 6

Presently, it is not feasible to commercially produce pueruli in hatcheries (Kittaka, 1988; Kittaka and Booth, 1994; Illingworth et al., 1997; Tong et al., 1997; Jeffs and Hooker, 2000), because survival rates are very low and it is also not economical. The technology for commercial hatchery production of pueruli is still many years away from full development and therefore, initial development of the industry is focusing on the collection of recently settled pueruli from the wild and on growing them in culture.

For more than twenty years researchers in New Zealand have been investigating the on-growing of J. edwardsii (Jeffs and Hooker, 2000). Utilising the settlement collectors used in the calculation of recruitment statistics in fisheries models, it may be possible to collect enough pueruli for commercial purposes in certain areas (Booth and Bowring, 1998; Booth et al., 1998). The potential impact of removing pueruli from the sea has not been estimated, and conflict with people in the wild fishery is likely. However, if the survival ofpueruli is low then the removal of some pueruli for aquaculture purposes will have little impact on overall yield (Kittaka and Booth, 1994; 2000).

After collection, techniques are required to on-grow the pueruli to a marketable size of 200-400g (Jeffs and Hooker, 2000). In the wild growth to this size has been estimated to be less than three years (McKoy and Esterman, 1981 ). It has been suggested that this can be reduced to less than two years in culture conditions (Booth and Kittaka, 1994; 2000). However, existing studies on growth rates in culture suggest otherwise and Hooker et al. (1997) and Jeffs and Hooker (2000) suggested that it is more likely to take three to four years to reach marketable size in captivity.

For the aquaculture of J. edwardsii to become commercially viable, comprehensive knowledge of the biological and environmental parameters and their subsequent influence on the growth of lobsters in captivity are required (Sheppard, 2001). Initial aquaculture research was aimed at understanding the general biology of spiny lobsters. Subsequent research has focused on developing techniques for maximising growth and survival. Many factors affect spiny lobster growth in captivity. These include photoperiod where increasing and decreasing night hours from the norm reduces growth rates (Brett, 1989). Increasing water temperature to a maximum (18°C) increases growth rates (Hooker et al., 1997; Thomas et al., 2000). Shelter maintains high survival rates but does not affect growth (James et al., 2001). Eyestalk ablation decreases growth Chapter One: General Introduction 7 rates (Bunter and Westaway, 1993). Increasing stocking density from 50 to 200 m-2 decreases growth rates (James et al., 2001) and altering the diet from mussels has resulted in decreased growth rates and survival (Sheppard, 2001; Crear et al., 2002; Ward et al., 2003).

While providing variable (higher and lower) growth rates compared to wild lobsters, the research summarised above indicates that optimal growth rate conditions for culture can be identified and maximised. This suggests that with future work, not only is the commercial rearing of spiny lobsters biologically feasible, but it is also economically viable (Sheppard, 2001).

Part of the present research focuses upon diet and nutrition which is two of the most important factors known to influence growth and survival of J. edwardsii (Conklin, 1975; 1977, Crear et al., 2000; Jeffs and Hooker, 2000; Crear et al., 2002; Ward et al., 2003). It investigates which carbohydrates juvenile J. edwardsii can efficiently utilise and the effect of different carbohydrates on growth. This research would help determine which carbohydrate would be best to incorporate into an artificial diet for the culture of these lobsters.

1.4. Nutrition

Nutrition is accepted as one of the key factors controlling survival, intermoult period and growth rates in culture (Mikami et al., 1993). There is major emphasis in lobster culture research on the development of a nutritionally adequate and economically feasible diet. This must be measured in terms of growth rates, quality yield of lobster and assessed in terms of the balance between production costs and market returns (Sheppard, 2001 ).

Digestive System

Morphologically and functionally the digestive system of spiny lobsters is similar to that of other decapod crustaceans (Mikami and Takashima, 1994; 2000). It appears that juvenile J. edwardsii are well equipped to ingest and mechanically process a broad range of food items (Jeffs et al., 2001 b). The first moult juvenile J. edwardsii use their calcified mandible for grinding large calcified material (Nishida et al,. 1990). The gastric mill, with its well developed gastric teeth Chapter One: General Introduction 8 and setae, are specialised for further masticating softer food materials into smaller pieces and mixing them with digestive fluids.

Little is known about the digestive capability or the types and concentrations of digestive enzymes produced by either adult or juvenile J. edwardsii (Jeffs et al., 2001b). Juvenile J. edwardsii have a similar capability to digest protein and lipids as other crustaceans, which suggests that a wide range of enzymes (e.g. proteases and lipases) are produced (Ward, 1999; Ward et al., 2003). In order to develop species specific diets and feeding practices an understanding of the digestive enzymes present in the digestive system is paramount.

Food Preferences

In the wild, early juvenile lobsters are omnivorous, selective, generalist feeders that consume a diverse spectrum of species, dominated by molluscs and some calcareous algae (Jemakoff et al., 1993; Lipcius and Cobb, 1994; James and Tong, 1998; Jeffs et al., 2001b; Sheppard, 2001). Lobsters prefer food of a marine rather than terrestrial origin, and prefer the feed to be fresh rather than decomposing or frozen (Fielder, 1965; James and Tong, 1997). Natural food, such as mussels, do not generally satisfy all of the requirements for an aquaculture feed, such as ease of handling, storage, year round availability and uneaten material affects water quality. If only one type of natural food is used the diet lacks the ability to change composition consistency to meet the varying demands of the different growth stages of lobsters (Van Olst et al., 1980). Therefore, the strict product specifications for lobster meat (dark red exoskeleton colour and taste) required by Asian markets cannot be met. If lobsters are raised on natural diets, then additional dietary supplements, such as carotenoid pigments may need to be added (Kittaka and Booth, 1994; 2000).

Previous nutritional trials for juvenile J. edwardsii have mainly used natural food such as mussels (Chittleborough, 1975; James and Tong, 1998; Ward, 1999; Sheppard, 2001). Mussels, in particular the Greenshell™ mussel (Perna canaliculus) promotes the fastest growth rates and food conversion ratios and are generally considered to be a suitable diet for J. edwardsii on a commercial scale (James and Tong, 1998). Mussel diets have resulted in faster growth rates compared to all artificial diets developed to date. Although mussels are exempt from the Chapter One: General Introduction 9

problems associated with natural feeds (as outlined above) this is offset by the high prices to obtain and store them. Also, using fresh mussels as a feed for lobsters increases labour costs associated with feeding and cleaning tanks to avoid bacterial contamination caused by decomposing feed (Jeffs and Hooker, 2000; Jeffs et al., 2001b). Furthermore, it is difficult to ensure that all individuals are receiving the same amount of nutritionally important parts of the mussel. One lobster may feed on mussel muscle and another may get gonads and this could affect the growth rates (Sheppard, 2001).

Artificial Diet

A purified artificial diet presents an inexpensive and more convenient option for a cultured spiny · lobster feed. An ideal formulated diet in which optimum nutrient levels would be supplied by an appropriate but varying mixture of feed stuffs could be derived by the knowledge of specific dietary requirements of the lobster. This has the potential to increase growth rates (Sheppard, 2001 ). The first artificial diets tested on lobsters were those which had been designed for fish and other crustaceans (Conklin, 1975). Little is known of the specific nutritional requirements of J. edwardsii, although this is critical to their culture success (Ward, 1999; Crear et al., 2000; Kanazawa, 2000; Jeffs et al., 2001b). Smith (1998) developed a set of nutrient specifications for formulated diets for spiny lobsters. The formulation of experimental diets for J. edwardsii nutritional research has largely been based on those specifications (Ward, 1999; Crear et al., 2000; Ward et al., 2003).

There may be differences in nutritional requirements of J. edwardsii as it grows from puerulus to marketable size (Jeffs et al., 2001b). For example, the requirements for post pueruli are likely to be specific because the pueruli use large quantities of stored lipid reserves during shoreward migrations (Jeffs et al., 1999; 2001c). The ontogenetic shift in diet that is observed in the wild may indicate a phase where changes in nutritional requirements occur (Jeffs et al., 2001b).

Many studies have investigated the protein and lipid requirements of spiny lobsters (Conklin et al., 1975, Crear et al., 2000; Calvert, 2000; Glencross et al., 2001; Phelger et al., 2001; Ward et al., 2003). There has been little research devoted to the carbohydrate requirements of J. edwardsii or any spiny lobsters for that matter. Carbohydrates can play an important role in Chapter One: General Introduction 10

artificial diets. Protein is the most expensive ingredient in the artificial diet, therefore maximising the use (building of tissue) of the protein is paramount. Protein and lipids are the main energy sources in J. edwardsii. Protein can be converted to carbohydrates via the gluconeogenic pathway (Rosas et al., 2000). When insufficient energy is available from non­ protein sources, protein may be catabolized to meet the energy requirements at the cost of somatic growth. Therefore, the most efficient diets contain sufficient non-protein sources (carbohydrate and lipid) that are metabolised preferentially to protein to meet general requirements, leaving the crustacean to direct the maximum level of available protein into somatic growth- 'the protein sparing effect' (Johnston et al., 2003).

There are specific combinations of ingredients, such as proteins, lipids, vitamins and carbohydrates, that affect growth. The majority of the experiments investigating artificial diets and nutrition have determined the percentage inclusion levels of various ingredients (Britz and Retch, 1997; Ward, 1999; Calvert, 2000; Cuzon et al., 2001; Glencross et al., 2001; Ward et al., 2003). Therefore, one of the aims of the present research was to investigate which types of carbohydrate are best utilised. Carbohydrates include sugars, starches, and fibers and generally contain only the elements carbon, hydrogen and oxygen. Although carbohydrates are usually the cheapest source of energy in foods and feeds (Shiau, 1997) there is little information about their utilisation by lobsters or crustaceans.

1.5. Feeding Energetics and Specific Dynamic Action

This is the first study to investigate the feeding energetics and the specific dynamic action (SDA) of juvenile J. edwardsii in relation to its aquaculture potential. Information about feeding energetics and metabolic rates are essential in the establishment of fishery facilities for aquaculture (Buesa, 1979; Kittaka and Booth, 1994; 2000). The models developed for SDA are of relevance to aquaculturists in two ways. Firstly, the post feeding oxygen requirements in an intensive culture system can be predicted where the ration and protein levels are known and this information can be used to ensure oxygen levels in the water are adequate and secondly, in energy budgeting the amount of energy wasted or expended as SDA can be considered (Chakraborty et al., 1992b). Chapter One: General Introduction 11

The juvenile stage in the life cycle is the most important stage in aquaculture because juveniles grow faster than adults. Hooker et al (1997) concluded that 0 +size classes undergo more moults and grow faster than 1 + and 2 + size classes. Therefore, it is important to understand the feeding physiology and energetics of juveniles to enhance growth rates and promote faster development to marketable size. If growth rates are optimised for earlier stages then a shorter period will be required to get juvenile lobsters to the markets.

1.6. Aims

Little is known about the feeding physiology and carbohydrate digestion in juvenile New Zealand rock lobsters, J. edwardsii. The aims of the present research were to measure the feeding response- specific dynamic action (SDA). Oxygen consumption rates and ammonia excretion rates were measured for a food source (squid). Chapter two investigates the effect of feeding time on the SDA of juvenile J. edwardsii. Energy utilisation was also determined in relation to feeding time. The results from this research will allow aquaculturists to maximise the energy content of the diet for growth. Diel rhythms in oxygen consumption and ammonia excretion were also investigated.

Chapter three investigates the effects of vanous carbohydrates, rangmg from complex polysaccharides to simple monosaccharides, on the SDA. The aim of this chapter was to determine which carbohydrates juvenile J. edwardsii could best utilise. The SDA response was used as an indicator of digestion. The present research also was the first study that has used this technique to determine if an ingredient of an artificial food can be digested.

Chapter four investigates further whether juvenile J. edwardsii can digest and utilise the various carbohydrates tested in chapter three. If digestion occurs then haemolymph glucose concentrations should increase because glucose is the end product of carbohydrate digestion and is the carbohydrate that is absorbed across the digestive tract. LnlZDE~~r One: General Introduction 12

Chapter five growth experiment was set up to determine whether night time feeding or day time feeding gave faster growth rates. Another growth experiment was set up to determine which of the carbohydrates were best utilised and promoted faster growth rates.

The present research concentrated on aspects likely to be important in the development of an artificial diet and rearing juvenile rock lobsters in captivity. Chapter Two: Specific Dynamic Action In Juvenile Jasus edwardsii 13

CHAPTER TWO

SPECIFIC DYNAMIC ACTION IN JUVENILE JASUS EDWARDS/I

2.1. Introduction

The essential feature of living organisms is their ability to capture, transform, and store various forms of energy according to the specific instructions carried by their individual genetic material (Peusner, 1974). Energy is derived from the food an eats. Therefore, food provides two vital commodities; firstly chemicals and secondly energy (Brafield and Llewellyn, 1982). The following is the equation that integrates the elements of energetic balance (Carter and Brafield, 1991; Rosas eta/., 1998).

C=P+R+U+F

Where; C = consumption, energy from food; P = production, energy retained from food in the form of chemical bonds of the growth material; R = energy lost as heat, results from respiration; U =energy lost through nitrogen metabolism; F =energy lost in faeces.

The food an animal eats (C) provides it with two essential ingredients. Firstly, elements and compounds needed to maintain and increase structure and secondly, energy required to drive metabolic processes and operate its muscles. The energy content of food must be sufficient to provide for the energy requirement~ of the animal (Brafield and Llewellyn, 1982). Production (P) is mainly channelled into increasing the animal's biomass or into the production of gametes and embryos. Faeces (F) are formed from food material, which has not been digested, or has been digested but not assimilated (Braefield and Llewellyn, 1982). Chapter Two: Specific Dyna~ic Action In Juvenile Jasus edwardsii 14

In order to enhance Jasus edwardsii culture it is important to understand its energetics, especially oxygen consumption associated with feeding (specific dynamic action). The increase in oxygen uptake that follows a meal is termed the "specific dynamic action" (SDA). The SDA is a component of R in the equation above. The SDA has been described in a variety of animals from vertebrates, such as fish and mammals to invertebrates, such as crustaceans and molluscs (Jobling, 1983; Houlihan et al., 1990; Boyce and Clarke, 1997; Whiteley et al., 2001). In crustaceans, SDA has been described in a number of species including brachyuran (Burggren et al., 1993), isopods (Robertson et al., 2001a), and shrimps (Rosas et al., 1992). In each case the SDA response is characterised by an increase in oxygen consumption following a meal. These respiratory rates are elevated above the normal rates of fasted animals. A gradual decline back to pre-feeding levels can take a period of several hours to days (Whiteley et al., 2001).

Aerobic regeneration of ATP stores involves oxygen as the terminal electron acceptor, thus the rate of oxygen consumption is a measure of the rate at which energy reserves are being consumed to fuel ATP regeneration (Clarke, 1998). ATP production and hence oxygen consumption, are driven by the requirements for energy to fuel mechanical and biochemical work, the costs of which must be met by food or stored reserves (Ware, 1999).

The SDA reflects energy requirements of numerous physical and biochemical processes involved with digestion (Jobling, 1983; Whiteley et al., 2001). These include; manipulation and handling of the food, absorption and storage of nutrients, deamination of amino acids, synthesis of excretory products and the increase in synthesis of lipids and protein associated with growth (Jobling, 1983; Whiteley et al., 2001). After feeding there is a marked increase in the motor activity of the stomach and the intestinal tract. Although increased muscular activity and secretion of digestive enzymes contributes to the increase in metabolic rate following feeding, this contribution is usually relatively small and most of the increase is due to post absorptive effects (Jobling, 1981; 1983).

Post-absorptive processes include storage of nutrients, the deamination of amino acids, the synthesis of proteins and lipids associated with growth (Lyndon et al., 1992). If the SDA is largely from amino acid catabolism, then the efficiency of the dietary protein utilisation and thus Chapter Two: Specific Dynamic Action In Juvenile Jasus edwardsii 15 production in aquaculture might be improved if the SDA was reduced. However, if the cost represents the costs of protein anabolism (i.e. growth), it should be directly related to protein utilisation and accepted as an unavoidable cost of feeding (Lyndon et al., 1992).

The contribution of increased muscular activity of the alimentary tract to the SDA was determined to be negligible in fish (Jobling and Davis, 1980). Studies using inert diets have estimated contributions of gut peristalsis to the SDA at 10- 30%, but these may be overestimates as absorption related increases in oxygen consumption still might occur (Jobling, 1981). Clearly, the mechanical and biochemical processes of absorption are not a major component ofthe SDA.

Post-prandial metabolic rates (oxygen consumption) and rates of growth are correlated in fish and isopods (Jobling, 1981; 1983; Carefoot, 1990b). Most proteins are indispensable nutrients, they are essential to the structure and overall function of all animals, including crustaceans (Kanazawa, 1994; 2000). Protein synthesis may contribute as much as 44% and 52% of the observed post-prandial rise in oxygen consumption in fish and isopods, respectively (Jobling, 1983; Carefoot, 1990a; Lyndon et al., 1992). Protein turnover occurs at all levels of feeding, even in fasting animals. However, following a feeding event, a positive protein balance is achieved by a combination of increased protein synthesis and a decrease in the rate of endogenous protein breakdown, with the increased energy consumption proportional to that ingested (Wieser, 1994). This implies that a substantial part of the SDA represents the unavoidable costs of growth related to the synthesis of new body tissue (Jobling, 1983; Lyndon et al., 1992, Wieser, 1994).

In addition to measuring oxygen uptake, ammonia excretion rates are also important in measuring metabolic response associated with feeding (Boyce, 1999). There are two main sources of nitrogenous waste in animals. Firstly, there are endogenous wastes which are derived from the continual metabolic turnover of cellular compounds such as proteins and nucleic acids. Secondly, there are exogenous wastes which are derived from the need to excrete excess dietary amino acids, as only limited amounts can be stored in tissue (Boyce, 1999). Crustaceans like most marine invertebrates, are ammonotelic; that is, nitrogenous waste is eliminated primarily in the form of ammonia, which represents 60 - 100% of total nitrogen excreted (Binns and Peterson, 1969; Kormanik and Cameron, 1981; King et al., 1985; Greenaway, 1991; Crear and Forteath, Chapter Two: Specific Dynamic Action In Juvenile Jasus edwardsii 16

2002). Relative to other forms of nitrogenous compounds excreted by aquatic organisms, ammonia is the smallest and simplest excretory product. Its highly soluble in water and therefore, ammonia can be excreted by passive diffusion across the gills, or it can be actively excreted (Greenaway, 1991) and importantly, unlike urea or urate production no further ATP is used.

Considerable amount of research has focused on different factors that affect the SDA. However, there has not been any research addressing the affect of different feeding times. Feeding time was expected to result in a difference because there is a large difference in the metabolism of lobsters during the day compared to the night. The primary aim of the present research was to determine if feeding either at night or in the day had differing effects on the SDA of juvenile J. edwardsii. There are four parameters of the SDA that are of interest. Three of them were described by Whiteley et al. (2001). These were factorial rise (ratio of peak metabolic rate to resting metabolic rate), magnitude (the integrated post-prandial increase in oxygen consumption), and duration (length of the SDA response). The fourth parameter was the SDA coefficient (the percentage of the energy content of the meal used metabolically in the SDA response), which was described in Robertson et al. (2001a). Another aim was to determine ammonia excretion rates following a meal. The results from the present research will determine the best time to feed these crustaceans in culture.

2.2. Methodology

Collection and Storage

Juvenile Jasus edwardsii (20--40 mm carapace length) were collected from Gisbome by NIWA (National Institute of Water and Atmosphere) using pueruli collectors (A. Jeffs pers comm). They were held in Wellington and fed green lipped mussels, Perna canaliculus, twice a week until transported to Christchurch by air in sealed insulated containers containing oxygenated seawater. On arrival, the lobsters were stored in 80 litre plastic recirculating seawater containers, containing filtered seawater (13±1 °C) in the Zoology aquarium at the University of Canterbury. Chapter Two: Specific Dynamic Action In Juvenile Jasus edwardsii 17

Lobsters were maintained in the recirculating seawater aquaria for a minimum of two weeks prior to experimentation. The water temperature was maintained at 13±1 oc, pH 8.0-8.3 and salinity 33±2 %o. Salinity was checked twice a week to avoid osmotic stress. The seawater was obtained from Lyttleton Harbour (Christchurch) and was replaced as necessary. Light was maintained on a 12:12 hour light cycle.

Lobsters were fed fresh blue mussels, Mytilus galloprovincialis, weekly to maintain base level metabolism. This species was chosen because J. edwardsii can utilise this species better compared to frozen P. canaliculus (James and Tong, 1997). However, when fresh mussels were not available lobsters were provided with frozen M galloprovincialis or P. canaliculis. Each lobster was fed half a mussel in its shell. The byssus threads were removed to minimise clogging and debris in the aquaria. Any uneaten parts of mussel were removed the next day to prevent bacteria and ammonia build up.

Experimental lobsters 20-30 mm carapace length were transferred to the aquaria in the constant temperature room (15 °C) one week before experimentation for acclimation. Light was controlled with a 12h light/dark photoperiod with the light period commencing at 0800 hrs. Only intermoult (stage C) lobsters were used in the experiments. One pleopod was removed from each lobster and examined using the moult index described by Turnbull (1989) for and Oliver (2000) for J. edwardsii.

Experimental Design

Metabolic changes associated with feeding were measured by comparing the rates of oxygen consumption and ammonia excretion of fed and unfed juvenile lobsters at 15 °C.

Four respirometers with a volume of 500 ml were simultaneously used in the experiment (Figure 2.1 ). The respirometry chamber was made from Perspex tube and was fitted with a square Perspex lid with bolts in the four comers to attach it to the chamber. The lids were grooved and were fitted with a rubber gasket to provide an airtight seal. An outer perspex tube provided a temperature controlled water jacket around the main chamber. Once the system was closed the Chapter Two: Specific Dynamic Action In Juvenile Jasus edwardsii 18

water temperature was maintained at the experimental temperature. Four submerged pumps 1 (Maxi-Jet MJlOOO) supplied the respirometers with a constant flow of water (721 h- ) providing sufficient flow and mixing in the chambers to prevent hypoxia and ammonia build up. Dall (1986) outlined problems associated with measuring oxygen consumption where crustaceans are placed in smoothed walled respirometers. The problem led to increased oxygen consumption due to the animals' increased activity in trying to find attachment points. Therefore, attachment points were built into the respirometers to provide a grasping surface so that the lobsters remained quiescent in the respirometers.

Lobsters (4 sampled at one time) were starved for 3 days prior to the commencement of the experiment. In fish starvation periods of two to four times the duration of the SDA are recommended to achieve a constant post-absorptive condition (base level metabolism) (Boyce and Clark, 1997). Because there is limited literature on this measurement for J. edwardsii and other palinurid lobsters, a starvation period of 3 days was allowed. Lobsters were transferred to the respirometers 48 hrs prior to the commencement of feeding. For the first twelve hours the lobsters were left to acclimate to the conditions and the following 36 hrs a base level for metabolism was determined. Base level metabolism is defined as the cost of maintaining the body tissues in the absence of factors such as activity, growth or SDA, which may cause elevations in oxygen consumption (Peck, 1998).

The cycle of the respirometer was 15 mm shut (measuring period) and 45 min open (reoxygenation period). One sample was taken at the start of the 15 min period when the respirometer was closed and another taken at the end of the 15 min period before the respirometer was opened again. Before each sample was taken, water was taken into the syringe and pumped back into the respirometer several times to ensure that the water was thoroughly mixed. Oxygen tensions did not fall below 80 % saturation at the end of the measuring period. After each experiment the system was thoroughly cleaned with chlorine to reduce bacterial oxygen consumption. Tests with a blank chamber showed that there was no need to correct for oxygen consumption not attributed to the lobsters. Chapter Two: SRecific Dynamic Action In Juvenile Jasus edwardsii 19

Determination of Oxygen Consumption

Partial pressure of oxygen (p02) in water samples were obtained by injection of 500 Ill sample of water taken from the respirometer into an oxygen electrode cell attached to a Strathkelvin 781B oxygen meter. The p02 of the sample was taken to be the first value that remained stable after two minutes following injection into the electrode.

Oxygen consumption was calculated from the change in p02 over time, while taking into account the volume of the respirometer, the weight of the animal and the solubility coefficient of oxygen in seawater at the experimental temperature (15 °C).

Oxygen consumption (M02) was determined from the following equation:

(p021 - p02f) * a * V * 60

txW

Where; p021 was the oxygen tension at the start of the measuring period (torr); p02f was the oxygen tension at the end of the measuring period (torr); V was the volume of the respirometer (L); W was the weight of the lobster (g); a was the solubility coefficient of oxygen in sea water at 15 oc (1.6218); twas the time of the measuring period (minutes); and 60 was a conversion factor from minutes to hours.

Determination of Ammonia Excretion

The ammonia concentration of samples (2 ml) was analysed using the phenol-hypochlorite method (Solorzano, 1969), modified for use with smaller volumes. In this method the ammonia reacts with phenol and hypochlorite in alkaline solution to form indophenol blue. Sodium nitroprusside was used to intensify the colour at room temperature. The intensity of the colour was proportional to the amount of ammonia present and was measured spectrophotometrically at 640nm. Chapter Two: Specific Dynamic Action In Juvenile Jasus edwardsii 20

1 Ammonia concentration of the water samples (mg L- ) was calculated as follows:

Abs(sample)- Abs(blank) NH3 (mg L- 1) =------* [standard] Abs(standard)- Abs(blank)

These concentrations were then used to calculate total ammonia-N excretion as follows:

1000 * V * 60 * (NH3(end)- NH3(start))

t X W

Where; NH3(end) was the concentration of ammonia in the water sample at the end of the 1 measuring period (mg L- ); NH3(start) was the concentration of ammonia in the water sample at the 1 start of the measuring period (mg L- ); 1000 was the conversion factor for mg to )!g; V was the volume of the respirometer (L), 60 was the conversion factor from minutes to hours; twas the time of the measuring period (minutes); and W was the weight of the animal.

Diel Rhythm

Oxygen consumption and ammonia excretion of 12 lobsters were recorded over a minimum of 48 hrs to establish if a nocturnal rhythm was present. Samples were taken every hour for oxygen consumption readings and every two hours for ammonia excretion. This allowed the calculation of routine oxygen consumption, which was the oxygen consumption of fasting animals over a 24 hr period, including oxygen consumption resulting from spontaneous activity (Crear and Forteath, 2000). Night time oxygen consumption was calculated on all readings taken in the dark period (2000 to 0800 hrs). Oxygen consumption during the day was described as standard oxygen consumption (Crear and Forteath, 2000), as some disturbance during the day was unavoidable. Chapter Two: Specific Dynamic Action In Juvenile Jasus edwardsii 21

8

E

D c

Figure 2.1: Photo of the respirometer used during the experiment. A= Reservoir syringe; B = Sampling syringe; C = Incurrent tube; D = Excurrent tube; E = Incurrent to water jacket. Chapter Two: Specific Dynamic Action In Juvenile Jasus edwardsii 22

SDA Determination

Base level metabolism was determined from oxygen consumption and ammoma excretion readings measured over two days at 1100 and 1600 hrs on the same lobster. These times were used so that any effects of the nocturnal rhythm were avoided (Robertson et al., 2001b). Blanks were run at the start of each experiment for three hours to determine any oxygen consumption that was not attributed to the lobsters. These test runs indicated there was no need to correct for bacterial oxygen consumption.

Two lobsters were fed half an hour before the start of any experiment with commercially caught squid (3% of their body weight) while the other two unfed lobsters acted as controls. If the lobsters did not consume all the squid, the experiment was discontinued.

In the first set of experiments, 12 lobsters were used, 6 lobsters fed at 0800 hrs and 6 unfed controls. The first water samples were taken at 0830 hrs. Two water samples were taken, 1ml was taken for determination of oxygen tension and 5 ml was taken for ammonia analysis. Oxygen tension samples were taken every hour for the first 12 hrs, every 6 hrs thereafter up to 42 hrs and then hourly again up to 48 hrs. An extra 5 ml sample every 2 hrs for the first 12 then 6 hourly thereafter was frozen for ammonia determination.

In the second set of experiments the lobsters were fed during the night at 2000 hrs and the first water samples were taken at 2030 hrs. Again 12 lobsters were used, 6 were fed and 6 unfed lobsters were used as controls. The same sampling period was employed in this set of experiments as in the first.

Parameters and Statistical Analysis

The parameters of the SDA are duration of the response (length of time that the rates of oxygen consumption were elevated above basal rates), factorial scope (peak oxygen consumption relative to the basal rate), magnitude of the response (the integrated post-prandial increase in oxygen consumption) and the SDA coefficient (percentage of the energy content of the meal used Chapter Two: Specific Dynamic Action In Juvenile Jasus edwardsii 23 metabolically in the SDA response) (Jobling, 1983; Robertson et a/., 2001a; Whiteley et a/., 2001). The magnitude of the response is calculated from the area under the graph and above the base level of metabolism. When converted into energy units and expressed as a percentage of the energy content of the food ingested, the magnitude is termed the SDA coefficient (Robertson et a/., 2001a). The calorific equivalent used for the conversion was 1 J.lmol 0 2 = 0.45 J (Wieser and Medgysey, 1990). The calorific value of squid used to calculate the SDA coefficient was (3 85 kJ 1 100g- ) (www.nal.usda.gov). The oxygen: nitrogen ratio was calculated from molar atomic ratios (Mayzaud and Conover, 1988).

Data were analysed for normality and homogeneity using Kolmogorov-Smirnov and the Barletts tests, respectively. Data that were not homogenous were compared using the Mann Whitney test. Data that were homogenous were compared using t-tests and one way ANOV As (Underwood, 1997; Zar, 1999). Paired t-tests were used to evaluate if there was a nocturnal rhythm in oxygen consumption by comparing average night time oxygen consumption rate to the standard oxygen consumption rate (Crear, 1998). Students t-test was used to compare fed oxygen consumption rates to unfed oxygen consumption rates at the same time to determine the end of the SDA response (Crear, 1998). ANOVA was used to determine if there were significant differences within fed and unfed oxygen consumption rates. Barlett's test was performed on the statistical package Statistica (Version 6) and all other analyses were performed using Statistica and Microsoft EXCEL with the a set at 0.05. All means are expressed as mean±S.E.

2.3. Results

2.3.1 Effect of Time on Oxygen Consumption

Average M02 of undisturbed rock lobsters over a 48 hr period starting at 0800 hrs demonstrated a clear nocturnal rhythm (Figure 2.2). The lobsters consumed significantly more oxygen at night (t

= 2.63, P<0.05).

1 1 Routine M02 was calculated as the average of the night time (0.074±0.003 mg 0 2 g- h-· ) and day 1 1 time oxygen consumption rates (0.066±0.004 mg 0 2 g- h-· ) (Crear 1998). Routine M02 was Chapter Two: Specific Dynamic Action In Juvenile Jasus edwardsii 24

6.1% higher than standard M02• Average night time M02 was 12.1% higher than standard day time M02. During night time recordings there were two peaks of M02. Video recordings during the night showed that these periods of increased M02 were associated with increased activity.

One period of increased activity and M02 was several hours after the onset of darkness and the other several hours prior to the lights coming on.

0.120 c 0 0.100 +=ic.- e~ j 'r'"..r:. 0.080 tn' c C) 0 0.060 0 d c C) ~ ..§. 0.040 ~ 0 0.020

0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 Time (hours)

Figure 2.2: Average oxygen consumption of undisturbed rock lobsters (n=6). Shaded areas represent night time. 0 and 24 hrs = 0800 hrs.

2.3.2 Effect of Feeding Time on Oxygen Consumption

The response of M02 following a meal in J. edwardsii is shown in Figures 2.3 and 2.4 and Table 2.1. The fasting metabolic rate of lobsters before they were fed in the morning (0.064±0.001 1 1 1 mg 0 2 g-th- ) was not significantly different to lobsters fed at night (0.063±0.002 mg Oz g- h- )

(t=1.55, P=0.154). M02 increased after feeding, reaching a maximum at 18 hrs after the meal when lobsters were fed in the morning (Figure 2.3). M02 peaked at 18±2.2 h after feeding 1 1 lobsters in the morning and the peak metabolic rate was 0.114±0.006 mg 0 2 g- h- • After feeding, M02 increased 1.58±0.36 times (factorial scope) the base level M02 rate when fed in the morning. When lobsters were fed at night, the factorial scope and peak metabolic rate were Chapter Two: Specific Dynamic Action In Juvenile Jasus edwardsii 25 significantly different compared to lobsters fed in the morning (t=2.86, P<0.05 and t=2.87,

P<0.05 respectively). The time it took M02 to peak when lobsters were fed at night was significantly shorter than lobsters fed in the morning (t=5.58, P<0.001).

Table 2.1: Summary of SDA parameters of lobsters fed in the morning and at night. * =values significantly different at P<0.05; **=values significantly different at P<0.001; t =significantly different to fasting metabolic rates; t = significantly different from lobsters fed at night; a = ratio of peak metabolic rate to fasting metabolic rate; b = total post-prandial oxygen consumed; c = the percentage of the energy content of the meal used metabolically in the SDA response. Means expressed as means± S.E.

SDA PARAMETERS Morning Night 1 1 Fasting Metabolic Rate (mg 0 2 g" h- ) 0.064±0.001 0.063±0.002 1 1 Peak Metabolic Rate (mg 0 2 g· h- ) 0.114±0.006t**t* 0.12l±0.008t**

Factorial Rise a 1.582±0.036t* 1.806±0.013

Magnitude (mg 0 2) b 38.66±4.52t** 46.38±5.3 Time to Peak (hours) 18±2.2t** 3±0.3 Duration (hours) 30t** 36 Food Consumed (%body weight) 3.012±0.031 2.981±0.054

SDA Coefficient(%) c 29.3±2t* 37.9±4

The duration of the SDA was determined as a point when fed M02 rates were not significantly different from unfed rates (P>0.05). This occurred at 30 hrs when lobsters were fed in the morning and was significantly shorter than when lobsters were fed at night (36 hrs) (t=5.58, P<0.05). The magnitude of the SDA was significantly different between the two feeding times,

38.66±4.52 mg 0 2 when fed in the morning and 46.38±5.3 mg 0 2 when fed at night (t=9.93, P<0.001). The SDA coefficient (proportion of the energy of the meal consumed metabolically in the SDA response) was 29.3±2 % when lobsters were fed in the morning and this was significantly smaller than lobsters fed at night (37.9±4 %) (t=3.86, P<0.05). Chapter Two: Specific Dynamic Action In Juvenile Jasus edwardsii 26

-+- Fed c::: 0.14 Post-Prandial - Unfed 0 - 0.12 ___...._ Base Level c.- E ....:.C: 0.10 ~ .... en' c::: C) 0.08 0 N 0 0 0.06 c::: C) CD E 0.04 C)- ~ 0.02 0 0. 00 +-----IL------,-----,-----L..r------r-..,...---J-,------,----,------l."'""""""'...... ,.-.....,....~ -4 0 4 8 12 16 20 24 28 32 36 40 44 48 Time (hours after feeding)

Figure 2.3: Oxygen consumption of lobsters fed in the morning (n=6). The star represents when fed lobsters' oxygen consumption was not significantly (P>0.05) different from unfed lobsters= 'duration of SDA'. The shaded areas represent the night periods.

A:lst-prandial -+- Fed 0.14 --Unfed 0.12 ---.- Base Level

0 +---~-,-----r----~--,---,-~-----r----,-----T---,--,--~ -4 0 4 8 12 16 20 24 28 32 36 40 44 48 Time (hours after feeding)

Figure 2.4: Oxygen consumption of lobsters fed at night (n=6). The star represents when fed lobsters' oxygen consumption was not significantly (P>0.05) different from unfed lob~ters = 'duration of SDA'. The shaded areas represent the night periods. Chapter Two: Specific Dynamic Action In Juvenile Jasus edwardsii 27

Lobsters consumed significantly (t=3.00, P<0.05) more oxygen in the first 12 hrs when fed at night compared to lobsters that were fed in the morning. Night fed lobsters also consumed more oxygen in the second 12 hrs (t=2.54, P<0.05). During this time period lobsters fed in the morning consumed more oxygen compared to lobsters fed at night. Therefore, the nocturnal rhythm had a significant effect on the SDA response.

2.3.3 Effect of Time on Ammonia Excretion

Average ammonia excretion of undisturbed lobsters starved over a 48 hr period had a nocturnal component and with ammonia excretion at night significantly higher than during the day (t=3.04, P<0.05) (Figure 2.5). Night time rates were up to three times there recorded day time rates. Average night time ammonia excretion was 37.6 % higher than standard day time ammonia excretion. Routine ammonia excretion was calculated as the average of standard (3.17±0.58 J..Lg 1 1 1 1 TAN g- h- ) and night time (5.08±0.41 J..lg TAN g- h- ) excretion rates and was 23.1 % higher than standard day time rates. Peak ammonia excretion occurred four hours after the onset of darkness in both dark periods.

11 10 •• '\I c:::: ·--0 9 C1) ....• 8 -... J: ><(,) ....• 7 w C) z 6 ca <( 5 c:::: t- 4 0 C) E :::1. 3 E - 2 <( 1 0 0 2 4 6 8 1012141618202224262830323436384042444648 Time (hours)

Figure 2.5: Average ammonia excretion of undisturbed rock lobsters (n=6). Shaded areas represent night time. 0 and 24 hrs = 0800 hrs. Chapter Two: Specific Dynamic Action In Juvenile Jasus edwardsii 28

2.3.4 Effect of Feeding Time on Ammonia Excretion

The response of ammonia excretion following a meal in J. edwardsii is shown in Figures 2.6 and 2.7 and Table 2.2. After feeding, ammonia excretion increased immediately. Fasting rates of ammonia excretion were similar for lobsters fed in the morning and at night (t=0.09, P=0.97), however, both were significantly different from peak ammonia excretion rates (t=9.87, P<0.001). Peak ammonia excretion of lobsters fed in the morning was significantly higher than lobsters fed at night (t=5.54, P<0.05). Day time feeders showed a greater factorial rise (16.2±1.73 cf 12.1±1.98) (t=2.86, P<0.05) and reached a peak earlier than night time feeders (t=7.00, P<0.001). After the peak, ammonia excretion rates slowly declined toward base level rates. The duration of the response was the length of time ammonia excretion rates take to return to pre-feeding levels. This was 18 h for morning feeders, which was significantly shorter (t=3.01, P<0.001) than for night time feeders (36 h). The magnitude of ammonia excretion of lobsters fed in the morning (11.18±3.3 mg TAN) and at night (14.28± 2.9 mg TAN) were similar (t=0.07, P=0.93).

Table 2.2: Summary of ammonia excretion parameters of lobsters fed in the morning and at night. *=values significantly different at P<0.05; **=values significantly different at P< 0.001 level; t = significantly different to fasting metabolic rates; t = significantly different from lobsters fed at night; a = ratio of peak metabolic rate to fasting metabolic rate; b = total post­ prandial ammonia excreted. Means expressed as mean± S.E.

SDA PARAMETERS Morning Night 1 Fasting Metabolic Rate (ug TANg-'h- ) 1.82±0.21 1.96±0.39 1 Peak Metabolic Rate (ug TANg-'h- ) 29.64±2.81 t** t* 23.83±2.18t**

Factorial Rise3 16.21±1.73t* 12.11±1.98 Magnitude (mg TAN)b 11.18±3.3 14.28±2.9 Time to Peak (hours) 4±0.9t** 6±1 Duration (hours) 18±** 36 Chapter Two: Specific Dynamic Action In Juvenile Jasus edwardsii 29

2.3.5 Oxygen: Nitrogen Ratio

Oxygen:Nitrogen ratios of lobsters following a meal in the morning and at night decrease to a minimum then slowly increase and return to base level metabolism (Figures 2.8 and 2.9 and Table 2.3). The fasted ratios for lobsters fed in the morning and at night were similar (20.29±1.97 and 20.68±1.67, respectively) (t=1.34, P>0.05). At both feeding times, post-prandial O:N ratios declined and reached their lowest values rapidly (O:N =2.95±1.21 within 4±0.9 hours when fed in the morning and O:N=2.8±0.73 within 6±1.34 hours when fed at night). When lobsters were fed in the morning, O:N ratios returned to fasted levels within 18 hrs, whereas lobsters fed at night returned to fasted levels significantly later (24±1.3 hours) (t=3.87, P<0.05).

Table 2.3: Summary of data for oxygen nitrogen ratio for lobsters fed during the day and at night. *=values significantly different at P<0.05; **=values significantly different at P<0.001; t = significantly different to fasting metabolic rates; t = significantly different from lobsters fed at night. Means expressed as mean± S.E.

Parameters DAY NIGHT Fasting Ratio 20.29 ±3.18 20.68±4.54 Peak Deflection 2.95±1.21 t** 2.8±0.73 t**

Time to Peak (hrs) 4±0.9 t** 6±1.3 Duration (hrs) 18 t* 24 Chapter Two: Specific Dynamic Action In Juvenile Jasus edwardsii 30

"iii :a Post-prandial 39 c 1 y ' ~ . ' ' .. '< ~\., -'. : 36 '?­ ,1' t: 33 ~ -+- Fed a.. .2- 30 --Unfed Cl) .....• -... J: 27 ___.....__ Base Level (J'";' >< C') 24 w z 21 ca

Figure 2.6: Ammonia excretion of lobsters fed in the morning (n=6). The star represents when fed lobsters' ammonia excretion was not significantly (P>0.05) different from unfed lobsters = 'duration of SDA'. The shaded areas represent the night periods.

A:>st-prandial [ij :g ~..,..,....IT"m"',.,....,~""'l !!! 9- 1!! a..

-+- Fed --Unfed ___.....__ Base Level

-3 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 Time (hours after feeding)

Figure 2.7: Ammonia excretion of lobsters fed during the night (n=6). The star represents when fed lobsters ammonia excretion was not significantly (P>0.05) different from unfed lobsters = 'duration of SDA'. The shaded areas represent the night periods. Chapter Two: Specific Dynamic Action In Juvenile Jasus edwardsii 31

60 55 Post-Prandial 50 -+- fed 45 ---..- Base Level 0 - Unfed ; 40 ns 35 ~ 30 z 25 0 20 . 15 10 5 0 -3 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 Time (hours after feeding)

Figure 2.8: Oxygen nitrogen ratios of lobsters fed in the morning (n=6). The star represents when the fed lobsters O:N ratio was not significantly different (P>0.05) from base level lobsters = 'duration of SDA'. The shaded areas represent the night periods.

1\j Fbs t- prandial 70 '6 -+- Fed c 65 ~ ---..- Base Level 60 9- 55 a.~ - Unfed 50 0 ; 45 ns 40 ~ 35 z 30 0 25 20 15 10 5 0 -3 0 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 Time (hours after feeding)

Figure 2.9: Oxygen nitrogen ratios of lobsters fed during the night (n=6). The star represents when the fed lobsters O:N ratio was not significantly different (P>0.05) from base level·Iobsters ='duration ofSDA'. The shaded areas represent the night periods. Chapter Two: Specific Dynamic Action In Juvenile Jasus edwardsii 32

2.4. Discussion

2.4.1 Oxygen Consumption

Juvenile J. edwardsii, like other subtidal decapods (Binns and Peterson, 1969; Ansell, 1973; Du Prez, 1983; Dall, 1986; Naylor, 1988; Crear, 1998; Crear and Forteath, 2000) exhibited an increase in oxygen consumption associated with activity at night. The nocturnal rhythm was consistent with their behaviour patterns (Ansell, 1973; Dall, 1986; Naylor, 1988; Crear and Forteath, 2000). The routine rate of oxygen consumption was 6.1 % above standard oxygen consumption. This was similar to the routine rate calculated by Dall (1983) for (6-12 %). However, Crear and Forteath (2000) calculated the routine oxygen consumption rate to be 24 % than standard oxygen consumption for adult J. edwardsii, which was quadruple the value calculated for juveniles.

Where diel rhythms are present in crustaceans, light is the main controlling factor (Dall, 1986; Williams and Dean, 1988). J. edwardsii commence foraging just before dusk and continue to forage throughout the night, ceasing at dawn (Fielder, 1965). This foraging pattern was not consistent with patterns of oxygen consumption in the present research. Juvenile J. edwardsii demonstrated a crepuscular oxygen rhythm, with a peak at dusk and dawn, and lower rates at night and during the day. These patterns were similar to the activity patterns of the western rock lobster, Panulirus cygnus (Jernakoff, 1987).

Post-prandial (after food ingestion) increases in oxygen consumption have previously been reported in fish (Jobling, 1981; 1983; Chakraborty et al., 1992b) and crustaceans (Houlihan et al., 1990; Carefoot, 1990a; 1990b; 1990c; Robertson et al., 2001a; 2001b). The increase in oxygen consumption following food ingestion is considered to be associated with the extra energy required for transportation of food down the alimentary tract, its digestion, absorption and post­ metabolic processing (Jobling, 1981; 1983). For the , the first peak in post-prandial oxygen consumption was due to activity and feeding processes, whilst the second peak was due to digestive and absorptive activity (Du Prez et al., 1992). In juvenile J. edwardsii, the initial peak of oxygen consumption at the start of the post-prandial period was Chapter Two: Specific Dynamic Action In Juvenile Jasus edwardsii 33 most probably related to the transport of the food down the alimentary tract. The next peak in oxygen consumption, which occurred a few hours later, was most likely associated with digestion and absorption processes (Jobling, 1981). Food elicited a strong locomotor response in juvenile J. edwardsii and this was also found in adult lobsters by Crear and Forteath (2000).

Many factors, including ration size, type of food, species and temperature, affect the size and duration of the SDA. Generally fish exhibit the classic SDA response, where oxygen consumption peaks at 2-3 times base level metabolism, with the peak occurring within 12 hrs after feeding and which has a duration of 24--36 hrs (Jobling, 1981; Boyce and Clarke, 1997). Both juvenile (present research) and adult (Crear and Forteath 2000) J. edwardsii exhibit a classic SDA response. There are many other decapods that also show a classic SDA. Panulirus cygnus, had a 2.19 fold increase in oxygen consumption 8-10 hrs following a meal, returning to normal within 46 hrs (Crear and Forteath, 2001a). Carcinus maenus had a 2.3 fold increase in oxygen consumption 3 hrs following a meal, returning to normal within 24 hrs (Houlihan et al., 1990) and had a 3.8 fold increase in oxygen consumption taking 6-9 hrs to reach the peak, and 24 hrs to return to normal (Ansell, 1973).

Post-prandial increases in oxygen consumption (1.5-1.8 times) reported in the present research were less than those reported in fish (Chakraborty et al., 1992b; Boyce and Clarke, 1997), small crustaceans Glyptonotus antarcticus (Robertson et al., 2001a) and Saduria entomon (Robertson et al., 2001b), which ranged between 2-3 times base level. However, the factorial scope (ratio of peak metabolic rate to fasting metabolic rate) was similar to that reported by Crear and Forteath (2000) for adult J. edwardsii. The factorial scope differed between lobsters fed in the morning (1.582±0.036) and at night (1.806±0.013). The duration of the SDA also differed between morning (30±1.2 h) and night (36±1 h). The difference was due to the time the lobsters were fed and this therefore, was another factor that affects the size and duration of the SDA. Lobsters were more active at night than day and consequently their oxygen consumption was greater. At night, the increased oxygen consumption is not only a result of the SDA, but also a result of the increased activity.

Duration of the SDA, peak oxygen consumption, ·magnitude and time to peak were all significantly affected by the time lobsters were fed. When the lobsters were fed in the morning, Chapter Two: Specific Dynamic Action In Juvenile Jasus edwardsii 34

the magnitude (38.66±4.52 mg 0 2) was lower than when they were fed at night (46.38±5.3 mg

0 2). When the magnitude of the SDA was converted into energy units, this showed that lobsters fed in the morning used less energy during the SDA compared to lobsters fed at night. Therefore, morning fed lobsters had more ingested energy available for growth and anabolic processes (Robertson et al., 2001a).

2.4.2 SDA Coefficient

This was the first study to measure the SDA coefficient of rock lobsters. The proportion of energy consumed in a meal that was utilised in the SDA represents the SDA coefficient (i.e. percent loss of energy from the meal) (Boyce and Clarke, 1997). Although the consumed rations were the same when fed in the morning and at night, the SDA coefficients were different (29.3±2.0% when fed in the morning, 37.9±4.1% when fed at night). The SDA coefficient for juvenile J. edwardsii was large compared with 2.1 % for Glyptonotus antarcticus (Robertson et al., 2001a) and 1 %for Parborlasia corrugatus (Clarke and Prothero-Thomas, 1997). In some fish the SDA coefficient ranges from 6-21 % (Peck and Veal, 2001). However, in fish the coefficient was affected by ration size; the smaller the ration the higher the SDA coefficient (Boyce and Clarke, 1997). In the present research the ration sizes were considerably smaller compared to the above studies, which could be a possible reason why the high values were calculated. This doesn't account for the differences between morning fed lobsters and night fed lobsters however, and implications can be taken from the results. When fed at night, lobsters used significantly more energy compared to when fed in the morning. Therefore, different feeding times affect the way juvenile J. edwardsii use the energy in the ingested food. Due to lobsters displaying increased activity at night compared with the day, more energy can be used for growth and other anabolic reactions if fed in the morning.

2.4.3 Ammonia Excretion and O:N Ratios

The nocturnal pattern of ammonia excretion in juvenile J. edwardsii showed the expected response. That was, night time oxygen consumption increased and was associated with a night time increase in ammonia excretion rates. Nocturnal increases in ammonia excretion rates have Chapter Two: Specific Dynamic Action In Juvenile Jasus edwardsii 35

been noted in several other crustacean species, P. cygnus (Crear, 1998), J. lalandii (Zoutendyk, 1987), Penaeus esculentus (Dall and Smith, 1986), P. japonicus (Marangos et al., 1990), (Carvalho and Phan, 1997) and adultJ. edwardsii (Crear, 1998).

The pattern of ammonia excretion in response to feeding was similar to that for oxygen consumption - a rapid increase up to a peak, followed by a slow decline back to pre-feeding levels over time. There has been limited research into ammonia excretion of juveniles of large decapods. However, in large adult lobsters J. lalandii, there was a 7.7 fold increase in ammonia excretion with a duration of 10 hrs (Zoutendyk, 1987). Homarus americanus had a 4 fold increase with a duration of 18 hrs (Wickins, 1987) and J. edwardsii had a 6.28 fold increase with a duration of 26 hrs (Crear, 1998). There was a major difference between the factorial rise in ammonia excretion and duration of the response between juvenile J. edwardsii and adults. The present research showed that juvenile J. edwardsii had a factorial rise of 16.21±1.74 with the duration being 18 hrs, when they were fed in the morning. Only the results from lobsters fed in the morning can be compared to Crear (1998), because he did not measure the effects of feeding at night. The factorial scope was greater and the duration of the SDA was shorter for juvenile J. edwardsii compared to adults. Therefore, juvenile J. edwardsii that were fed in the morning assimilate the same percentage of food much more quickly compared to adults that were fed at the same time. This suggests that juveniles grow faster than adults. The author obtained a specific growth rate of 1.64 %bwd- 1 for juvenile J. edwardsii in the weight range 0.77-2.83 g. Hooker et al (1997) obtained a specific growth rate of0.12 %bwd-1 for J. edwardsii in the weight range 115.7- 180g. This confirms that as J. edwardsii increase in body mass, their growth rate slows.

When the factorial rise and the duration of the response were compared between juvenile J. edwardsii fed in the morning and at night, the duration was shorter and the factorial rise was larger when fed in the morning. Thus it takes less time to assimilate the same size meal when fed in the morning. Lobsters remain inactive during the day so all the extra oxygen associated with the SDA was used in digestion, which explains why the duration of the SDA will be reduced. Lobsters were fed similar sized meals regardless of when they were fed, therefore total ammonia excretion would be expected to be similar despite the different feeding times. The results supported this. Chapter Two: Specific Dynamic Action In Juvenile Jasus edwardsii 36

In fasted juvenile J. edwardsii the O:N ratios were constant following 3 days starvation (20.29±3.18 during the day and 20.68±4.54 at night). This contrasts with other fasted crustaceans where the O:N ratios have been high and variable, Glyptonotus antarcticus (51.40±14.61) (Robertson eta/., 2001a) and Saduria entomon (42.63±6.74) (Robertson eta/., 2001b). Mayzaud and Conover (1988) reported O:N ratios of between 3-15 for total protein digestion, ratios between 16-60 as equal protein and lipid digestion, while ratios >60 represent carbohydrate digestion. The fasted O:N ratios suggest that juvenile J. edwardsii rely on a mixture of lipid and protein for their energy requirements. After feeding, the O:N ratio fell immediately and remained low until the response had returned to normal. This suggests that protein was the major substrate used for energy during the SDA and thereafter with lipids and carbohydrates becoming more important as metabolic substrates.

In conclusion, feeding time has important implications on the SDA of juvenile J. edwardsii, particularly on the duration of the response, factorial rise and the SDA coefficient. When juvenile J. edwardsii were fed in the morning the duration of the SDA, the factorial rise and the SDA coefficient were smaller than lobsters fed at night. Digestion proceeded faster when lobsters were fed in the morning and they utilised the meal more efficiently. The present research is of importance to lobster aquaculture. It is suggested that lobster culturists should feed their animals in the morning because the SDA is shorter and the lobsters utilise the meal more efficiently. These results need to be confirmed with a growth experiment (see chapter five). More research needs to be conducted on the SDA of juvenile J. edwardsii, particularly what effects different meal sizes, different food types and the effects of temperature have. Further research is also needed on adults of this species on all of the above, particularly the effects of different feeding times. This could be of importance for the storage and maintenance of this species in aquaculture, prior to live export. Chapter Three: Oxygen Consumption & Ammonia Excretion in Relation to Feeding 37 Different CHAPER THREE

OXYGEN CONSUMPTION & AMMONIA EXCRETION IN RELATON TO FEEDING DIFFERENT CARBOHYDRATES

3.1. Introduction

A non invasive way to determine whether a vertebrate or invertebrate is able to digest particular food is to measure its oxygen consumption and ammonia excretion after ingesting a meal. The increase in oxygen consumption that follows a meal is known as the 'specific dynamic action' (SDA) (Jobling, 1981; 1983; Houlihan et al., 1990; Boyce and Clarke, 1997; Whiteley et al., 2001). In crustaceans, SDA has been described in a number of species, including brachyuran crabs (Burggren et al., 1993), isopods (Robertson et al., 200la), and shrimps (Rosas et al., 1992). Oxygen consumption rates that follow feeding are elevated above that of fasted animals and then they gradually decline back to pre-feeding levels over several hours to days (Whiteley et al., 2001).

SDA reflects the energy requirements of numerous physical and biochemical processes involved with digestion (Jobling, 1983; Whiteley et al., 2001). These include; manipulation and handling of the food, absorption and storage of nutrients, deamination of amino acids, synthesis of excretory products and the increase in synthesis of lipids and protein associated with growth

(Jobling, 1992; Whiteley et al., 2001). The growing evidence linking post-prandi~l oxygen consumption with growth rates of crustaceans (Robertson et al., 2001a; 2001b) suggests the possibility that measurements of metabolic rate could be used as a research technique in the estimation of nutritional value of different dietary formulations (Job ling, 1981 ). Formulated diets can be manipulated by excluding certain key components such as protein, lipid or carbohydrates. Chapter Three: Oxygen Consumption & Ammonia Excretion in Relation to Feeding 38 Different Carbohydrates.

In addition to measuring oxygen uptake, ammonia excretion rates are also important in measuring metabolic response associated with feeding (Boyce, 1999). Therefore, oxygen consumption and ammonia excretion following a meal of a manipulated diet could indicate whether the certain ingredients in the meal are being digested an metabolised.

Ammonia accounts for 60-100 % of the total excreted nitrogen and in decapods the majority of this is excreted via the gill epithelium (Binns and Peterson, 1969; Kormanik and Cameron, 1981; Greenaway, 1991; Rosas et al., 2000; Crear and Forteath, 2002). Along with an increase in oxygen consumption associated with feeding there . is also increased ammonia excretion. Ammonia results from the catabolism of protein particularly from deamination and transamination reactions (Kormanik and Cameron, 1981; Greenaway, 1991).

The utilisation of carbohydrates by aquatic species varies and seems to be less efficient than in terrestrial domesticated animals (Shiau, 1997). However, crustaceans exhibit better ability to degrade carbohydrates compared to other classes of invertebrates (Kristensen, 1972). Simple sugars (monosaccharides) are poorly utilised in shrimp (Andrews et al., 1972; Deshimaru and Yone, 1979; Abel-Rahman et al., 1979; Alava and Pascual, 1987; Shiau and Peng, 1992). The utilisation of different carbohydrates and their dietary protein sparing effect in Penaeus monodon was investigated by Shiau and Peng (1992). They found survival rates of prawns fed starch and dextrin were higher than those fed glucose. Best protein utilisation was obtained with starch while there was poor protein utilisation in diets containing glucose. Therefore, it appears that starch has a better protein sparing effect than dextrin or glucose. Based on these studies, starch has become the typical carbohydrate source in formulated diets for crustaceans (Verri et al., 2001).

Lobsters, shrimps and prawns can utilise proteins as a source of energy. Proteins can be converted to carbohydrates by the gluconeogenic pathway (Rosas et al., 2000). Knowing which, and how much, carbohydrate to add to formulated feeds can cause a protein sparing effect. This results in the carbohydrate being used for energy and the protein being used for somatic growth, therefore optimises the use of the protein and will reduce feed costs. Chapter Three: Oxygen Consumption & Ammonia Excretion in Relation to Feeding 39 Different

The general trend in crustaceans is that as the carbohydrates get more complex (monosaccharides to disaccharides to polysaccharides) survival and utilisation increase. However, there are species-specific effects. In fish, the trend is reversed, simple sugars are used much more efficiently than complex ones (Shiau, 1997). Starch is not hydrolysed to the same extent in rock lobsters and is poorly utilised compared to prawns and shrimps (Verri et at., 2001). Therefore, there needs to be more research to determine which sources of carbohydrates can be utilised by spiny lobsters and which carbohydrates result in a protein sparing effect.

Crustacean hyperglycaemic hormone (CHH) produced by neurosecretory cells in the X-organ sinus gland in the eyestalk, is the major regulator of carbohydrate metabolism (Santos and Colares, 1986; Santos and Keller, 1993; Hall and Van Ham, 1998; Verri et at., 2001). It was first discovered over 50 years ago, by Abramowitz and Hisaw (1944). The main target tissues of CHH are the hepatopancreas and the abdominal muscles. It is believed that the main actions of CHH are the interconversion of phosphorylase b to phosphorylase a, which results in the release of glucose into the blood and amylases from the midgut gland (Santos and Keller, 1993; Hall and Van Ham, 1998). There are also external factors, such as photoperiod, temperature, nutrition and stress that affect the release of CHH (Santos and Colares, 1986; Santos and Keller, 1993; Hall and Van Ham, 1998).

Although dietary carbohydrates are the most economical source of energy in food (Shiau and Peng, 1992; Cruz-Suarez et at., 1994; Shiau, 1997; Rosas et at., 2000; Gonzalez-Pena et at., 2002), little is known about their utilisation or digestion by rock lobsters. This was the first study to determine which carbohydrates juvenile J. edwardsii were able to digest, and to use the SDA as a technique to assess digestibility. There have been a few studies on shrimps and prawns (Rosas et at., 1995; 2001; Cuzon et at., 2001b). However, these studies have focused only on the dietary inclusion level, for example, whether 10%, 15%, 25% or 50% carbohydrate levels in the diet promoted faster growth and not types of dietary carbohydrate sources. The carbohydrates used in the present research were glycogen, maltose, sucrose, fructose and glucose and these were termed 'general carbohydrates'. Agar, carrageenans and alginate were also used and were described as 'algal carbohydrates'. Digestion was measured by measuring the SDA (oxygen Chapter Three: Oxygen Consumption & Ammonia Excretion in Relation to Feeding 40 Different Carbohydrates.

consumption and ammonia excretion) in relation to feeding juvenile J. edwardsii a meal containing the various carbohydrates.

3.2. Methodology

Diet Formulation

Five general carbohydrate jelly diets were formulated from sucrose, fructose, maltose, glucose and glycogen. Glycogen was from greenshell mussels (Perna canaliculis), sucrose from sugar cane, maltose from grain, glucose and fructose from plant sources. Three carbohydrates extracted from algal were also used, agar ( Gracilaria and Gelidium ), alginate (brown algal) and carrageenans (red algal). All carbohydrates were supplied by NIWA. The diets were made into a jelly using gelatine (protein) as the gelling agent. A plain gelatine jelly was used as the control for the experiment so therefore there were six diets to analyse for the general carbohydrates. The jellies were made by adding 4.5 g of gelatine to 100 ml of hot water, followed by 4.5 g of the carbohydrate once the gelatine had dissolved. This produced a 50:50 ratio of gelatine to carbohydrate. Once the carbohydrate had dissolved, the mixture was then placed in ice cube trays and placed into a refrigerator where the jellies were allowed to set and were stored.

The carbohydrates extracted from the algal were not mixed with gelatine because they are gelling agents themselves. These carbohydrates were mixed with differing quantities of water to form a gel to get a consistent firmness. The agar (4.5 g) was dissolved in 60 ml of water; 1.5 g of carrageenans dissolved in 40 ml of water; and 3 g of alginate was dissolved in 80 ml of water. Agar and carrageenans were allowed to set in ice cube trays placed in the refrigerator. Alginate was extruded through a 3 mm diameter tube into a water bath of 2% calcium chloride for five minutes and allowed to set. This allowed the jelly to set firmer. All the prepared gels were stored in a refrigerator. Chapter Three: Oxygen Consumption & Ammonia Excretion in Relation to Feeding 41 Different

Experimental Design

The experimental design used here was the same as described in Chapter 2 (Figure 2.1 ).

Experimental Procedure

Base level metabolism was determined by taking oxygen consumption readings over two days at 1100 hrs and 1600 hrs. These times were used to avoid the effects of any nocturnal rhythm (Robertson et al., 2001b). The control was an empty respirometer without a lobster in it Controls were run at the start of each experiment for three hours to determine any oxygen consumption that was not attributed to the lobsters. These test runs indicated there was no need to correct for bacterial oxygen consumption.

Three lobsters were fed jelly meals (approximately 3 % body weight) half an hour before the start of the experiment at 0800 hrs and a fourth lobster was unfed. Lobsters were stimulated to eat the meal by coating the jelly in the seawater from an opened blue mussel (Mytilus galloprovincialis). If the lobsters did not consume all the available food the results were not included. For each of the diets, 6 lobsters were used to measure oxygen consumption and ammonia excretion. The first water samples were taken at 0830 hrs and two samples were taken. A 1 ml sample was taken for determination of oxygen tension and 5 ml was taken for ammonia analysis. Further oxygen tension samples were taken every hour for the first 12 hrs. The next sample was then taken 12 hrs later at 0830 hrs, which was the end of the experiment. Ammonia samples were taken every 2 hrs for the first 12 hrs with the next sample taken 12 hrs later at 0830 hrs.

Determination of Oxygen Consumption

The same procedure was used here as described in Chapter 2. Chapter Three: Oxygen Consumption & Ammonia Excretion in Relation to Feeding 42 Different

Determination of Ammonia Excretion

The same procedure was used here as described in Chapter 2.

Statistical Analysis

Before any statistical analyses were conducted on the data, Kolmogorov-Smirnov test were used to test for normality. No significant differences were recorded, therefore transformations were not necessary. Homogeneity of variances was verified with the Bartlett's test and again transformations were not necessary. Repeated measures ANOVA was used to test for significant differences between diet, time and the interaction (Diet x Time). Comparisons of means following ANOVA were done using the Tukey-HSD when significant differences were observed (Underwood, 1997; Zar, 1999). The general and algal carbohydrates were analysed separately at first. Then the data were combined to test for differences between the general and algal carbohydrates. All analyses were performed using the Statistica (Version 6) statistical package with the a set at 0.05 except where stated otherwise. All means were expressed as mean ± SE unless otherwise stated.

3.3. Results

3.3.1 General Carbohydrates

Oxygen Consumption

Oxygen consumption following a meal of a general carbohydrate diet in juvenile J. edwardsii was elevated above base level metabolism (Figure 3.1 and Table 3.1). The amount of food consumed was similar for each different carbohydrate diet (F0.05). Base level metabolism was not significantly different among treatment groups (F0.05). For each of the meals, oxygen consumption decreased from 0 to 1 hr after feeding. After this oxygen consumption increased significantly to a peak then decreased over time (F=6.81, P

The repeated measures interaction (Diet x Time) was not significantly different (F=1.128, P>0.05) (Table 3.3). Overall, repeated measures analysis indicated there was a significant difference in oxygen consumption between diets (carbohydrate type) (F=3.237, P<0.05) (Table 3.3). Lobsters oxygen consumption following a meal of glycogen, sucrose and maltose were significantly higher than gelatine, glucose and unfed lobsters (Table 3.4). Following a meal of glycogen or sucrose oxygen consumption was significantly higher than fructose. After a meal of glucose or fructose oxygen uptake was similar to the controls of gelatine and unfed lobsters. Time after feeding was also significantly different (F=9.05, P< c:: i;l'< (l)(JQ 0, 0.10 Sucrose 1-1 (1) ., g ::l ·- .... ----.- Glucose ..... (1 a.- 0.09 (jO ---+--- Maltose ~ ::l e-:J J:: 0.08 g-~ -t- Unfed ::rS ~n-1 c:: 0) 0.07 '<"S.c...... 1-1 0 J 01 IP\tLi ;i>f \f ±~ -Mf/ .L ~ ~= f - pre feeding e;. ::l 8 cS 0.06 &1l Ro ~ e o.os ~- 0.04 fr;;· ~ 0.03 ~ 0.02 a0 0.01 §' s· 0.00 ~ ~...... -4 -2 0 2 4 6 8 10 12 14 16 18 20 22 24 g Time (hours after feeding) s a~ Figure 3.1: Oxygen consumption rates over time of lobster fed with different general carbohydrates. Means± S.E., n=6. The control ~· was unfed lobsters (n=6). The solid horizontal line represents base level metabolism.

"'" Chapter Three: Oxygen Consumption & Ammonia Excretion in Relation to Feeding 45 Different Carbohydrates.

Ammonia Excretion

Ammonia excretion following a meal of a general carbohydrate diet in juvenile J. edwardsii increased above base level metabolism (Figure 3.2). Base level metabolism was the same for all diets (F<0.001, P>0.05) (Table 3.2). Ammonia excretion increased significantly (F=35.29, P<0.001) after feeding to a peak except for the unfed animals. Peak ammonia excretion rates ranged from 12.82±0.98 f.lg TAN g- 1 h-1 for glycogen fed lobsters to 5.12±1.01 f.lg TAN g-1 h- 1 for unfed lobsters. All the dietary treatments had significantly higher peak rates compared to unfed lobsters. The time taken for ammonia excretion to peak for each of the diets was significantly different (F=34.17, P<0.001). Ammonia excretion of lobsters fed sucrose was significantly longer to all other diets, while ammonia excretion following a meal of gelatine, glucose, fructose and glycogen were similar. Time taken for ammonia excretion to peak ranged from 2±0.5 hrs for maltose and unfed lobsters to 12±0.4 hrs for sucrose fed lobsters. The factorial rise in ammonia excretion was also significantly different (F=5.12, P<0.05) with glycogen and glucose fed lobsters being higher to all other treatments. Lobsters fed a meal of glycogen had the largest factorial rise in ammonia excretion (7.04±1.28) and unfed lobsters had the smallest (2.81±1.81). The magnitude of ammonia excretion ranged from 1.09±0.07 mg TAN for unfed lobsters to 3.10±0.06 mg TAN for lobsters fed a meal of glycogen (Table 3.2). The magnitude also differed (F=99.57, P<0.001) between treatments, with unfed lobsters having a smaller magnitude to all other treatments. Lobsters fed a meal of glycogen and sucrose had a similar magnitude but was higher to all other treatments.

Overall, there was an insignificant interaction (Det x Time) (F=0.610, P>0.05), a significant difference in lobster ammonia excretion between diets (carbohydrate type) (F=2.831, P<0.05) and time after feeding (F=16.739, P< Q. ~'< 13 ~()Q (1) (1) c: 12 -n::I ::I -+--- Gelatine no .2..,_ - 11 ~ ::I -e-- Fructose §"~ ~ 10 _ .....><._ :c - ~ ::r.§ ~ '< ...... u-)(' ---.- Glycogen C) 9 ' ' =r s· w ~- Sucrose ~::I " ' ns z 8 ' ~ Ro " ----.- Glucose ·-c: 1-<( 7 ~ 0 C) ------Maltose 3 6 0 E :::l --+- Unfed ::I..... E- 5 ~ <( - Pre Feeding (")~ 4 1-t (1) 3 ...... s· ::I...... 2 ::I ~ 1 (1) -...... ~ 0 0 ::I ...... -4 0 2 4 0 -2 6 8 10 12 14 16 18 20 22 24 I ~ (1) (1) Time (hours after feeding) ...... 0.. I ~

Figure 3.2: Aminonia excretion rates over time oflobsters fed with different general carbohydrates. Means± S.E., n=6. The control

~ was unfed lobsters (n=6). The solid horizontal bar represents base level metabolism. 0"1 Table 3.1: Summary of oxygen consumption results comparing lobsters fed general carbohydrate diets. a = The ratio of peak Q -§ oxygen consumption to base level metabolism. b = total oxygen consumed up to 12 hrs after feeding. Means followed by the same letter ~ are not significantly different (P>0.05). * = P<0.05; ** = P

Time to Peak (hrs) 4±0.5c 6±0.2d 1±0.3f 3±0.5c 12±0.2a 4±0.5c 4±0.6c 40.56*** J::; Magnitude (mg 0 2)b 22.09± 24.19 ± 24.60± 33.29 ± 32.72± 26.82 ± 24.30± 173.7*** ~ tn-· 0.52b 0.34c 0.12c 0.24a 0.19a 0.21d 0.51c ~ ""! Food Consumed 3±0.2a 3±0.1a 3±0.1a 3.1±0.3a 3±0.2a 3±0.3a <0.001 !l (%Body Weight) g· ::r a~ 0::;-· 0 >'!j (!) (!) 0.. ~-

~ -.) Table 3.2: Summary of ammonia excretion results comparing lobsters fed general carbohydrate diets. a = The ratio of peak ammonia Q excretion to base level metabolism. b = total ammonia excreted up to 12 hrs after feeding. Means followed by the same letter are not -§ ~ significantly different (P>0.05). * = P<0.05; ** = P<0.01; *** = P

~ 00 Chapter Three: Oxygen Consumption and Ammonia Excretion in Relation to Feeding 49 Different

Table 3.3: Oxygen consumption and ammonia excretion of juvenile lobsters fed different carbohydrates. Summary of (a) repeated measures univariate analysis of variance model (F values) between diets through time and, (b) repeated measures multivariate analysis (F values) within subjects through time and interactions between time and treatments within subjects. % Variance percentage of the variance explained. * P<0.05; ** = P<0.01; *** = P

Diet Between MS F Variance 02 General 6,35 8.49 3.23* 32 Carbohydrate 0 2 Consumption Algal 3,20 <0.01 0.14 2 Carbohydrate NH3 Excretion General 6,35 121.26 2.83* 30 Carbohydrate NH3 Excretion Algal 3,20 88.80 7.69** 54 Carbohydrate 02 Consumption all diets 9,50 6.95 2.54* 27 NH3 Excretion - all diets 9,50 105.63 3.06** 37 (a)

TIME TIME* DIET Within Subjects Wilks df F % Wilks df F % Variance Variance 02 Consumption 0.163 13,23 9.05*** 20 0.065 78,132 1.12 15 General Carbohydrate 02 Consumption 0.161 13,8 3.20 10 0.077 39,24 0.86 9 Algal Carbohydrate NH3 Excretion 0.198 7,29 16.73*** 23 0.057 42,139 0.Q1 14 General Carbohydrate NH3 Excretion 0.342 7,14 3.84* 17 0.167 21,40 1.67 12 Algal Carbohydrate 02 Consumption 0.222 13,38 10.2*** 33 0.057 117,297 1.24 19 all diets NH3 Excretion - all 0.251 7,44 18.7* 21 0.085 63,253 0.45 17 diets (b) Chapter Three: Oxygen Consumption and Ammonia Excretion in Relation to Feeding 50 Different

Table 3.4: Tukeys HSD test comparing the effect of diet (carbohydrate type) (Table 3.3) on oxygen consumption oflobsters fed general carbohydrates.

Gelatine Glucose Fructose Glycogen Sucrose Maltose Unfed Gelatine N.S. N.S. <0.05 <0.01 <0.05 N.S. Glucose N.S. <0.05 <0.01 N.S. N.S. Fructose N.S. <0.05 N.S. N.S. Glycogen N.S. N.S. <0.05 Sucrose N.S. <0.01 Maltose N.S. Unfed

Table 3.5: Tukeys HSD test comparing the effect of diet (carbohydrate type) (Table 3.3) on ammonia excretion oflobsters fed general carbohydrates.

Unfed Gelatine Glucose Maltose Sucrose Fructose Unfed <0.01 <0.01 <0.05 <0.05 <0.01 Gelatine N.S. N.S. N.S. N.S. Glucose N.S. N.S. N.S. Maltose N.S. N.S. Sucrose N.S. Glycogen N.S. Fructose

Table 3.6: Tukeys HSD test comparing the effect of time (Table 3.3) on ammonia excretion of lobsters fed general carbohydrates.

0 2 4 6 8 12 24 0 <0.0001 <0.001 <0.01 N.S. N.S. N.S. <0.05 2 N.S. N.S. <0.01 <0.0001 <0.001 <0.0001 4 N.S. <0.001 <0.0001 <0.0001 <0.0001 6 N.S. <0.01 <0.05 <0.0001 8 N.S. N.S. <0.001 10 N.S. <0.05 12 <0.01 24 Table 3.7: Tukeys HSD test comparing the effect of time (Table 3.3) on oxygen consumption oflobsters fed general carbohydrates. Q -§ &' 0 1 2 3 4 5 6 7 8 9 10 11 12 24 ~ <0.01 <0.0001 <0.01 <0.05 <0.0001 <0.001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.01 <0.0001 0 ~ 1 N.S. N.S. N.S. N.S. N.S. N.S. <0.01 <0.01 <0.0001 <0.05 N.S. <0.0001 ~ 2 N.S. N.S. N.S. N.S. N.S. <0.05 <0.05 <0.001 N.S. N.S. <0.0001 oo 3 N.S. <0.05 N.S. N.S. <0.001 <0.001 <0.0001 <0.01 N.S. <0.0001 -·~'< :>< 4 <0.05 N.S. N.S. <0.001 <0.001 <0.0001 <0.01 N.S. <0.0001 CD(Jq '"I CD 5 N.S. N.S. N.S. N.S. <0.05 N.S. N.S. <0.0001 CD ::I 6 N.S. <0.01 <0.01 <0.001 <0.05 N.S. <0.0001 S.n (')0 7 <0.05 <0.05 <0.01 N.S. N.S. <0.0001 ~ ::I 8 N.S. N.S. N.S. <0.001 <0.01 &~ 0 3 9 N.S. N.S. <0.001 <0.05 ::r'U '< ...... 10 N.S. <0.0001 N.S. 0.. -· 11 <0.01 <0.01 E.§ 12 <0.0001 ~ Ro 24

i§ ~-· ~ ag· s· ~ g·~ s >:rj CD CD 0.. JJ"

V'l..... Chapter Three: Oxygen Consumption & Ammonia Excretion in Relation to Feeding 52 Different Carbohydrates.

3.3.2. Algal Carbohydrates

Oxygen Consumption

Oxygen consumption of juvenile J. edwardsii following a meal of an algal carbohydrate diet did not increase significantly (F=2.17, P>0.05) (Figure 3.3 and Table 3.8). The amount of food consumed of each carbohydrate diet was similar (F0.05). Base level metabolism was similar for all three carbohydrate diets and unfed lobsters (F0.05). As with general carbohydrates, oxygen consumption of lobsters fed algal carbohydrates decreased over the first hour after feeding. Subsequently, oxygen consumption increased to a peak (6±1.5 hrs for agar; 4±0.5 hrs for carrageenans; 5±0.6 hrs for alginate) for all three diets. The time taken for oxygen consumption to peak and the factorial rise was not significantly different among diets (F=l.72, P>0.05; F=2.11, P>0.05 respectively).

-+- Agar 0.11 Post-Prandial ----- Carrageenen ";:"- .c 0.1 ----A- Alginate ...I 0') N 0.09 -- unfed 0 ~ Pre Feeding 0') 0.08 E 0.07 -s::::: 0 :,;::; 0.06 0.. E 0.05 ::::J tn s::::: 0.04 0 0 0.03 s::::: C1) 0') 0.02 >o >< 0.01 0 0 -4 -2 0 2 4 6 8 10 12 14 16 18 20 22 24 Time (hours after feeding)

Figure 3.3: Oxygen consumption rates over time of lobsters fed with different algal carbohydrates. Means ± S.E., n=6. The control was unfed lobsters (n=6). The solid horizontal bar represents base level metabolism. Chapter Three: Oxygen Consumption & Ammonia Excretion in Relation to Feeding 53 Different

There was a significant difference (F=6.28, P

23.85±0.17 mg 0 2 for carrageenan fed lobsters.

Overall, there was no significant difference in the interaction (Diet x Time) (F=0.861, P>0.05) for oxygen consumption. Diet (carbohydrate type) (F=0.861, P>0.05) and time after feeding (F=3.205, P>0.05) also exhibited no significant difference (Table 3.3). Faeces were found in the respirometers approximately 3 hrs after feeding when fed alginate and carrageenan diets, and 8 hrs after feeding when fed the agar diet compared to no faeces observed for the general carbohydrates.

Table 3.8: Summary of oxygen consumption results comparing lobsters fed algal carbohydrate diets. Means followed by the same letter are not significantly different (P>0.05). * P<0.05; ** P

Agar Carrageenans Alginate Unfed F Base Level 0.064±0.003a 0.061±0.004a 0.065±0.002a 0.064±0.0033a <0.001 Metabolism 1 1 (mg Ozg" h" ) Peak Oxygen 0.084±0.008a 0.077±0.007a 0.071±0.005a 0.076±0.004a 2.17 Consumption 1 1 (mg Ozg- h" ) Factorial Rise 1.31±0.09a 1.2±0.12a 1.11±0.15a 1.19±0.03a 2.11 Time to Peak (hrs) 6±1.5a 4±0.5a 5±0.6a 4±0.6a 1.72 Magnitude (mg Oz) 25.85±0.31a 23.85±0.17b 24.30±0.30b 24.30±0.51 b 6.28** Food Consumed 3±0.3a 3.1±0.1a 2.9±0.4a <0.001 (%Body Weight)

Ammonia Excretion

In juvenile J. edwardsii ammonia excretion following a meal of an algal carbohydrate diet was increased (Figure 3.4 and Table 3.9). Base level metabolism was similar between all three carbohydrate diets and the unfed lobsters (F0.05). For all three algal carbohydrate diets, ammonia excretion increased for the first 2 hrs after feeding. After 4 hrs, Chapter Three: Oxygen Consumption & Ammonia Excretion in Relation to Feeding 54 Different Carbohydrates. ammonia excretion peaked and differed between treatments (F=44.74, P<0.001), with agar being significantly higher to alginate and unfed lobsters. Alginate and carrageenans fed lobsters were similar, however, they differed from unfed lobsters. Peak ammonia excretion ranged from 8.94±0.76 )lg TAN g-1 h- 1 for agar fed lobsters to 5.12±1.01 )lg TAN g-1 h- 1 in unfed lobsters. The time taken for ammonia excretion to peak differed between treatments also (F=13.43, P

11 Post prandial -+- Agar 10 ---- Carrageenans --6--- Alginate 1: 9 ·--0 8 --"'-- Unfed -...Q)• .....c: ____...._ Pre Feeding (J..- 7 >< I w C) z 6 ca <( 5 1: 1- 0 C) 4 E :I E 3 <( - 2 1 0 ' --- -4 -2 0 2 4 6 8 10 12 14 16 18 20 22 24 Time (hours after feeding)

Figure 3.4: Ammonia excretion rates over time of lobsters fed with different algal carbohydrates. Means ± S.E., n=6. The control was unfed lobsters (n=6). The solid horizontal bar represents base level metabolism. Chapter Three: Oxygen Consumption & Ammonia Excretion in Relation to Feeding 55 Different Carbohydrates.

Overall, the interaction (Diet x Time) (F=1.677, P>0.05) was insignificant. However, there was a significant difference in ammonia excretion amongst diets (carbohydrate type) (F=7 .699, P<0.001) and time after feeding (F=3.844, P<0.05) (Table 3.3). Lobsters ammonia excretion following a meal of agar was significantly higher than for alginate and unfed lobsters. Carrageenans and alginate fed lobsters also had significantly higher ammonia excretion than unfed lobsters (Table 3.10). Time after feeding significantly affected ammonia excretion rates, (Table 3.11 ), with 24 hrs after feeding being significantly different to all the other times.

Table 3.9: Summary of ammonia excretion results comparing lobsters fed algal carbohydrate diets. Means followed by the same letter are not significantly different (P>0.05). * = P<0.05; ** = P<0.01; *** = P

Agar Carrageenans Alginate Unfed F Base Level Metabolism 1.81±0.03a 1.85±0.02a 1.82±0.04a 1.82±0.02a <0.001 1 1 (J.tg NH3g- h- )

Peak Oxygen Consumption 8.94±0.76a 7.07±0.64b 6.65±0.41b 5.12±1.01c 44.74*** 1 1 (J.tg NH3g- h- )

Factorial Rise 4.91±0.46a 3.88±0.17b 3.65±0.34bc 2.8±1.81c 11.69***

Time To Peak (H) 6±1.2a 2±0.7b 2±0.4b 2±0.6b 13.43***

Magnitude (mg NH3) 2.56±0.06a 1.55±0.17b 2.05±0.05c 1.09±0.07d 91.92***

Table 3.10: Tukeys HSD test comparing the effect of diet (carbohydrate type) (Table 3.3) on ammonia excretion of lobsters fed algal carbohydrates.

Agar Carrageenans Alginate Unfed Agar <0.01 N.S. <0.0001 Carrageenans N.S. <0.05 Alginate <0.05 Unfed Chapter Three: Oxygen Consumption & Ammonia Excretion in Relation to Feeding 56 Different

Table 3.11: Tukeys HSD test comparing the effect of time (Table 3.3) on ammonia excretion of lobsters fed algal carbohydrates.

Time 0 2 4 6 8 10 12 0 N.S. N.S. N.S. N.S. N.S. 2 <0.01 N.S. <0.01 <0.01 <0.01 <0.0001 4 N.S. N.S. N.S. N.S. <0.05 6 N.S. N.S. N.S. <0.001 8 N.S. N.S. <0.05 10 N.S. <0.05 12 <0.05 24

3.3.3. General and Algal Carbohydrates

Oxygen Consumption

The time course in oxygen consumption following a meal of a carbohydrate diet in juvenile J. edwardsii can be seen in Figure 3.5 and Table 3.12. Base level metabolism was similar for all ten treatments (F0.05). The amount of diet consumed was also similar (F<0.01, P>0.05). Peak oxygen consumption was significantly different (F=50.789, P<0.05). Peak oxygen consumption of lobsters fed glycogen and sucrose were significantly higher from the other 8 treatments. The time taken for oxygen consumption to peak differed between treatments (F=66.257, P<0.05). Sucrose was significantly longer to all other treatments. Sucrose fed lobsters peaked at 12±0.2 hrs and all the other treatments peaked within 6 hrs. The factorial rise in oxygen consumption was also significantly different (F= 56.749, P<0.05). Lobsters fed a meal of glycogen had the greatest factorial rise (1.74±0.06) and was significantly higher compared to all the other treatments. The magnitude of oxygen consumption differed (F=146, P

There was no significant effect exhibited by the interaction (Diet x Time) (F=1.24, P>0.05). However, diet (carbohydrate type) had a significant effect (F=2.547, P<0.05) on oxygen Chapter Three: Oxygen Consumption & Ammonia Excretion in Relation to Feeding 57 Different Carbohydrates. consumption over time. There were only two treatments that were significantly higher than the unfed treatment (Table 3.3), glycogen and sucrose fed lobsters (Table 3.13). Oxygen consumption following a meal of glycogen and sucrose were also significantly different from the algal carbohydrates carrageenans, agar, and alginate. Time after feeding had significant effects on oxygen consumption (F=l0.2, P<0.001) (Table 3.14) (Table 3.3).

Ammonia Excretion

The time course for ammonia excretion following a meal of a carbohydrate diet in juvenile J. edwardsii can be seen in Figure 3.6 and Table 3.15. Base level metabolism was similar between treatments (F<0.01, P>0.05). Peak ammonia excretion was significantly different (F=84.078, P<0.001), with all treatments being significantly higher (P<0.05) compared to unfed lobsters. Peak ammonia excretion for lobsters fed carrageenans and alginate were lower than all the general carbohydrate treatments. The time taken for ammonia excretion to peak was significantly different (F=28.011, P<0.001). Time taken for ammonia excretion to peak following a meal of sucrose took the longest (12±0.4 hrs) and was significantly longer than all the other treatments. Lobsters fed a meal of glycogen showed the greatest factorial rise (7.04±1.28) and this was significantly higher (F=14.74, P<0.05) than all the algal treatments, unfed, gelatine and sucrose treatments. Agar was the only algal carbohydrate treatment that was not significantly different from some of the general carbohydrate treatments. The magnitude of ammonia excretion differed (F=93.29, P< 0 ~'< ---.- Maltose (llfJQ ;:::;...... -... '"I (II c...... 0.080 (II 1:1 -+- Agar an E :C ()0 Sll 1:1 :::l.,.... 0.070 - Carrageenans '"I Cll tn• c:t't: c: C) 0.060 - Alginate 0 s 0 N J··~ · ·· · ~"S.0...... Unfed '"I 0 ~ 1:1 0c: ~ 0.050 ---pre feeding ~ Re C1) C) §. 0.040 ~ >< ig 0 0.030 Sll-· tyj 0.020 Q (II ::t. 0 0.010 1:1 s· 0. 0 0 0 -t--,---+--,-----,----,------,-----,---,-----,------,------,----,------,-- ~ -~...... -4 -2 0 2 4 6 8 10 12 14 16 18 20 22 24 0 1:1 ...... Time (hours after feeding) 0 "!j (II (II Figure 3.5: Combined oxygen consumption rates over time oflobsters fed with both algal and general carbohydrates. See earlier 0.. ~- figures for error bars they have been removed for ease of viewing, n=6. The control was unfed lobsters (n=6). The solid horizontal bar represents base l~vel metabolism.

Vl 00 g -§ --+-- Gelatine 14 ro Post-Prandial ~ '6 c - Glucose ~ a..~ I I \ ------..- Fructose ~ 12 ~ I t~O a.. - -- Glycogen ..... ;.d c: ~<...::: (l)(Jq .2 ~10 --- sucrose '-I (1) .... I g :::3 Cl).c: __._ Maltose .-+-('1 .... ('10 (.) ."';" 8 -+- Agar ~ :::3 >< C) a.~ - Carrageenans g..g wz 'a ::t. - Alginate '-I 0 .~

Vl \0 Table 3.12: Summary of oxygen consumption results comparing lobsters fed both algal and general based carbohydrate diets. Means in g each row followed by the same letter are not significantly different (P>0.05). * = P<0.05; ** = P<0.01; *** = P

Gelatine Glucose Fructose Glyco~en Sucrose Maltose A~ar Carra~eenans Al~nate Unfed F ...... tiO :>< Base Level Metabolism 0.062± 0.061 ± 0.064± 0.064± 0.065± 0.063 ± 0.064± 0.061 ± 0.064± 0.064± <0.001 (l){Jq:::::S'< 1 1 (mg 0 g· h. ) 0.003a 0.002a 0.001a 0.001a 0.006a 0.005a 0.003a 0.004a 0.002a 0.003a "'1 (1) 2 g ::l Peak Oxygen Consumption 0.069± 0.078± 0.089± 0.111 ± 0.098 ± 0.086± 0.084± 0.077± 0.071 ± 0.076± 50.78* 1 1 -n (mg 0 g· h. ) 0.008b 0.006bc 0.001c 0.01a 0.005a O.Ollb 0.008b 0.007b 0.005b 0.004b (")0 2 ~ ::l Factorial Rise 1.08 ± 1.22± 1.39± 1.74± 1.53 ± 1.34± 1.31 ± 1.20± 0.12d 1.11 ± 1.19± 56.74* &~ 0.02e 0.05d 0.01f 0.06a 0.07b 0.01c 0.09c 0.15e 0.03d 0 ::r'-os Time to Peak (h) 4±0.5c 6±0.2b 1±0.3d 3±0.5ce 12±0.2a 4±0.5be 6±1.5b 4±0.5de 5±0.6ce 4±0.6ce 66.25* '<0.. -...... "'1 0 Magnitude (mg 0 2) 22.09± 24.19± 24.60± 33.29 ± 32.72± 26.82± 25.85± 23.85 ± 24.30± 24.30± 146*** e_::l 0.31b 0.34 c 0.12c 0.24a 0.19a 0.21d 0.31d 0.17bc 0.17c 0.30bc ~ Ro Food Consumed(% BW) 3±0.2a 3±0.1a 3±0.la 3.1±0.3a 3±0.2a 3±0.3a 3±0.3a 2.9±0.1a 3.1±0.4a <0.001 . i 0 Table 3.13: Tukeys HSD test comparing the effect of diet (carbohydrate type) (Table 3.3) on oxygen consumption oflobsters fed both ::l...... ~ the general and algal carbohydrates. "'1~ ...... ~ 0 Gelatine Glucose Fructose Glycogen Sucrose Maltose Agar Carageenens Alginate Unfed s·::l Gelatine N.S. N.S. <0.05 <0.01 <0.05 N.S N.S N.S N.S. Glucose N.S. <0.05 <0.01 N.S. N.S N.S N.S N.S. ~ -a...... Fructose N.S. <0.05 N.S. N.S N.S N.S N.S. 0 Glycogen N.S. N.S. <0.05 <0.05 <0.05 <0.05 ::l 0 Sucrose N.S. <0.05 <0.01 <0.05 <0.01 - ~ Maltose N.S N.S N.S N.S. (1) 0.. Agar N.S N.S N.S ~- Carageenens N.S N.S Alginate N.S Unfed

0'\ 0 Table 3.14: Tukeys HSD test for the effect of time (Table 3.3) on oxygen consumption oflobsters fed general and algal carbohydrates. Q -§ &' 0 1 2 3 4 5 6 7 8 9 10 11 12 24 ~ 0 <0.001 <0.01 N.S. N.S. <0.05 <0.01 <0.05 <0.01 <0.0001 <0.0001 <0.0001 <0.05 <0.0001 ~ 1 N.S. N.S. <0.05 N.S. N.S. N.S. N.S. N.S. <0.05 N.S. <0.01 <0.0001 ~ 2 N.S. N.S. N.S. N.S. N.S. N.S. <0.05 <0.05 N.S. <0.05 <0.0001 oo...... ;>< 3 N.S. N.S. N.S. N.S. <0.05 <0.001 <0.001 <0.05 N.S. <0.0001 i;l'< (I)(JQ 4 N.S. N.S. N.S. <0.001 <0.0001 <0.0001 <0.01 N.S. <0.0001 '""! (I) (I) ::s 5 N.S. N.S. N.S. <0.05 <0.05 N.S. <0.05 <0.0001 an 6 N.S. <0.05 <0.05 <0.001 N.S. N.S. <0.0001 ()0 $l:> ::s 7 N.S. <0.05 <0.05 N.S. <0.05 <0.0001 '""! c:ll N.S. N.S. N.S. <0.0001 <0.0001 d"'t:: 8 0 s 9 N.S. N.S. <0.0001 <0.05 ::T"' '<0...... 10 N.S. <0.0001 <0.05 '""! 0 11 <0.0001 <0.0001 a ::s 12 <0.0001 &g Ro 24 . i 0 ::s...... Table 3.15: Summary of ammonia excretion results comparing lobsters fed both algal and general carbohydrate diets. Means in each row $l:>

followed by the same letter are not significantly different (P>0.05). * = P<0.05; ** = P

...... 0'1 Table 3.16: Tukeys HSD test comparing the effect of diet (carbohydrate type) (Table 3.3) on ammonia excretion oflobsters fed general and Q algal carbohydrates. >§ ~ ;;~ Gelatine Glucose Fructose Glyc()gen Sucrose Maltose Carageenens Alginate Unfed ~ Gelatine N.S. N.S. N.S. N.S. N.S. <0.05 <0.05 <0.05 <0.05 uo Glucose N.S. N.S. N.S. N.S. <0.05 <0.05 <0.05 <0.001 ..... :X (1)!]1:1~'< Fructose N.S. N.S. N.S. <0.05 <0.05 <0.05 <0.001 ""'! (!> N.S. N.S. <0.05 <0.05 <0.05 <0.001 g...... ()= Glycogen no Sucrose N.S. <0.05 <0.05 <0.05 <0.001 ~ = Maltose N.S. N.S. <0.001 <0.001 &~ 0 s Agar N.S. N.S. <0.001 ~~c...... Carageen ens N.S. <0.05 ""'! 0 !!! = Alginate <0.05 ~ Ro Unfed . i 0 Table 3.17: Tukeys HSD test comparing the effect of time (Table 3.3) on ammonia excretion oflobsters fed general and algal = -·~ carbohydrates. ~ .....~ =-·0 0 2 4 6 8 10 12 24 ::r 0 <0.001 <0.001 <0.01 N.S. N.S. N.S. <0.0001 fi: 2 N.S. N.S. <0.0001 <0.0001 <0.0001 <0.0001 !!!..... 4 N.S. <0.01 <0.001 <0.01 <0.0001 0 6 <0.05 <0.01 <0.01 <0.0001 0= 8 N.S. N.S. <0.0001 ~ 10 N.S. <0.0001 8.. 12 <0.0001 JJ. 24

""N Chapter Three: Oxygen Consumption & Ammonia Excretion in Relation to Feeding 63 Different Carbohydrates.

There was no significant interaction observed (Diet x Time) (F=0.415, P>0.05). Diet (carbohydrate type) had a significant (F=3.062, P<0.01) effect on ammonia excretion (Table 3.3) . .The unfed treatment was significantly different to all the treatments except carrageenans and alginate treatments (Table 3.17). Time after feeding (Table 3.17) also exhibited a significant effect (F=18.702, P<0.001).

3.4. Discussion

It is known from studies on the specific dynamic action (SDA) (Jobling, 1981; Houlihan et al., 1990; Chakraborty et al., 1992a; Boyce and Clarke, 1997; Robertson et al., 2001; Whiteley et al., 2001) that ingestion of a meal is followed by an increase in oxygen consumption and ammonia excretion. This was the first study to use oxygen consumption and ammonia excretion rates as indicators of digestion and metabolism in crustaceans fed different carbohydrates.

Oxygen Consumption

The gelatine control diet did not affect oxygen consumption of lobsters. However, the inclusion of different carbohydrates with the gelatine did significantly effect oxygen consumption. It was postulated that this was related to the digestion and metabolisation of the carbohydrate components. Each of the general carbohydrate diets resulted in a rise in oxygen consumption of lobsters following feeding. Oxygen consumption remained high for up to 8 hrs following feeding on the glycogen diet. Feeding on the sucrose diet resulted in oxygen consumption remaining high for up to 12 hrs. The maltose diet resulted in high oxygen consumption rates for up to 4 hrs after feeding with gelatine and glucose not having a great effect. Carefoot (1990a) showed that altering the amino acid quantity in the diet of Ligia pallasii resulted in an increase in oxygen consumption. Coulson and Hernandez (1979) showed that increase in oxygen consumption of alligators following feeding varied with different types of protein and their amino acid balances. The present research showed that feeding lobsters different types of carbohydrates resulted in varying oxygen consumption rates. Gelatine fed lobsters did not result in increased oxygen consumption and this was consistent with the results obtained for humans and alligators (Garrow, 1970; Coulson and Hernandez, 1979). Chapter Three: Oxygen Consumption & Ammonia Excretion in Relation to Feeding 64 Different Carbohydrates.

The pattern for the general carbohydrates suggest that as the complexity of the carbohydrate in a meal increases, oxygen consumption increases. Glucose and fructose (monosaccharides) were the simplest carbohydrates and therefore had the smallest oxygen consumption effect. Maltose and sucrose (disaccharides) were the next simplest and had an intermediate oxygen consumption effect. Glycogen (polysaccharide) was the most complex and resulted in the greatest oxygen consumption effect. Glucose and fructose do not need to be hydrolysed (Shiau, 1997; Rosas et al., 2001a) and can be absorbed straight across the digestive tract resulting in a minimal increase in oxygen consumption. Only enough oxygen was consumed to provide extra ATP to actively transport glucose across the digestive tract (Randal et al., 1997). This contrasts with the glycogen oxygen consumption profile, which was the largest of all the carbohydrate diets tested. It was postulated that the large increase in oxygen consumption was a result of glycogen needing to be hydrolysed and broken down to its simplest form (glucose) which was then absorbed across the digestive tract.

The SDA responses of lobsters fed the algal carbohydrates were not significantly different from the oxygen consumption profile of unfed lobsters. To show that digestion has occurred, there would be a rise in oxygen consumption that was significantly different from unfed animals (Jobling, 1981; Robertson et al., 2001a; Whiteley et al., 2001). The algal carbohydrates, based on the SDA response, appeared to have not been digested by the lobsters. However, by calculating the oxygen consumption magnitude, which was a more sensitive way of measuring digestion, a significant difference was observed for lobsters fed a meal of agar. Lobsters that were fed carrageenans and alginate still showed no difference and thus it could be postulated that digestion does not occur for lobsters fed those particular algal carbohydrates.

There were faeces in the respirometer after the first 3 hrs of the experiment for lobsters fed alginate and carrageenan. For lobsters fed agar 8 hrs had passed before faeces was observed. Faeces represent undigested material (Randell et al., 1997) and can be used as an indicator of indigestible diets. It is fair to conclude that with faeces appearing in the respirometer after only 3 hrs, that the diets of alginate and carrageenan were indigestible. Thus, the production of faeces and the minimal rise in oxygen consumption further support that juvenile J. edwardsii do not have the ability to digest carrageenans and alginate. Chapter Three: Oxygen Consumption & Ammonia Excretion in Relation to Feeding 65 Different Carbohydrates.

When the results were analysed together statistically (general and algal carbohydrates), glycogen and sucrose were the carbohydrates that were significantly different from all the other treatments. Glycogen and sucrose produced the largest SDA response in juvenile J. edwardsii. This large response suggests that J. edwardsii use more energy to utilise glycogen and sucrose than any of the other carbohydrate treatments. The algal carbohydrates did not produce a significant SDA in J. edwardsi, however, the magnitude of oxygen consumption showed that agar was significantly higher than unfed lobsters thus suggesting that this was the only algal carbohydrate able to be digested. This also suggests that oxygen consumption magnitude be used to calculate whether digestion is occurring, as opposed to using the SDA response, because it produces more sensitive results.

Ammonia Excretion

The presence of ammonia indicates that digestion is occurring. Ammonia is the by-product of protein metabolism, chiefly from deamination and transamination reactions (Kormanik and Cameron, 1981; Greenaway, 1990). Gelatine was the only source of protein in the diets developed in these experiments and was present as the binder in the general carbohydrate diets. Ammonia excretion in the lobsters fed general carbohydrates was significantly higher than unfed lobsters suggesting that digestion of gelatine (protein) was occurring. To determine whether the combination of gelatine with the general carbohydrates affected the quantity of ammonia excreted, a comparison was made between lobsters fed general carbohydrate jellies and those fed only gelatine. Ammonia excretion of lobsters fed the general carbohydrates was similar to gelatine, indicating that the general carbohydrates used in the present research did not affect the digestion of gelatine. This was important in terms of developing an artificial diet because protein is the most expensive ingredient. Therefore protein metabolism in the general carbohydrate diets used in the present research was being optimised.

There was no protein present in the diets containing algal carbohydrates. Gelatine was not included because the algal carbohydrates used in the experiments were binding agents themselves (Pearce et al., 2002). All the algal carbohydrate treatments exhibited significantly higher ammonia excretion rates compared to unfed lobsters with agar fed lobsters exhibiting the highest Chapter Three: Oxygen Consumption & Ammonia Excretion in Relation to Feeding 66 Different rates. It is unclear why there was a significant difference observed as there was no protein in the diet (Boyce, 1999).

When the results were analysed together statistically (algal and general carbohydrates) all of the general carbohydrates including the gelatine treatment had significantly higher ammonia excretion rates compared to lobsters fed algal carbohydrates. This was consistent with the lack of protein (amino N) in the algal carbohydrate diets.

In conclusion, glycogen and sucrose produced the only significant SDA and appeared to be the carbohydrates that require the most energy to digest and utilise. Agar was the only algal carbohydrate that could be digested and metabolised. Ammonia excretion of lobsters fed gelatine was not affected by the presence of a general carbohydrate in the diet. There needs to be further research on ammonia excretion of lobsters fed algal carbohydrates. Gelatine fed lobsters did not produce a significant rise in oxygen consumption following feeding, which was consistent with other studies (Gan-ow, 1970; Coulson and Hernandez, 1979). Therefore, the rise in oxygen consumption of lobsters fed the general carbohydrate diets must have been attributed to the carbohydrate in the feed. The magnitude of oxygen consumption was the only SDA parameter to exhibit a significant difference when lobsters were fed the algal carbohydrate agar. This therefore, shows that the magnitude of oxygen consumption was a more sensitive indicator of digestion. In future evaluations of artificial diets, oxygen consumption magnitude can be used as an indictor in whether or not the animal can digest certain ingredients. If all the ingredients can be digested in a diet, this will make it cost effective because the animal will utilise the majority of the ingredients. In relation to the aquaculture of J. edwardsii, the present research supports the choice of glycogen as the carbohydrate component of an artificial diet. Although sucrose displayed a large SDA as well, glycogen is the storage carbohydrate in mussels which are natural prey to lobsters making this a more desirable choice. Chapter Four: Haemolymph Glucose 67

CHAPTER FOUR

HAEMOLYMPH GLUCOSE

4.1. Introduction

Blood (haemolymph) in crustaceans, as in other coelomates, distributes oxygen, nutrients, hormones, and other metabolites throughout the body. Animals need to quantitatively and temporally regulate their glucose concentration to ensure that adequate glucose levels are available for all cells and that excess glucose can be stored (Verri et al., 2001). Haemolymph glucose reflects changes in anabolic and catabolic processes. More specifically, it represents a dynamic equilibrium between glycogenesis, glucogenolysis and gluconeogenesis (Hall and Van Ham, 1998). Glycogenesis in crustaceans is the synthesis of glycogen. Glucogenolysis is the breakdown of glycogen to glucose 6-phosphate (Randall, et al., 1997). Gluconeogenesis is a biosynthetic pathway for de novo synthesis of glucose from the precursors lactate and alanine (Rosas, et al., 2001; Cuzon et al., 2001).

In crustacean haemolymph, a variety of monosaccharides, disaccharides and polysaccharides are found. The main reducing sugar is the hexose monosaccharide glucose (Meenakshi and Scheer, 1961; Dean and Vernberg, 1965; Telford, 1968a; 1968b; Williams and Lutz, 1975; Hall and Van Ham, 1998; Verri, et al., 2001). Haemolymph glucose may vary depending on moult stage (Telford, 1968b; Chang and O'Connor, 1983), hormonal state (Hall and Van Ham, 1998), nutritional status (Meenakshi and Scheer, 1961) and stress (Telford, 1968a). Therefore, when reporting experiments conducted on haemolymph glucose in crustaceans it is important to standardise the conditions of the experiment with regard to these parameters. Chapter Four: Haemolymph Glucose 68

In crustaceans, a constant supply of glucose in the blood is paramount to maintain the regular functions of certain organs, such as the brain and muscles (Verri et al., 2001). Therefore, haemolymph glucose levels need to be constantly replenished in order to maintain normal physiological conditions. In chapter three, it was proposed that differences in the SDA (and ammonia production) associated with feeding different carbohydrates could be used to indicate the relative efficiencies of the processes of digestion and assimilation. If this is the case, we might expect to observe parallel increases in haemolymph glucose concentrations following feeding. This was the primary focus of the study reported here.

Several authors have demonstrated that in shrimps and prawns, a meal containing pure glucose cannot be used efficiently by the animal. It has. been claimed that feeding pure glucose produces a negative physiological effect caused by hyperglycaemia resulting from its higher rate of absorption across the digestive tract (Abdel-Rahman et al., 1979; Rosas, et al., 2001; Cuzon et al., 2001 ). If abnormal levels of haemolymph glucose are maintained, the control of crustacean hyperglycaemic hormone (CHH) is suppressed, maintaining glycogen synthesis for long periods. This results in the poor regulation of carbohydrate metabolism and hence a negative physiological effect (Cuzon et al., 2001). This is reported to manifest itself in reduced growth rates and general reduction in the overall health of the animaL The aim of the present research was to quantify the hyperglycaemic response to feeding different carbohydrates (glucose, fructose, maltose, sucrose, glycogen, agar, carrageenan and alginate). Such data will be useful in assessing whether juvenile Jasus edwardsii are similarly impaired

4.2. Methodology

Experimental Design

Glucose haemolymph concentrations were measured after feeding lobsters with different carbohydrate diets used in chapter three. These were glucose, fructose, maltose, · sucrose, glycogen, gelatine (general carbohydrates), agar, carrageenans and alginate (algal carbohydrates). The diets were formulated as described in chapter three and the experiments were conducted in a constant temperature room (15 °C). Chapter Four: Haemolymph Glucose 69

Lobsters were held in 500 ml plastic holding containers as shown in Figure 4.1. Two 5 ml holes were drilled in each end of the container, one high near the lid and the other low near the bottom. Two taps with rubber '0' rings were placed in the holes forming an airtight seal. The two taps provided a mechanism for an incurrent (bottom tap) and an excurrent (top tap) flow of water. Each container was connected to a submerged pump (Maxi-Jet MJlOOO), which supplied a 1 constant flow of water (72 L h- ). Dall (1986) described that lobsters were stressed when held in smoothed wall respirometers and Telford (1968a) described that stressed lobsters have increased haemolymph glucose concentrations. Therefore, stones were placed in the bottom of the holding containers to provide attachment points for the lobsters.

Experimental Procedure

Four lobsters (moult stage C, Turnbull, 1989; Oliver, 2000) were removed from the main aquarium and placed in recirculating seawater tanks in the 15 °C constant temperature room and allowed to acclimate for one week. Prior to the commencement of the experiments, lobsters were starved for 4 days to achieve a constant post-absorptive condition (base level metabolism) between all four lobsters. This time was based on that recorded for fish experiments where, to achieve post-absorptive conditions starvation, periods of two to four times the duration of the SDA are recommended (Boyce and Clarke, 1997). The SDA recorded in the present research for juvenile J. edwardsii was between 36 and 42 hrs; a starvation period of 4 days was chosen.

Lobsters were allowed 48 hrs to acclimate to the holding containers and was included in the 4 day starvation period. Base level haemolymph glucose was determined by taking two 100 J.tl haemolymph samples over 36 hrs at 1100 hrs and 1600 hrs. These initial samples were taken to determine base level haemolymph glucose concentration, which is defined as the haemolymph glucose concentration during pre-absorptive conditions after 4 days of starvation.

Three lobsters were fed one of the carbohydrate diets half an hour (0800 hrs) prior to the commencement of the experiment at 0830 hrs. Lobsters were fed a meal the size of 3 % of their body weight. The results from lobsters that did not consume all the meal were not used. g .§ (i) ""'! ~ ~

g.f

~;:::T' 0 2"" (") 0 rn ('1)

Figure 4.1: Photo of the container used for holding juvenile J. edwardsii during the haemolymph glucose experiments. A= Incurrent; B =

Excurrent; C = Stones used for attachment points for the lobsters.

-....] 0 Chapter Four: Haemolymph Glucose 71

Lobsters were stimulated to eat the meals by coating them in seawater from an opened blue mussel (Mytilus galloprovincialis). The fourth lobster was unfed and used as the control. For each of the diets, six lobsters were used to determine haemolymph glucose concentrations.

Haemolymph was sampled at 0, 1, 3, 6, 12 and 24 hrs after feeding. At each time 100 ~1 of haemolymph was sampled.

Haemolymph Sampling

Prior to the lobsters being transferred to the holding containers a small hole was made through the carapace into the pericardia! cavity with a 21 gauge (Terumo 21G*1W') needle. The hole was blocked with grease to prevent any bleeding and seawater entering the pericardia! cavity. This hole was used to take all haemolymph samples. All haemolymph samples were taken with an ice chilled 1ml syringe (Tuberculin) using a 23 gauge (Terumo 23G*1W') needle. The haemolymph was immediately placed in an ice chilled 1 ml eppendorf tube and glucose analysis performed immediately.

Glucose Determination

The eppendotf tubes containing the 100 ~~ of haemolymph were centrifuged for 5 min at 9000 g on a Force 13 centrifuge (Denver Instruments). Glucose was determined using a Sigma glucose test kit (No. 51 0), using the glucose-oxidase method. The enzyme solution was made by adding 1 capsule of PGO enzyme to 100 mls of distilled water. Colour reagent was made by adding 1 vial of 0-Dianisidine Dihydrochloride to 20 ml of distilled water. Adding 1.6 ml of colour reagent to 100 ml of enzyme solution made the combined enzyme colour reagent.

Combined enzyme colour reagent (1 ml) was added to each of the cuvetts. A blank was prepared by adding 100 ~1 of distilled water to the combined enzyme colour reagent. The standard was prepared by adding 95 ~1 of distilled water and 5 ~~of 1 g L" 1 glucose standard to the combined enzyme colour reagent. Samples were prepared by adding 50 ~1 of the supernatant from the eppendorf tube and 50 ~1 of distilled water to the combined enzyme colour reagent. Chapter Four: Haemolymph Glucose 72

Samples were inverted to mix, then incubated for 30 mins at 37 °C. The absorption was measured at 450 nm on a Unicam SP1800 Ultraviolet spectrophotometer.

Haemolymph glucose concentrations were calculated as follows:

Abs (sample)- Abs (blk) 1 Glucose (mg dL- ) = * 10 Abs (standard)- Abs (blk)

1 Glucose (mg dL- ) 1 Glucose (mmol L- ) = 18

Statistical Analysis

Before any statistical analysis was conducted on the data, normality tests were carried out using the Kolmogorov-Smimov test. Homogeneity of variances was verified with the Bartlett's test. Repeated measures ANOVA was used to test for significance between diet, time and the interaction (Diet x Time). Comparisons of means following ANOVA were done using the Tukey-HSD test (Underwood, 1997; Zar, 1999). The general and algal carbohydrate sources were analysed separately at first. The data were then combined to test for differences between the different sources. All analyses were performed using the Statistica (Version 6) statistical package with the a set at 0.05. All means are expressed as mean± S.E. unless otherwise stated.

4.3. Results

4.3.1 General Carbohydrate

In all the experiments, lobsters exhibited a rise in haemolymph glucose concentrations when fed one of the carbohydrate treatments (Figure 4.2). There were no significant differences among treatments in base level metabolism (F=1.987, P>0.05) (Table 4.2). Glucose fed lobsters had significantly higher peak haemolymph glucose concentrations than all the other treatments (F=33.59, P<0.001). lobsters fed a meal of fructose also had significantly higher haemolymph Chapter Four: Haemolymph Glucose 73 glucose concentrations than all the other treatments except glucose. The glycogen, sucrose and maltose fed lobsters were significantly higher than gelatine and unfed treatments, but were similar to each other. Peak haemolymph glucose concentrations ranged from 1.78±0.08 mmol L" 1 fed with glucose to 0.72±0.02 mmol L- 1 in unfed lobsters. There was a significant difference in the time that haemolymph glucose peaked (F=l3.5, P0.05). The duration of glucose haemolymph concentration of lobsters in response to glucose and fructose diets was inconclusive because they never returned to pre-feeding levels by the end of the experiment (24 hrs after feeding). Glycogen, sucrose and maltose returned to pre-feeding levels at 24 hrs after feeding and gelatine returned to pre-feeding levels 3 hrs after feeding. Factorial rise in haemolymph glucose concentrations differed between treatments (F=14.43, P

Overall, the interaction (Diet x Time) (F= 11.67, P§ ro -+- Glucose ~ 2 "0 c:: ""' co s::: ..... - Glycogen ;::~ 1.8 0 :-t 0 Q)..... -..- Maltose ·-..... 1.6 a.. ::I: ca - Sucrose § ...... -. 1.4 ----.- Fructose g. S:::";" Q) ..J 1.2 ---+- Unfed ~ s::: - --+- Gelatine t:r' 0 0 9 -+-- Pre Feeding =(') o E ~ Q) 0.~ j Vl ~ < (I) tn E 0 ~ 0.6 0 :::s 0.4 (!)- 0.2 0 -4 -2 0 2 4 6 8 10 12 14 16 18 20 22 24 Time (hours after feeding)

Figure 4.2: Haemolymph glucose concentrations oflobsters in response feeding different carbohydrate diets, glucose, glycogen, maltose, fructose, sucrose, gelatine and unfed. Means ± S.E., n=6. The solid horizontal line represents base level metabolism.

-....) ..&:>. Chapter Four: Haemolymph Glucose 75

Table 4.1: Haemolymph glucose results. Summary of (a) repeated measures univariate analysis of variance model (F values) between treatments through time and, (b) repeated measures multivariate analysis (F values) within subjects through time and interactions between time and treatments within subjects. * = P<0.05, ** = P<0.01, *** = P

Treatment Between Subjects Df MS F % General Carbohydrates 6,35 2.42 143.33*** 58 Algal Carbohydrates 3,20 0.59 6.96** 23 Combined general and algal 9,50 2.12 46.94*** 45 Carbohydrates (a)

Time Time x Treatment Within Subjects Wilks Df F % Wilks Df F % General 0.037 5,31 157.67*** 28 0.004 30,126 11.67*** 18 Carbohydrates Algal Carbohydrates 0.165 5,16 16.1 *** 24 0.348 15,44 1.38 6 Combined general 0.107 5,46 76.35*** 24 0.018 45,208 6.7*** 16 and algal carbohydrates (b) Table 4.2: Summary of haemolymph glucose parameters (means± S.E., n=6) of lobsters fed either glucose, glycogen, maltose, sucrose, Q fructose, gelatine or unfed control. Means followed by the same letter are not significantly different. a = peak haemolymph glucose divided -§ &' by base level metabolism. ~ ~ :1 ::r: Glucose Glycogen Maltose Sucrose Fructose Gelatine Unfed F p ~ 3 Base Level Metabolism 0.72±0.04a 0.61±0.03a 0.734±0.04a 0.69±0.02a 0.54±0.02a 0.58±0.05a 0.53±0.01a 1.98 >0.05 0 1 (mmol L- ) i Peak [Glucose] 1.78±0.08a 1.14±0.01b 1.26±0.08b 1.17±0.05b 1.39±0.08c 0.78±0.03d 0.72±0.02d 33.59 <0.001 Cl 2" 1 (") ) 0 (mmol L- 00 ('1)

Duration (hrs) >24 24 24 24 >24 3 Time to Peak (hrs) 6a 3b 3b 3b 3b 1c 3b 13.5 <0.001 Factorial Risea 2.51±0.21a 1.89±0.08b 1.81±0.1b 1.72±0.07b 2.57±0.19a 1.39±0.13c 1.35±0.04c 14.43 <0.001

-...) 0'1 Chapter Four: Haemolymph Glucose 77

Table 4.3: Tukeys HSD test comparing the effect of diet (carbohydrate type) (Table 4.1) on haemolymph glucose concentrations of lobsters fed different general carbohydrates.

Glucose Glycogen Maltose Sucrose Fructose Gelatine Unfed Glucose <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 Glycogen N.S. N.S. <0.0001 <0.0001 <0.0001 Maltose N.S. <0.0001 <0.0001 <0.0001 Sucrose <0.0001 <0.0001 <0.0001 Fructose <0.0001 <0.0001 Gelatine N.S. Unfed

Table 4.4: Tukeys HSD test comparing he effect of time (Table 4.1) on haemolymph glucose concentrations of lobsters fed general carbohydrates combined.

0 1 3 6 12 24 0 <0.0001 <0.0001 <0.0001 <0.0001 N.S. 1 <0.0001 <0.0001 <0.0001 <0.0001 3 N.S. <0.0001 <0.0001 6 <0.0001 <0.0001 12 <0.0001 24

4.3.2 Algal Carbohydrates

Lobsters fed a meal of agar and alginate exhibited a rise in haemolymph glucose concentrations (Figure 4.3). Base level metabolism was similar (F=l.83, P>0.05) (Table 4.5). Peak haemolymph glucose concentrations differed between treatments (F=7.15, P<0.01), with agar fed lobsters being significantly higher than all the other treatments. Peak haemolymph glucose concentrations ranged from 1.11±0.11 mmol L- 1 for agar to 0.63±0.09 mmol L-1 in carrageenans. The time taken for haemolymph glucose concentrations to peak were similar (3 h for all · Chapter Four: Haemolymph Glucose 78

treatments). Factorial rise in haemolymph glucose concentrations differed among treatments (F=8.88, P<0.05), with agar and alginate fed lobsters being significantly higher than carrageenans and unfed lobsters. Duration times were different for all three carbohydrate treatments. Agar fed lobsters returned to pre-feeding levels after 24 hrs, carrageenans after 2 hrs and alginate after 12 hrs (Table 4.5).

Overall there was no significant difference in the interaction (Diet x Time) (F=1.38, P>0.05) but there was a significant difference in haemolymph glucose concentrations between diets (F=6.96, P<0.01) and time (F=16.1, P<0.001) (Table 4.1). The only difference between diets was agar, it differed to all the other treatments (Table 4.6). All measuring times (hrs after feeding) differed from each other except between times 0 and 24 hrs after feeding and 3 and 6 hrs after feeding (Table 4.7).

Table 4.5: Summary of haemolymph glucose parameters (means ± S.E., n=6) of lobsters fed either agar, carrageenan, alginate or unfed control. Means followed by the same letter are not significantly different. a =peak haemolymph glucose divided by base level metabolism.

Agar Carrageenan Alginate Unfed F p Base level Metabolism 0.63±0.1la 0.56±0.05a 0.55±0.2a 0.53±0.01a 1.88 >0.05 1 (mmol L- ) 1 Peak (mmol L- ) l.ll±O.lla 0.63±0.09b 0.88±0.03b 0.72±0.2b 7.15 <0.01

Duration (hrs) 24 2 12 Time to Peak (hrs) 3a 3a 3a 3a 0.98 >0.05 Factorial Risea 1.76±0.25a 1.15±0.14b 1.60±0.11a 1.35±0.14b 8.88 <0.05

Table 4.6: Tukeys HSD test comparing the effect of diet (carbohydrate type) (Table 4.1) on haemolymph glucose concentrations of lobsters fed different algal carbohydrates.

Agar Carrageenan Alginate Unfed Agar <0.05 <0.05 <0.01 Carrageenan N.S. N.S. Alginate N.S. Unfed Chapter Four: Haemolymph Glucose 79

Table 4.7: Tukeys HSD test comparing the effect of time (Table 4.1) on haemolymph glucose concentrations of lobsters fed the algal carbohydrates combined.

0 1 3 6 12 24 0 <0.05 <0.001 <0.05 <0.05 N.S. 1 <0.05 <0.001 <0.001 <0.0001 3 N.S. <0.001 <0.001 6 <0.01 <0.0001 12 <0.05 24

Post prandial ]! -+-Agar .... 1.3 "0 -I c -J ro 1.2 L... - Carrageenans 0 a 1.1 ~ _....__ Alginate E a... E 1 ¥- Unfed -c:::: 0.9 ___....__ Pre Feeding 0 :;::::; 0.8 ...ns 0.7 c:::: -Q) 0.6 CJ c:::: 0.5 0 (.) 0.4 Q) 0.3 In 0 0.2 CJ :I 0.1 C) 0 -4 -2 0 2 4 6 8 10 12 14 16 18 20 22 24 Time (hours after feeding)

Figure 4.3: Haemolymph glucose concentrations of lobsters in response to feeding different algal carbohydrate sources, agar, carrageenan, alginate and unfed. Means ± S.E., n=6. The solid horizontal bar represents base level metabolism.

4.3.3 Combined General and Algal Carbohydrates

Haemolymph glucose concentrations increased after feeding in all the treatments (Figure 4.4), but base level metabolism was similar (F=l.88, P>0.05) (Table 4.9). Peak haemolymph glucose concentrations differed among treatments (F=24.96, P<0.001), glucose fed lobsters had the Chapter Four: Haemolymph Glucose 80

1 highest haemolymph glucose concentrations (1.78±0.08 mmol L" ) and carrageenans had the 1 lowest (0.63±0.09 mmol L" ). Lobsters fed glucose diets differed from all the other diets. The time required for haemolymph glucose concentrations to reach the peak was also significantly different (F=l2.88, P

Overall, the interaction (Diet x Time) (F=6.7, P<0.001), diet (carbohydrate type) (F=2.12, P

Table 4.8: Tukeys HSD test comparing the effect of time (Table 4.1) on haemolymph glucose concentrations of lobsters fed the combined algal and general carbohydrates.

0 1 3 6 12 24 0 <0.001 <0.001 <0.001 <0.001 N.S. 1 <0.001 <0.001 <0.001 <0.001 3 <0.05 <0.001 <0.001 6 <0.001 <0.001 12 <0.001 24 Q {3 2 Post prandial ~ ]'! "'C -+-- Glucose ~ c ~ 1.8 ~ ~ 0 ------Glycogen ~ --.- Maltose ~ c: 1.6 _j c.. ·-...0 -+---- Sucrose ~ ...ctJ.... 1.4 ~ Fructose [ c:-'I"" -+-- Gelatine 1.2 C1) .:..J Agar CJ ~0 c: - rll 0 0 1 Carrageenans C1l u E - Alginate C1) E 0.8 f/)- -+-- Unfed 0 -Pre Feeding CJ 0.6 :::l (!)- 0.4 0.2 0 -4 -2 0 2 4 6 8 10 12 14 16 18 20 22 24 Time (hours after feeding)

Figure 4.4: Haemolymph glucose concentrations of lobsters in response to feeding different carbohydrates glucose, glycogen, maltose, sucrose, fructose, gelatine, agar, carrageenan, alginate and unfed. Means ± S.E., n=6. The solid horizontal line represents base level metabolism. -00 Table 4.9: Summary of haemolymph glucose parameters (means± S.E., n=6) of lobsters fed either glucose, glycogen maltose, sucrose, Q -§ a= peak fructose, gelatine, agar, carrageenan, alginate or unfed. Means followed by the same letter are not significantly different. ~ "'1: haemolymph glucose divided by base level metabolism. ;:~ :'! ::c: Glucose Glycogen Maltose Sucrose Fructose Gelatine Agar Carrageenan Alginate Unfed F p $l:> s(!> Base Level 0.72±0.04 0.61±0.03 0.73±0.04 0.69±0.02 0.54±0.02 0.58±0.05 0.63±0.11 0.56±0.05 0.55±0.2 0.53±0.01 1.88 >0.05 0 Metabolism a a a a a a a a a a - 1 ~::r" (mmol L" ) C1 <0.001 ~ Peak 1.78±0.08 1.14±0.01 1.26±0.08 1.17±0.05 1.39±0.08 0.78±0.03 1.11±0.11 0.63±0.09 0.88±0.03 0.72±0.2 24.96 -n 0 CZl [Glucose] a b b b e cd bd c cd c (!> 1 (mmol L- )

Duration >24 24 24 24 >24 3 24 2 12 (hrs) Time to Peak 6 3 3 3 3 1 3 3 3 3 12.88 <0.001 (hrs) a b b b b c b b b b Factorial 2.51±0.21 1.89±0.08 1.81±0.1 1.72±0.07 2.57±0.19 1.39±0.13 1.76±0.25 1.15±0.14 1.60±0.11 1.35±0.04 10.74 <0.001 Risea a b b b a c b d b c

00 N Table 4.10: Tukeys HSD test comparing the effect of diet (carbohydrate type) (Table 4.1) on haemolymph glucose concentration oflobsters Q fed algal and general carbohydrates. -§ ~ ::::~ Glucose Glycogen Maltose Sucrose Fructose Gelatine Agar Carrageenan Alginate Unfed :1 ::r:: Glucose <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 s:>:> <0.001 <0.001 3 Glycogen N.S. N.S. <0.01 <0.01 N.S. <0.001 <0.001 <0.001 0 Maltose N.S. <0.05 <0.001 N.S. <0.001 <0.001 <0.001 i Sucrose <0.05 <0.001 N.S. <0.001 <0.001 <0.001 ~n 0 Fructose <0.001 <0.001 <0.001 <0.001 <0.001 Cil (I) Gelatine <0.001 N.S. N.S. N.S. Agar <0.001 <0.05 <0.001 Carrageenan N.S. N.S. Alginate N.S. Unfed

00w Chapter Four: Haemolymph Glucose 84

4.4. Discussion

Digestion in vertebrates and invertebrates is dependent on the anatomy and mechanical functions of the digestive system, the production and location of digestive enzymes, and the composition of diets (Glass and Stark, 1995). The ability of J. edwardsii to digest different types of carbohydrates depends on the carbohydrases it possesses. To date there have been no studies on the carbohydrases in the digestive system and on the ability of juvenile J. edwardsii to digest different carbohydrates.

Crustaceans display a diurnal rhythm in haemolymph glucose concentrations (Hall and Van Ham, 1998). The moult cycle (Telford, 1968b; Chang and O'Connor, 1983), nutritional state (Meenakshi and Scheer, 1961) and stress (Telford, 1968a) also affect haemolymph glucose concentration. The present research controlled these conditions by maintaining a constant post­ absorptive condition by starving the lobsters for 4 days prior to the experiment. Only lobsters in the intermoult stage C (as described by Turnbull, 1989; Oliver, 2000) were used, and unfed lobsters were used as controls for stress.

Changes in haemolypmh glucose concentrations due to feeding in juvenile J. edwardsii were predicted. Hepatopancreas digestive enzyme secretion begins immediately after ingestion of food (Dall and Moriarty, 1983). In Orconectes limnosus, glucose derived from the diet was rapidly absorbed, with about 9 % of the derived glucose passing straight into the haemolymph (Ulrich et al., 1973). The introduction of pure glucose into the gut of Cancer magister resulted in the doubling of haemolymph glucose concentrations and a fivefold increase in the concentration of oligosaccharides (Meenakshi and Scheer, 1961). Presumably, the oligosaccharides were synthesised from glucose precursors. The product of the first metabolic reaction by a hexokinase-catalysed transphosphorylation in glucose metabolism is glucose-6-phosphate (Hall and Van Ham, 1998). Glucose-6-phosphate is not detected by the glucose oxidase method employed in the present research (Sigma Diagnostics). Other research, in particular research on penaeides ( Hall and Van Ham, 1998; Gonzalez-Pena et al., 2002) and homarids (Telford, 1968b), also suggest that the increase in total haemolymph sugar concentration is undoubtedly much greater than for glucose alone. Chapter Four: Haemolymph Glucose 85

In the blue , transport of glucose across the midgut occurs via two routes; one is a sodium-dependent, saturable mechanism; the other is a sodium-independent nonsaturable pathway that probably represents simple diffusion into the cells (Chu, 1986; Hall and Van Ham, 1998). Most of the transported glucose into the haemolymph is free glucose, whereas, most of the glucose in the midgut is phosphorylated (glucose-6-phosphate) and this represents various stages of metabolic degradation (Adhem, 1982; Chu, 1986). The majority of glucose uptake from the digestive tract takes place in the hepatopancreas. Dall and Moriarty (1983) suggested that different organs of the digestive tract have different affinities for glucose absorption and that there was a biphasic appearance of glucose in the haemolymph. Hall and Van Ham (1998) have shown that Penaeus monodon has a biphasic time course of haemolymph glucose uptake. After the initial peak 1-2 hrs after feed intake, there was a further peak in the later stages of digestion. However, juvenile J. edwardsii in the present research did not exhibit a biphasic time course of haemolymph glucose uptake. This could be due to the measuring period in the later stages after feeding being to long.

In the present research, glucose that appeared in the haemolymph presumably originated from dietary uptake. In food deprived controls, basal concentrations of haemolymph glucose remained relatively stable. A rise in haemolymph glucose was induced only after feeding. In juvenile J. edwardsii, haemolymph glucose concentrations remained above pre-feeding levels for several hours after feeding on glycogen, maltose, and sucrose and did not return to pre-feeding levels within 24 hrs for fructose and glucose. Similarly in (Dall, 1974), Carcinus maenas (Williams and Lutz, 1975), Cancer magister and Hemigrapus nudus (Meenakshi and Scheer, 1961), Penaeus japonicus (Abdel-Rahman, et al., 1979), P. monodon (Hall and Van Ham, 1998), P. setiferus (Rosas et al., 1995) and Litopenaeus stylirostris (Rosas et al., 2000; Cuzon et al., 2001) haemolymph glucose concentrations were significantly elevated for several hours following a single meal.

The different carbohydrates fed to juvenile J. edwardsii produced different haemolymph glucose concentration profiles over time. The simple monosaccharides resulted in the greatest peaks in haemolymph glucose concentrations followed by the next simplest carbohydrates, the disaccharides. Working down through the complex polysaccharides, the general carbohydrate glycogen had a greater peak in haemolymph glucose concentration than agar, alginate and Chapter Four: Haemolymph Glucose 86

carrageenan, respectively. Only lobsters fed the simple monosaccharides glucose and fructose showed haemolymph glucose concentration profiles that did not return to pre-feeding levels within 24 hrs. It has been reported in species Penaeus japonicus (Abdel-Rahman et al., 1979) and P. monodon (Shiau and Peng, 1992) that when fed a simple monosaccharide such as glucose, haemolymph glucose concentrations do not return to pre feeding levels within 24 hrs. The reason for this apparently poor utilisation of glucose is not yet fully understood. Abdel­ Rahman et al. (1979) reported that haemolymph glucose concentrations in Penaeus japonicus increased rapidly after administration of glucose, and remained at high concentrations for 24 hrs. He also reported that when they were fed disaccharides and polysaccharides, haemolymph glucose concentrations increased to a maximum concentration at 3 hrs and then decreased to pre­ feeding levels. A similar response was observed in the present research on juvenile J. edwardsii.

Several authors have suggested that glucose and other monosaccharides cannot be used directly by prawns or shrimps. It is suggested that they produce a negative physiological effect caused by abnormal haemolymph glucose saturation resulting from a higher rate of absorption across the digestive tract (Abdel-Rahman et al., 1979; Cuzon et al., 2001; Rosas et al., 2001). If abnormal levels ofhaemolymph glucose are maintained, the control of CHH is suppressed. This maintains glycogen synthesis for long periods and results in the poor regulation of carbohydrate metabolism and thus results in a negative physiological effect (Cuzon et al., 2001). The present research showed results that were similar to those reported by Abdel-Rahman et al. (1979) for prawns fed a meal of glucose and fructose. It is postulated that the same effect would occur in juvenile J. edwardsii. The prolonged elevation of glucose in the haemolymph of J. edwardsii may have occurred because glucose requires no digestion and was absorbed immediately across the digestive tract. Glucose may therefore enter the haemolymph faster than it can be used, or stored as glycogen by glycogenesis. A similar time course was observed for J. edwarsdii fed fructose, although the effect was not as great. Fructose is readily converted to glucose and requires less energy compared to glycogen which has to be hydrolysed to glucose.

Juvenile J. edwardsii displayed lower haemolymph glucose concentrations as the carbohydrates they were fed got more complex (disaccharides and polysaccharides). Concentrations were relatively lower than when they were fed monosaccharides (glucose and fructose). Presumably this is because the more complex carbohydrates take longer to hydrolyse to glucose. Glass and Chapter Four: Haemolymph Glucose 87

Stark (1995) reported that the European lobster, , can digest maltose, sucrose and glycogen. The , orienta/is, can digest maltose and glycogen (Johnston and Yellowlees, 1998) and C. maenas and can digest maltose, sucrose and glycogen (Kristensen, 1972). Wigglesworth and Griffith (1994) reported that the prawn, P. monodon can digest maltose, sucrose and glycogen and P. vannamei can digest maltose and glycogen (Chevalier and VanWormhoudt, 1998). The results in the present research suggest that juvenile J. edwardsii can digest and efficiently utilise sucrose, maltose and glycogen as sources of carbohydrates.

Low concentrations of the digestive enzyme necessary for the hydrolysis of carrageenan and alginate are found in other decapod carnivores, Homarus americanus, Cancer borealis, C. irroratus, C. sapidus and Thenus orienta/is (Johnston and Yellowlees, 1998). The European lobster, H. gammarus, has limited ability to digest alginic acid (Stark and Glass, 1995). Alginate is a salt of alginic acid (Kristensen, 1972) therefore it is postulated that H. gammarus would not be able to digest this either. Kristensen (1972) has reported that C. maenas has the ability to digest alginate and C. crangon has the ability to digest agar and alginate with neither animal able to digest carrageenans. Both animals are omnivores, but C. maenas prefers animal food. This could account for the difference in the ability to digest different algal based carbohydrates. Wigglesworth and Griffith (1994) reported that the prawn P. monodon can digest agar and carrageenans. The results in the present research suggest that juvenile J. edwardsii have limited ability to digest alginate and carrageenans but were able to digest agar. Keeping other research results in mind, their ability to digest carbohydrates from algal sources may be limited because they are carnivores and might not possess, or have low levels of, the enzymes necessary to digest them.

In conclusion, haemolymph glucose concentrations increased in response to feeding different carbohydrates in juvenile J. edwardsii. The different carbohydrates used in the present research resulted in different haemolymph glucose concentration profiles over time. The simple monosaccharides (glucose and fructose) increased haemolymph glucose to higher concentrations and took longer to return to pre-feeding concentrations compared to disaccharides (maltose and sucrose), and polysaccharides (glycogen, agar, carrageenans, and alginate). The monosaccharides flood and saturate the haemolymph and a resulting negative physiological Chapter Four: Haemolymph Glucose 88 effect is postulated to occur. It appears that the uptake of glucose in the haemolymph exceeds the rate at which it can be used, or be converted into glycogen and stored. It is for this reason that many researchers, including the author, suggest that more complex carbohydrates be used to prepare lobster diets. Glycogen or agar must undergo enzymatic hydrolysis before assimilation and would permit glucose to be absorbed at a much slower rate than by using free glucose or fructose (Pascual et al., 1983; Alava and Pascual, 1987; Rosas et al., 2001a). However, juvenile J. edwardsii poorly digested two out of the three algal binder carbohydrates with agar being the only one that resulted in a haemolyrnph glucose concentration profile that differed from gelatine and unfed controls. Therefore, the author would propose that agar be used as a source of carbohydrate and binder in the formulation of artificial diets for juvenile J. edwardsii. Chapter Five: Growth Experiments 89

CHAPTER FIVE

GROWTH EXPERIMENTS

5.1 Introduction

Feeding strategies are important in the development of rock lobster aquaculture. Studies on shrimp species indicate that more frequent feeding of smaller rations of formulated diets improves growth rate and feed utilisation (Thomas et al., 2003). Results from Chapter 2 indicate that when juvenile Jasus edwardsii were fed in the morning they utilised the ration more efficiently compared to lobsters that were fed at night. From those results an aim was derived, which was to investigate if juvenile J. edwardsii grow faster when fed in the morning compared with feeding at night over 80 days.

The development of feed for the on-growing of captive New Zealand rock lobsters, Jasus edwardsii is seen as one of the key steps for the successful establishment of a viable rock lobster aquaculture venture in New Zealand. There are numerous studies on diet development for the American clawed lobster, Homarus americanus (Conklin, 1975), however, few have been reported for rock lobsters. The rock lobster aquaculture industry in New Zealand has largely used mussels (Perna canaliculus and Mytilus galloprovincialis) as a diet for on-growing operations (James and Tong, 1997; Crear et al., 2001; Crear et al., 2002).

Integral to the development of a feed for any species of crustacean is the identification of their protein, lipid and energy requirements. Protein provides the fundamental amino acids for growth, while dietary lipids provide both essential fatty acids and some of the energy needed for metabolic processes (Glencross et al., 2001). Further energy can also be derived from the metabolism of carbohydrates and protein and it is therefore important to know which carbohydrate lobsters best utilise to develop an artificial diet. Chapter Five: Growth Experiments 90

Another aim of the present research was to determine which carbohydrates juvenile J. edwardsii best utilise over an 80 day period. The National Institute of Water and Atmosphere (NIWA) (Dr Andrew Jeffs and Michelle Devey) carried out the growth experiment using the general carbohydrates glycogen, sucrose, maltose, fructose and glucose. The present research tested the algal derived carbohydrates agar, carrageenans and alginate. These carbohydrates are binding agents and if they can be utilised efficiently then there would be cost reduction in the formulation of the artificial diet because extra carbohydrates would not need to be added.

5.2 Methodology

Collection and Storage

. Early juvenile J. edwardsii (J 1'sand J 2's) for this study were supplied by NIWA in Wellington, New Zealand. Lobster juveniles were collected from Gisbome by NIWA using pueruli collectors (A. Jeffs pers comm) and were held at Mahanga Bay laboratory in Wellington. The lobsters were transported to Christchurch by air in sealed insulated containers containing fresh oxygenated seawater. On arrival, the lobsters were removed from the transporting containers and put into 80 litre plastic holding containers, containing seawater (18±0.5°C) in the Zoology aquarium at the University of Canterbury.

Experimental System and Water Quality

This experiment was conducted in a recirculating seawater system (Figure 5.1). The system consisted of a biofilter, protein skimmer and two Tronic Electronic Aquarium Heaters (300W). Seawater for the system was collected from Lyttleton Harbour. Light was maintained on a 10:14 hour light cycle. Seawater temperature was maintained at 18 ± 0.5 oc. It is generally accepted that 18 - 20 oc is the optimal temperature range for the on-growing of J. edwardsii (Booth and Kittaka, 2000; Hooker et al., 1997). Recent studies suggest that fluctuations in water quality impact negatively upon lobsters in culture, causing retarded growth and making them more Chapter Five: Growth Experiments 91

Protein Skimmer

Aquaria

(A) (B)

(C) Figure 5.1: Equipment used during the experiments: A, recirculating sea water system; B, 2 x 1000 L sump tanks used to hold the lobsters for each experiment; C, containers used to house the lobsters during the experiments. Chapter Five: Growth Experiments 92

susceptible to disease and moulting difficulties (Jeffs and Hooker, 2000). Water quality parameters were checked once a week. Salinity was maintained at 33-35%o, ammonia 0.1-0.3 mg 1 1 L- , nitrates <0.1 mg L- and pH 8.1-8.4. Half the seawater in the recirculating system was changed twice a week to keep these parameters to within their levels.

Two 1000 L plastic sump tanks were used (see Figure 5.1). One experiment was conducted in each of the tanks. All the lobsters were housed in individual containers within the larger tanks (Figure 5.1). Phillips et al., (1977) has demonstrated that growth rates of young juveniles of Panulirus longipes cygnus were not affected by being held in isolation. Within these containers stones and mussel shells provided shelter for the lobsters. The larger tanks and lobster houses were washed every 20 days when lobsters were weighed.

Preparation of Diets

Food cubes were prepared by mincing up mussels (Mytilus galloprovincialis) and adding to a solution of the algal carbohydrates. Agar diet was prepared by adding 1. 7 5 g of agar powder to 30 ml of boiling water. Once the agar had dissolved in the water, 45 g of minced mussel was added to the agar solution. The mixture was then poured into ice cube trays. The carrageenan diet was prepared by adding 1.25 g of carrageenan powder to 20 ml of boiling water and allowing it to dissolve. Minced mussel (32 g) was then added to the carrageenan solution, mixed and poured into ice cube trays. Both the agar and carrageenan diets were placed in a fridge and allowed to set. Once set, the diets were removed from the ice cube trays and placed into the freezer until they were needed. The alginate diet was prepared by adding 2 g of alginate powder to 30 ml of boiling water and allowing it to dissolve. Minced mussel (51 g) was added and mixed. This mixture was then extruded through a 3 mm diameter tube into a 2 % calcium chloride bath and allowed to set for 5 min. Once set, the alginate diet was placed into the freezer until it was needed. Chapter Five: Growth Experiments 93

Experiment One

Twenty four lobsters in individual containers were randomly placed in the sump tank. There were two treatments with twelve lobsters per treatment; lobsters fed squid at 0800 hrs (morning) and lobsters fed at 1800 hrs (night). Treatments were randomly assigned to the lobsters and lobsters were allowed one week to acclimate to their new environment. If any deaths occurred during the acclimation week individuals were replaced with similar sized lobsters that were held in identical conditions. At the end of the acclimation week lobsters were blotted dry using a paper towel, weighed and carapace length measured and recorded.

All lobsters were fed approximately 5% of their body weight per day at each feed. If any of the lobsters consumed all the food given to them then the amount of feed was increased so that they were always fed in excess. Lobsters that were fed during the day had excess food removed at 1800 hrs. Lobsters that were fed at night, had any excess food removed at 0800 hrs. All lobsters were fed 6 days a week with a double ration given on the sixth day to cover for the seventh.

Experiment Two

Forty eight lobsters in individual containers were randomly placed into the second sump tank. There were twelve lobsters per treatment and there were five treatments. These were, an agar artificial diet, a carrageenan artificial diet, an alginate artificial diet and a blue mussel (Mytilus galloprovincialis) diet, which acted as the control. The squid treatment in experiment one was also used in this experiment as another diet control. Lobsters were randomly assigned to treatments. Lobsters that were assigned the artificial diets were fed alternatively with mussel meals. Due to the experimental nature of the diets the feeding schedule was of a supplementary nature. They had three days of artificial diet and four days of mussels. The week started with two days of artificial diet followed by two days of mussels, then another day of artificial followed by two more days of mussels. A fresh mussel control was used because the base of the artificial diet was ground up mussel (see diet preparation) and was fed in the half shell. All the diets were fed at night (1800 hrs) only. Chapter Five: Growth Experiments 94

Lobsters were allowed one week to acclimate to their new environment and if any deaths occurred individuals were replaced with similar sized lobsters held in identical conditions. Following acclimation, lobsters were blotted dry using paper towels, weighed and their carapace measured prior to the commencement of the experiment. Lobsters that were fed the artificial diets were fed approximately 5 % of their body weight and this was always in excess. If the lobsters consumed all the diet, then the next feed was. increased so that they were always fed in excess. The artificial diets were removed from the freezer every morning at 0800 hrs to defrost prior to the lobsters being fed at 1800 hrs. Excess food was removed at 0800 hrs the following morning.

Experimental Procedure

Every morning at 0800 hrs moults were recorded by collecting and counting excuvia. Not all moults could be recorded because sometimes the lobster consumed the entire excuvia. At this time dead lobsters were also recorded and removed. The carapaces were examined microscopically to see if death was the result of any known exoskeleton diseases.

Individual lobsters were weighed and their carapace length measured on days 20, 40 and 60. Lobsters were blotted dry on paper towels, weighed on a Mettler PE 360 balance and their carapace length recorded using vernier callipers (±0.3 mm). The weighing procedure was done quickly to minimise any stress. Adjustments to feed allocation for the increase in biomass were made every 20 days following the weight check.

The growth experiments were conducted over 80 days to allow for an approximate 200 % increase in the weight of the control group oflobsters (mussel fed) (Ward, 1999). Before the start 0 of the experiment 10 lobsters (moult stage C and D ) were sacrificed to determine biochemical composition. This allowed comparisons between lobster biochemical composition at the start of the experiment and at the end. At the conclusion of the experiment all lobsters were starved for 24 hrs .to clear their gastrointestinal tract of ingested food. Cockcroft (1997) showed that moult stage can significantly affect biochemical composition. Therefore, individual lobsters at moult stage C and D0 (Turnbull, 1989; Oliver, 2000) were identified using a microscope and Chapter Five: Growth Experiments 95 were sacrificed by placing them in a freezer for 10 mins. The hepatopancreas was removed by dissecting over pre-weighed aluminium foil to prevent any loss of tissue. The abdomen was also removed and weighed, then the carapace removed and the abdomen tissue weighed. All dissected tissue was wrapped in aluminium foil and weighed, rapidly frozen in liquid nitrogen and stored at -18 oc for later analysis.

Leaching

Food cubes containing the different diets were weighed and placed into empty housing containers. After 14 hrs the cubes were removed, rinsed to remove any salt residue and gently dried with a paper towel to remove any excess water then weighed again. For the squid treatment half the cubes were removed after 10 hrs and the other half after 14 hrs. This was due to the amount of time each treatment was submerged during the experiment. The cubes were placed in an oven and dried at 100 oc for 12 hrs. The weight difference between the final diet weight and the initial diet weight was considered to be food loss into the water via leaching.

The fraction of diet lost into the water via leaching was taken into account when calculating the amount of food consumed by lobsters. The fraction of food leached into the water was calculated as follows:

L = 1 -

Where; L = the fraction of food leached into the water; F0 = the amount of food taken out of the housing containers (g); Fi =the initial amount of food put into the housing containers (g).

Food Intake

In the experiments it was essential to determine whether lobsters consumed more food during the day than at night, and also to determine which algal carbohydrates lobsters preferred. The amount of food consumed was gravimetrically measured daily for each treatment. All uneaten Chapter Five: Growth Experiments 96 food collected from each individual lobster was rinsed in freshwater to remove any salt residue then pattered dry with a paper towel to remove any excess water and dried at 100 oc (Ward, 1999) and the dry matter weight recorded. Feed intake was calculated as follows:

Cg = (Fi-L)-Fr

Where; Cg =consumption (g); Fi =initial food weight; L =feed loss due to leaching; Fr =weight of food remaining after feeding. All consumption rates were estimated as dry weight. This enabled a direct comparison between all dietary treatments.

An estimate of the dry matter of all the dietary treatments was obtained by drying ten pre­ weighed amounts of diet at 100 oc for 12 hrs. Then the percentage of the dry weight to the wet weight will give the percentage of dry matter. Table 5.1 gives the percentage of dry matter in each diet.

Glycogen and Lipid Analysis

Glycogen and lipid were measured using the same sample. Approximately 0.05 - 0.1 g of hepatopancreas was placed in a glass centrifuge tube from each lobster. 2.5 ml of 10M H2S04 was added to the sample and it was homogenised using an Ultra Turrex tissumiser. Samples took approximately 5 min to homogenise. Following homogenising a further 2.5 ml of 1OM H2S04 was added. The tubes were stored on ice. From this 5 ml of tissue homogenate, 3 ml was removed and put into a lipid analysis test tube. These tubes were stored on ice for separate analysis.

The remaining 2 ml of tissue homogenate was used for glycogen analysis using a D-glucose kit (No 510) from Sigma Diagnostics. The sample was centrifuged (EppendorfCentrifuge 5403) for 20 min at 5000 rpm. 1 ml ofthe supernatant ('sample') was pipetted into 2 eppendorftubes. One tube was put onto ice and the other was put into an eppendorf Thermoheater 5436 at 95 oc for 4 hrs. 10M NaOH (100 f.tl) was added to the eppendorftubes after 4 hrs then vortex mixed for 1 min. Samples were then centrifuged for 10 min at 15000 rpm. Chapter Five: Growth Experiments 97

Enzyme colour reagent (1 ml) was pipetted into 2 ml cuvettes. Sample blanks had 100 J.tl of distilled water added. Sample standards had 95 J.tl of distilled water plus 5 J.tl of 1 mg L-1 glucose standard. Samples had 50 J.tl of distilled water plus 50 J.tl of supernatant added. Samples were then placed in an incubator at 37 oc for 30 min. Absorbances were measured at 450 nm on a spectrophotometer (UV 601 PC Shimadzu) and the sample concentrations were calculated from the absorbances. Percent hepatopancrease glycogen was calculated as:

1 Cgtycogen (g L - ) %glycogen = X 100

Where Cgtycogen is the difference between the heated and the ice sample; Cassue is the tissue homogenate concentration.

Lipid was measured using a modification of the method described by Bligh and Dyer (1959). Chloroform:methanol (2:1) (5 ml) was added to the 3 ml of tissue homogenate. Samples were vortex mixed for 1 min each, facilitating the mixing of the lipid layer into the chloroform layer. Samples were centrifuged for 10 min at 2000 rpm to separate the chloroform/methanol layers. Following centrifugation, the top layer containing the methanol was removed using a pasteur pipette. The bottom layer containing the mixed chloroform/lipid was removed (2 ml) and put into a pre weighted Kimble test tube. Samples were placed in a water bath of boiling water then dried for approximately 30 min using a steady flow of nitrogen. The lipid residue was reweighed. Percentage hepatopancrease lipid was calculated as:

1 [Lresidue (mg L- ) X Vch/oroform (ml)] %lipid = X 100 Cassue

Where Lresidue is the lipid residue; V chloroform is the initial volume of chloroform added to the sample; Cassue is the tissue homogenate concentration. Chapter Five: Growth Experiments 98

Total Protein Analysis

Total protein was measured using a modification of the Kjeldahl method. The Kjeldahl method is the most common method used in determining organic nitrogen. Since most proteins contain approximately the same percentage of nitrogen, multiplication of this percentage by a suitable factor (6.25 for meats) gives the percentage of protein in a sample (AOAC, 1990).

Tail muscle was freeze dried before analysis. Approximately 12-16 mg of dried tissue from each lobster was weighed and put into a Kjeldahl flask containing potassiumsulphate-mercury (1.0 g:0.05 g) and 1 ml H2S04. Samples were digested (heated to approximately 350 oq in a fume cupboard for 2 hrs. This results in the production of ammonia from organic nitrogen. After cooling for approximately an hour, samples were diluted with distilled water (2-3 ml). Distillation involved the diluted digested sample being treated with 10M NaOH-Na thiosulphate. This released the ammonia from the ammonia salts. The distillate was collected in a beaker containing 5 ml boric acid. The ammonia absorbed in the boric acid as ammonium borate was determined by titration with standard H2S04 (0.0025 M). The endpoint was determined when the sample turned pink. From the titration volume (ml) used, the quantity of Kjeldahl nitrogen and subsequent meat protein was calculated:

Titration volume X 70j.tg NH4-N mg nitrogen = 1000

mgN %nitrogen = ------X 100 initial sample weight (mg)

%protein = %N X 6.25

Calculations

Growth was evaluated as percent body weight gain per day (specific growth rate, SGR) and percent weight gain (%WG) in the manner of Crear et al. (2000). Chapter Five: Growth Experiments 99

(lnWr-lnWi) X 100 T

Where; Wr= final body weight (g); Wi =initial body weight (g); and T =number of days.

%WG = ----x 100

Where; Wr =final body weight (g); and Wi =initial body weight (g).

The digestive gland (hepatopancrease) index (DGI) was also calculated to determine nutritional condition (Ward, 1999; Cuzon et al., 2001; Crear et al., 2002; Thomas et al., 2003).

DGI = X 100

Where; DGw = wet weight of digestive gland (g); and WBw = whole body wet weight (g).

Intermoult period (INT) was the period (days) between consecutive moults where INT2 =refers to number of days between the first and second moult, INT3 = number of days between the second and third moults (Thomas et al., 2003).

The percentage moult increment (WI) was calculated as the weight increase between consecutive moults and expressed as a percentage of pre-moult weight. Where WI1 = refers to weight increment of the first moult, Wh = refers to weight increment of the second moult (Thomas et al., 2003).

Statistical Analysis

Repeated measures analysis of variance (ANOVA) was used to test for differences between treatments. Normality was tested by the Kolmogorov-Smimov test and where appropriate

THE LIBRARY UNIVERSITY Of CANTERBURY CHRISTCHURCH, N.Z. Chapter Five: Growth Experiments 100 transformations were made. The homogeneity of variance was evaluated with the Bartletts test and again transformations were made where necessary. Percentage data was arcsine transformed prior to analysis and these data were analysed using one way ANOVA. All growth parameters for experiment one were compared using T tests (unbalanced variances). All other data were compared using one way ANOVAs for experiment two. Comparisons between the means following ANOVA (if significant P<0.05) were performed using Tukey's HSD test (Underwood, 1997; Zar, 1999). All analyses were conducted on the STATISTICA (Version 6) statistical package with the a set at 0.05. All means are expressed as mean± S.E.

5.3 Results

5.3.1 Diets

Squid and mussel had considerably more dry matter compared to the artificial diets of agar, carrageenans and alginate (Table 5.1).

Table 5.1: Summary of dry matter percentages for each of the diets used for growth experiments. Diet Dry Matter (%) Squid 24.2 Mussel 20.4 Agar 13.6 Carrageenans 11.4 Alginate 10.0

5.3.2 Experiment One

Lobster Growth and Feed Consumption

Lobsters that were fed in the morning were superior in all aspects of growth (SGR, %WG) compared to lobsters fed at night (Table 5.2). There was no difference in average lobster weight at the start of the experiments (t=1.09, P>0.05), 0.85±0.04 g for lobsters fed during the day and ChtWtE~r Five: Growth 101

0.80±0.07 g for lobsters fed at night. Increase in mean weight of lobsters fed in the morning was 0.88±0.01 g over 80 days. This resulted in a 107.4±3.1 %weight gain and an average growth 1 rate of0.92±0.07 %bw d- , with lobsters reaching a final mean weight of 1.76±0.10 g. This was significantly smaller (t=2.85, P<0.05) to lobsters fed at night, which had a mean weight increase of0.631±0.01 g. This resulted in a 78.7±3.1% (t=3.09, P<0.05) increase in body weight and an average growth rate of 0.76±0.01 %bw d" 1 (Figure 5.2 and Table 5.2). The mean increase in average carapace length was 4.60±0.21 mm for lobsters fed in the morning and 3.78±0.45 mm for lobsters fed at night (Figure 5.2).

Table 5.2: Growth, feeding rates and survival (mean± S.E.) ofjuvenile J. edwardsii fed either in the morning or at night over 80 days. Treatment Initial Final SGR (%bw d"1) %WG FI (% bwd-1) Survival Weight (g) Weight (g) (%) Morning 0.85±0.04 1.76±0.10 0.92±0.07 107.8±3.1 4.67±0.04 50 Night 0.80±0.07 1.43±0.12 0.76±0.01 78.7±3.1 4.06±0.06 25

T Stat 1.09 5.09 2.85 3.09 4.98 p N.S. <0.05 <0.05 <0.05 <0.05

SGR specific growth rate; FI = apparent feed intake; % WG = percent weight gain.

Repeated measures ANOVA performed on the wet weight and carapace length results showed there was no interaction (Diet x Time) (F=3.202, P>0.05; F=l.609, P>0.05). However, there was a difference between treatment and time for wet weight results, and for carapace length results there was a difference between treatment only (Table 5.3). Morning fed lobsters had a significant increase in body weight compared to lobsters that were fed at night (F=4.678, P

Growth of morning fed lobsters followed a steady exponential curve (Figure 5.2). Growth (weight gain) was not correlated with feed consumption rates (Figure 5.3). Repeated measures ANOVA showed there was no interaction (Diet x Time) for weight gain and consumption rate Chapter Five: Growth Experiments 102 results (Table 5.3). Lobsters fed in the morning had a significant increase in weight gain (F=7.18, P<0.05) but time had no affect (Table 5.3). Both diet and time did not significantly affect consumption rates (Table 5.3). One way ANOVAs were conducted on each individual treatment to test for differences between weight gain and consumption rates over time. Consumption rates were similar for lobsters fed in the morning (Table 5.4 and Figure 5.3) but differed for night fed lobsters (Table 5.4 and Figure 5.3). Night fed lobsters consumed significantly more at day 60 compared to days 20, 40 and 80 (Table 5.4 and Figure 5.3). Weight gain for morning fed lobsters was significantly higher at days 60 and 80. Weight gain for lobsters fed at night was similar (Table 5.4 and Figure 5.3).

Table 5.3: Growth experiment results. Summary of repeated measures univariate analysis of variance model (F values) between treatments through time (a) and, repeated measures multivariate analysis (F values) within subjects through time and interactions between time and treatments within subjects (b). * = P<0.05, ** = P<0.01, *** = P

Diet Between Subjects DF MS F % Wet Weight 1,4 0.1546 4.678** 9 Carapace Length 1,4 0.778 4.781 * 10 Weight Gain 1,4 0.004 7.180* 9 Consumption Rates 1,14 <0.001 1.550 10 (a)

Time Time x Treatment Within Subjects Wilks DF F % Wilks DF F % Wet Weight <0.001 4,1 401.5* 76 0.072 4,1 3.202 4 Carapace Length 0.047 4,2 2.917 68 0.237 4,2 1.609 8 Weight Gain 0.264 3,2 1.850 16 0.1 3,2 1.950 42 Consumption 0.839 3,12 0.764 3 0.731 3,12 1.460 4 (b) Chapter Five: Growth Experiments 103

2 1.8 * 1.6 c; 1.4 -:c 1.2 C) ~ 1 G) 0.8 -+--- morn 3: 0.6 - night 0.4 0.2

0 +------,------.------~------. 0 20 40 60 80 Time (days)

(a)

18 * 16 *

-+--- morn 4 - night 2 0 +------,------,------,------. 0 20 40 60 80 Time (days)

(b)

Figure 5.2: Mean growth ((a): wet weight; (b): carapace length) of juvenile J. edwardsii fed either at night or in the morning over 80 days (means± S.E.). *denotes significantly different. Chapter Five: Growth Experiments 104

1 1 Table 5.4: Summary of mean(± S.E.) consumption rates (g d.ay- ) (a) and weight gain (g daf ) (b) for lobsters fed in the morning or at night over 80 days. Values followed by the same letter are not significantly different.

20 40 60 80 F p Morning 0.083±0.015a 0.074±0.004a 0.103±0.009a 0.096±0.007a 0.93 N.S. Night 0.066±0.008b 0.070±0.004b 0.112±0.018a 0.078±0.010b 4.20 <0.05 (a)

20 40 60 80 F p Morning 0.144±0.048b 0.161±0.068ab 0.228±0.027a 0.258±0.093a 5.33 <0.05 Night 0.118±0.054a 0.258±0.079a 0.207±0.021a 0.165±0.045a 1.00 N.S. (b)

Ic::::J CONSUWTION -+-- BODY WSGHT I Ic::::J CONSUwrtON -+-- BODY W8GHT I

0.9 0.8

20 40 60 80 20 40 60 80 Time (days) Time (days)

(a) (b)

Figure 5.3: Mean (± S.E.) consumption and weight gain of juvenile lobsters fed either in the morning (a) or at night (b) over 80 days. Chapter Five: Growth Experiments 105

Moults and Survival

1--+- Squid Mom ----- Squid Night I 100 U) .:: j 80 0 ~ 0 60 -G) C) ca 40 c -G) ...c,) G) 20 D.. 0 20 40 60 80 Time (days)

(a)

1--+- Squid Morn ----- Squid Night I

C) c 100 "> -~ 80 j rn ...U) G) 60 0 u; -G).C e»O 40 CU..J c -G) 20 c,)... G) D.. 0 0 20 40 60 80 Time (days)

(b) Figure 5.4: (a): Percentage of moults for both morning and night fed treatments over 80 days; (b): mean survival of lobsters fed either in the morning or the night over 80 days. Chapter Five: Growth Experiments 106

Table 5.5: Percent moult increment and intermoult period (mean± S.E.) of juvenile lobsters fed either in the morning or at night over 80 days.

WI I WI2 Morning 15.36±0.93 34.91±2.42 30.75±1.03 36.75±0.85 Night 32.74±3.11 25.01±2.57 22.23±4.8 30±3.48

F 28.56 11.18 2.99 3.53 p <0.01 <0.05 N.S. N.S.

For lobsters fed in the morning, approximately 50% had moulted at each 20 day interval. This contrasted with lobsters that were fed at night, where a variable number of lobsters moulted during the 20 day intervals (Figure 5.4a). The highest percentage of lobsters moulting for morning fed animals was between 20 and 40 days with the lowest percentage of moults between 40 and 60 days. Lobsters that were fed at night had the highest percentage of moults between 40 and 60 days and the lowest percentage between 60 and 80 days. Intermoult periods were similar between morning and night fed lobsters (Table 5.5) and increased from INT2 to INT3. Lobsters that were fed at night had shorter intermoult periods than lobsters fed during the day. Moult increment differed between treatments. Lobsters that were fed in the morning had a significantly smaller WI1 than lobsters fed at night. However, WI2 reversed with lobsters fed in the morning having a significantly larger moult increment than lobsters fed at night (Table 5.5).

Overall, experimental mortalities were high (Figure 5.4b). Most mortalities were in the last moult stage D2 (as described by Turnbull, 1989; Oliver, 2000). Microscopic examination of lobster carcasses indicated that mortalities were not attributed to the known exoskeleton diseases, e.g. carapace erosion or fungal infections (Evans et al., 2000). Final mean survival percentages differed between treatments with lobsters fed at night (25 %) having a lower survival rate than lobsters fed in the morning (50%). There were no mortalities for both treatments during the first 20 day period. During the next 20 day period both treatments had the same percentage of lobster deaths (25 %). In the following 20 day period more lobsters died in the night fed treatment (33 %) compared to the morning fed treatment, which had, 18% die. During the last 20 day period Chapter Five: Growth Experiments 107 both treatments had the same percentage of lobster deaths (12 %). Survival percentages during this growth trial do not appear to be related to feed consumption or growth rates.

Biochemical Composition

Table 5.6: Percent lipid and % glycogen of the hepatopancreas, % protein of the abdomen muscle and the digestive gland index (DGI) of juvenile lobsters fed either in the morning or at night over 80 days. Means ± S.E. Means followed by the same letter are not significantly different (P>0.05). Control lobsters = lobsters that were sacrificed at the start of the 80 day growth experiment.

Morning Night Control F p % 22.2±1.47a 26.6±0.87a 23.2±1.19a 0.675 N.S. %Glycogen 0.043±0.014a 0.033±0.003a 0.012±0.026b 4.316 <0.05 %Protein 75.4±2.05a 73.3±2.88a 68.3±1.17b 3.333 <0.05 DGI 3.69±0.27b 2.72±0.87b 4.20±0.14a 5.779 <0.01

Hepatopancreas percent lipid was similar among lobsters fed in the morning, at night and control lobsters (Table 5.6). Hepatopancreas glycogen levels were 0.043±0.014% and 0.033±0.003 % for morning .fed and night fed lobsters respectively and were significantly higher than control lobsters (F=4.316, P<0.05). Abdomen protein levels were high at 75.48±2.05% and 73.35±2.88 %for morning and night fed lobsters and these were higher than the controls (F=3.333, P<0.05). Digestive gland index (DGI) was similar between both the morning and night fed treatments, but both treatments were significantly lower than the control lobsters (F=5.779, P

5.3.3 Experiment Two

Lobster Growth and Food Consumption

Lobsters on the diet where blue mussel (Mytilus galloprovincialis) was partially substituted with the formulated carbohydrate diets did not perform as well as the mussel only diet. However, lobsters fed the partial substitution diets performed better than the squid diet. Average lobster weights at the start of the experiment were similar (mussel= 0.78±0.09 g; squid= 0.80± 0.08g; agar= 0.90± 0.07 g; carrageenan= 0.86± 0.05 g; alginate= 0.86± 0.07 g) (Table 5.7) (F=1.654, P>0.05). The average mean weight increase for lobsters fed the mussel diet was 2.07±0.01 g. This resulted in a wet weight increase of 265.2±42.7% and an average SGR of 1.65±0.02 %bw d" 1 • This was significantly different to all the other diets tested. The next best performing diet was carrageenans, which had a mean weight increase of 1.30±0.02 g with an average wet weight gain 1 of 130.7±20.6 % and a SGR of 1.14±0.07 %bw d- • Carrageenans was the best performing artificial diet, however, growth on the artificial diets were similar. Lobsters fed the squid diet had a mean weight increase of 0.64±0.01 g. This resulted in a 78.7±3.1% increase in body weight and an average growth rate of 0.76±0.01 %bw d" 1 (Table 5.7 and Figure 5.5). The c~rrageenan diet resulted in the greatest effect on carapace length increase which was also greater than the mussel diet.

Repeated measures ANOV A performed on the wet weight results showed that there was a difference in the interaction (Diet x Time) (F=2.35, P<0.05), diet (F=4.976, P<0.05), and time (F=33.1, P<0.001) for wet weight. Mussel fed lobsters had a significantly greater increase in body weight over time compared to all the other treatments. All the carbohydrate diets had a significant increase in body weight over time compared to the squid diet, but there were no differences between the carbohydrate diets. Wet weight differed significantly over time except for 0 days and 20 days. For carapace length, however, only time (F=39.95, P<0.001) differed (Table 5.8). There was no difference between carapace length at day 20. However, from 20 days onwards carapace length was significantly different between all times (Table 5.11 ). Chapter Five: Growth Experiments 109

3.2 - squid c; 2.8 --...-.- mussel - agar J: 2.4 -en ___....._ carrageenan G) 2 ___.__ alginate ~ -G) 1.6 ~... 1.2 G) 1/) 0.8 -.c 0 ...J 0.4 0 0 20 40 60 80 Time (days)

(a)

- squid --...-.- mussel agar ___....._ carrageenan ___.__ alginate

0 20 40 60 80 Time (days)

(b)

Figure 5.5: Mean growth ((a): wet weight; (b): carapace length) of juvenile J. edwardsii fed either squid, mussels, agar, carrageenans or alginate over 80 days (means± S.E.). Table 5.7: Growth, feeding rates and survival of juvenile J. edwardsii fed either squid, mussel, agar, carrageenans or alginate over 80 days Q (mean± S.E.). .§ ~ ~ Squid Mussel Agar Carrageenan Alginate F p ~ Initial Body Weight (g) 0.80±0.08a 0.78±0.09a 0.90±0.07a 0.86±0.05a 0.86±0.07a 1.654 N.S. ~ ~ Final Body Weight (g) 1.43±0.02b 2.83±0.19a 1.88±0.27c 2.17±0.15c 1.89±0.12c 5.783 <0.05 5" ti:! 1 SGR (%bw d- ) 0.76±0.01c 1.65±0.02a 0.91±0.10b 1.14±0.07b 0.97±0.35b 4.729 <0.05 ~ ~ %WG 78.7±15.4b 265.2±42.7a 108. 7±22.8b 130.7±20.5b 119.7±12.6b 7.316 <0.05 g· 1 FI (%bw d- ) 4.06±0.06b 7.1±0.45a 6.578±0.48a 6.94±0.36a 5.12±0.85a 6.546 <0.05 atil Survival (%) 25 87 50 37.5 25 SGR = specific growth rate; FI = apparent feed intake; % WG =percent weight gain.

...... 0 Chapter Five: Growth Experiments 111

Table 5.8: Growth experiment results. Summary of repeated measures univariate analysis of variance model (F values) between treatments through time (a) and, repeated measures multivariate analysis (F values) within subjects through time and interactions between time and treatments within subjects (b). * = P<0.05, ** = P<0.01, *** = P

Diet Between Subjects DF MS F % Wet Weight 4,12 1.496 4.976* 49 Carapace Length 4,12 15.27 1.330 30 Weight Gain 4,13 0.234 0.980 38 Consumption Rates 4,35 0.011 6.833*** 43 (a)

Time Time x Treatment Within Subjects Wilks DF F % Wilks DF F % Wet Weight 0.063 4,9 33.10*** 33 0.074 16,28 2.35* 17 Carapace Length 0.053 4,9 39.95*** 65 0.103 16,28 1.930 16 Weight Gain 0.565 3,11 2.81 23 0.112 12,29 0.633 9 Consumption Rates 0.343 3,33 21.00*** 38 0.531 12,87 0.960 18 (b)

Table 5.9: Tukey's post hoc test comparing the effect of diet (Table 5.8) on final wet weight of juvenile lobsters fed either squid, mussel, agar, carrageenans or alginate. Squid Mussel Agar Carrageenan Alginate Squid <0.01 <0.05 <0.05 <0.05 Mussel <0.05 <0.05 <0.05 Agar N.S. N.S. Carrageenan N.S. Alginate Chapter Five: Growth Experiments 112

Table 5.10: Tukey's post hoc test comparing the effect of time (Table 5.8) on wet weight of lobsters fed either squid, mussel, agar, caiTageenans or alginate. 0 20 40 60 80 0 N.S. <0.01 <0.001 <0.001 20 <0.05 <0.001 <0.001 40 <0.05 <0.001 60 <0.001 80

Table 5.11: Tukey's post hoc test comparing the effect of time (Table 5.8) on carapace length of lobsters fed either squid, mussel, agar, caiTageenans or alginate. 0 20 40 60 80 0 N.S. <0.001 <0.001 <0.001 20 <0.001 <0.001 <0.001 40 <0.01 <0.001 60 <0.001 80

Table 5.12: Tukey's post hoc test comparing the effect of diet (Table 5.8) on consumption rates ofjuvenile lobsters fed either squid, mussel, agar, caiTageenans or alginate. Squid Mussel Agar Carrageenan Alginate Squid <0.05 N.S. <0.05 N.S. Mussel N.S. N.S. <0.05 Agar N.S. N.S. Carrageenan <0.05 Alginate ChtWtE~r Five: Growth 113

Table 5.13: Tukey's post hoc test comparing the effect of time (Table 5.8) on consumption rates

oflobsters fed either squid~ mussel, agar, carrageenans or alginate. 20 40 60 80 20 <0.05 N.S. <0.001 40 N.S. <0.001 60 <0.001 80

Growth in all treatments followed a steady exponential curve except for the squid treatment. Weight gains for mussel, agar and carrageenan treatments were closely correlated with consumption rates (Figure 5.6). For all the treatments except squid~ weight gain increased or were similar between each time period (Figure 5.6). Weight gain between treatments was similar for diet, time and the interaction (Table 5.8). There was no significant interaction (Diet x Time) (F=0.96, P>0.05) between consumption rates, however, there was a significant difference for diet (F=6.833, P

One way ANOVAs were conducted on each individual treatment to test for significant differences between weight gain and consumption rates at the different time periods. Weight gain and consumption rates were similar for the squid treatment (Table 5.14). Weight gain was significantly different for the mussel treatment with weight gain at 20 days being significantly less compared to 40, 60 and 80 days. There were significant differences in weight gains for each of the artificial diet treatments. Alginate 80 day weight gain was significantly different to 20, 40 and 60 day weight gains. The opposite occurred for agar and carrageenans, with a significant difference between the 20 day weight gain and those for 40, 60 and 80 days. Consumption rates differed for lobsters fed the alginate treatment, with 40, 60 and 80 days being significantly greater than 20 days. Consumption rates between the different time periods in the mussel treatment were all significantly different. In the agar and carrageenan treatments only the 80 day consumption rate differed from 20,40 and 60 day consumption rates. Chapter Five: Growth Experiments 114

1 1 Table 5.14: Summary of mean(± S.E.) consumption rates (g daf ) (a) and weight gains (g daf ) (b) of lobsters fed either squid, mussel, agar, carrageenan or alginate over 80 days. Values followed by the same letter are not significantly different. 20 40 60 80 F p Squid 0.066±0.008a 0.070±0.004a 0.112±0.018a 0.078±0.010a 0.20 N.S. Mussel 0.041±0.013d 0.123±0.013c 0.259±0.047b 0.413±0.025a 28.89 <0.05 Agar 0.019±0.011b 0.092±0.027b 0.127±0.043b 0.256±0.083a 3.79 <0.05 Carrageenan 0.064±0.017b 0.151±0.089b 0.157±0.027b 0.329±0.064a 9.91 <0.05 Alginate 0.022±0.009b 0.092±0.021a 0.083±0.024a 0.126±0.044a 5.82 <0.05 (a)

20 40 60 80 F p Squid 0.118±0.054a 0.258±0.079a 0.207±0.081a 0.165±0.085a 1 N.S. Mussel 0.258±0.124b 0.507±0.079a 0.560±0.142a 0.588±0.178a 4.34 <0.05 Agar 0.059±0.028b 0.328±0.074a 0.301±0.056a 0.374±0.195a 3.47 <0.05 Carrageenan 0.024±0.043b 0.304±0.151a 0.317±0.077a 0.447±0.157a 3.86 <0.05 Alginate 0.032±0.068b 0.044±0.054b 0.180±0.120b 0.588±0.001a 6.14 <0.05 (b)

Table 5.15: Percent moult increment and intermoult period (mean± S.E.) of juvenile lobsters fed squid, mussel, agar, carrageenan or alginate over 80 days. Values followed by the same letter are not significantly different.

Squid Mussel Agar Carrageenan Alginate F p

WI 1 32.74±3.11b 72.07±10.28a 52.07±0.78b 37.8±8.12b 53.97±6.74b 5.23 <0.01 Wh 23.08±2.57b 33.06±4.57ab 26.52±1.15b 50.52±7.53a 14.83±4.69b 8.35 <0.001 INT2 22.25±4.8a 30±3.48a 30±3.24a 28.75±1.49a 26±0.91a 1.12 N.S. INT3 30±3.48a 36.25±1.93a 35±2.04a 33.75±1.49a 28±0.91a 2.61 N.S. Chapter Five: Growth Experiments 115

Ic=::::J CONSUMPTION --+- BODY WEIGHf I

20 40 60 80 20 40 60 80 Time (days) Time (days)

{a) {c)

Ic=::::J CONSUMPTION --+- BODY WEIGHf I Ic=::::J CONSUMPTION --+- BODY WEIGHf I 0.5 0.9 0.45 0.9 >;0.45 0.8 >: 0.4 0.8 .g 0.4 0.7 >: .g 0.35 0. 7 >: :90.35 0.6 .g :9 0.3 0.6.g s 0.3 0.5:9 :s_0.25 s 0.25 0.5:9 E 0.2 0.4i :s. o.2 o.4 i ii:0.15 0.3 ...0 ~ 0.15 0.3 ~ Ill s 0.1 0.20 c 0.1 0.2 0 0 0 0.05 0.1 0 0.05 0.1 0 0 0~--±-<-+---'---'------1----'----'--+-'-~-+0 20 40 60 80 20 40 60 80 Time (days) Time (days

{b) {d)

IC=:JCONSUM PTION --+- BODY WEIGHf I

20 40 60 80 Time (days)

{e) Figure 5.6: Mean(± S.E.) consumption and weight gain ofjuvenile lobsters fed either squid (a), mussel (b), agar (c), carrageenan (d) or alginate (e) over 80 days. Chapter Five: Growth Experiments 116

Moults and Survival

Moulting was variable amongst all treatments (Figure 5.7). The mussel treatment was the most regular as lobsters moulting ranged from 75 % to 55 % per 20 days. The squid and agar treatments had the highest percentage of moult at any one time (100 %), both occurring between 40 and 60 days. Lobsters in the alginate treatment had the lowest percentage of moults (0 %), this occurred at 20 days. The intermoult period increased from INT2 to INT3 for all of the treatments and was similar between treatments (Table 5.15). Percent weight increase at moult, decreased from WI1 to Wh for all treatments except for carrageenans, which increased. There was a significant difference between lobsters at moult increment. Lobsters in the mussel treatment were significantly higher than all the other treatments at WI1• Artificial treatments had a similar weight gain at WI 1, however, carrageenans differed to both agar and alginate treatments at WI2.

Overall, mortalities were high (Figure 5.8), however, there were no deaths during the first 20 days of the experiment. Lobsters from the mussel treatment had the best survival (87 %) followed by agar (50%). The rest of the treatments had less than 40% lobster survival. The mussel diet had some lobster deaths between 20 days and 40 days then there were no further mortalities. The majority of the deaths occurred in moult stage D2 (Turnbull, 1989; Oliver, 2000) for squid, carrageenan and alginate treatments. In these treatments lobsters had trouble getting out of the excuvia and were found half in the old excuvia. Lobsters from the mussel treatment did not have any mortalities in the last moult stage. Examination of the carcasses showed that deaths did not occur via any of the known exoskeleton diseases, e.g. carapace erosion or fungal infection (Evans eta!., 2000). From 20 days onward there were a steady number of lobsters dying in each artificial and squid treatment. Highest lobster mortality was in the squid treatment between 40 and 60 days where 38% died. Lobster survival percentages during this experiment do not appear to be related to consumption rates or growth rates. Chapter Five: Growth Experiments 117

100 --squid 90 ___.....__ Mussel ~ 80 :::::s Agar 0 70 :e ----..- Carrageenans Cl) 60 ---+-- Alginate C) .f! 50 c Cl) 40 ...(,) 30 Cl) D. 20 10 0 0 20 40 60 80 Time (days)

(a)

100 90 80 c; > 70 ·~ :::::s 60 en 50 --+-- Squid c -Cl) 40 --Mussel (,)... Cl) 30 ___...._ Agar D. 20 Carrageenans 10 ----..- Alginate 0 0 20 40 60 80 Time (days)

(b) Figure 5.7: (a): percentage of moults for squid, mussel, agar, carrageenan and alginate treatments over 80 days; (b): mean survival of lobsters for squid, mussel, agar, carrageenan and alginate treatments over 80 days. Chapter Five: Growth Experiments 118

Biochemical Composition

Hepatopancreas lipid ranged from 19.93±1.37 %in the agar treatment lobsters to 36.82±3.88% in alginate treated lobsters (Table 5.16). There was a significant difference between treatments (F=11.78, P<0.001) with alginate differing from all treatments including the control lobsters. Glycogen levels in the hepatopancreas ranged from 0.012±0.002 % in control lobsters to 0.075±0.016 % in mussel treated lobsters (Table 5.16). Glycogen levels differed between treatments (F=4.40, P<0.01) with the mussel treatment having significantly higher levels than all the other treatments including the control. Abdomen protein levels ranged from 65.16±1.34% in alginate fed lobsters to 80.13±0.63 % in mussel fed lobsters (Table 5.16). The percentage of protein in the abdomen was significantly (F=8.54, P<0.001) higher when lobsters were fed mussel and carrageenan diets compared to control lobsters. Squid and agar fed lobsters had similar percentage of protein in the abdomen muscle as control lobsters, but lobsters fed alginate resulted in significantly reduced protein percentages. The DGI differed between treatments (F=4.26, P<0.05), with lobsters fed squid, agar, carrageenans and alginate all significantly less than mussel fed lobsters and the control. Mussel fed lobsters had a similar DGI to control lobsters. The DGI ranged form 2.24±0.32 for alginate fed lobsters to 4.23±0.28 in mussel treated lobsters (Table 5.16).

5.4 Discussion

5.4.1 Experiment One

The present research investigated whether feeding time had a significant effect on growth and survival rates of juvenile J. edwardsii that were fed squid. Thomas et al. (2003) recommended that J. edwardsii be fed once daily to excess, with the delivery coinciding with the change from light to dark. This time is consistent with their natural feeding behaviour where nocturnal activity is stimulated by the onset of darkness (Fielder, 1965; Thomas et al., 2003). However, the results of the present research showed that lobsters fed in the morning had superior SGR and %WG than lobsters fed at night and therefore suggest that juvenile J. edwardsii be fed in the morning with the delivery coinciding with the change from dark to light. Table 5.16: Percent lipid and % glycogen of the hepatopancreas, % protein of the tail muscle and the digestive gland index (DGI) of 9 juvenile lobsters fed either squid, mussel, agar, carrageenan, alginate over 80 days and a control group of lobsters. Means ± S.E. Values -§ ~ ~ followed by the same letter are not significantly different. Control lobsters = lobsters that were sacrificed at the start of the 80 day growth ~ experiment. ~ ~ ~ Squid Mussel Agar Carrageenan Alginate Control F p g. m %Lipid 26.6±0.87b 19.98±1.15b 19.93±1.37b 18.92±1.59b 36.82±3.88a 23.28±1.19b 11.78 <0.0001 ~ ~ %Glycogen 0.033±0.003b 0.075±0.016a 0.038±0.011b 0.023±0.008b 0.031±0.006b 0.012±0.026c 4.40 <0.01 s·

%Protein 73.35±2.88bc 80.13±0.63a 68.75±1.81c 76.63±2.75ab 65.16±0.34d 68.39±1.17c 8.54 <0.001 ac;ll DGI 2.72±0.87b 4.23±0.28a 3.58±0.37b 3.27±0.6b 2.24±0.32b 4.20±0.14a 4.26 <0.05

...... \0 Chapter Five: Growth Experiments 120

Previous experimental growth estimates for J. edwardsii held in captivity have been summarised in Table 5.17, which shows that stocking density and type of feed influence growth and mortality. Compared to other research under comparable conditions, the growth in the present research ranged from high, for morning fed lobsters, to average for night fed lobsters. James et al. (2001) reared juvenile J. edwardsii at stocking densities of 50, 100, 150, and 200 lobsters m·2 and noted 2 maximum growth was obtained at 50 lobsters m· • However, in the present research, density was not a determining factor influencing growth or mortality because lobsters were held individually, and almost all the mortalities occurred at moult stage D2• When nutritional intake is inadequate, cultured lobsters are susceptible to limb loss, deformities, moult death syndrome and reduced survival (Bowser and Rosemark, 1981 ). Examination of the carcases indicated that the lobsters were not dying of any known lobster diseases. This, along with the fact that growth rates were not low, supports the postulation that deaths were attributed to moult death syndrome and not as a result of poor nutrition. It was not clear what factors resulted in the moult death syndrome but it could be that there was a specific ingredient(s) in the diet that were missing which were preventing lobsters completing the moult.

The digestive gland is the major storage site of energy reserves in decapod crustaceans (Gibson and Barker, 1979). It appears to be the most responsive organ to physiological stress (Trendall and Prescott, 1989) and has been shown to be a good indicator of the condition of spiny lobsters (Glencross and Smith, 1997; Musgrove, 1997; Cockcroft, 1997; Ward, 1999; Crear et al., 2002; Ward et al., 2003). Lobsters that were fed at night had a lower DGI (2.72±0.87) than lobsters fed in the morning (3.69±027) with both treatments having a lower DGI than the control lobsters (4.20±0.14). Previous research (Crockcroft, 1997; Crear et al., 2000) have reported DGis of around 5% for adult Cape rock lobsters, J. lalandii and juvenile J. edwardsii. The low DGI for juvenile J. edwardsii in the present research suggests that they could be in relatively poor condition. The growth results suggest otherwise and therefore the reason for the low DGI is unclear. It may however, be associated with the low survival rates that were observed in the experiment. Five: Growth 121 Table 5.17: Comparisons among several studies of the growth, survival, feed intake of 0+ J. edwardsii in culture. Arranged from highest SGR to lowest SGR. Study Lobster Study Density Mean Feed Food intake Survival SGR 1 weight duration (lobsters temperature (%BW day- ) (%) range {g} {days} m-2} {oC} Kington 1.51-13.5 90 25 na pc na 100 2.44 (1999) Thomas et a!. 1.02-5.97 92 89 18 me,pj 2.08 90 1.92 (2000) James eta!. 1.3-10.8 118 50 18 me na 89 1.80 (2001 Kington 1.51-7 90 25 na fd na 39 1.71 (1999) Thomas et a!. 0.99-4.79 92 89 24 me,pj 2.21 51 1.70 (2000) Present 0.77-2.83 80 Na 18 mg 7.1 87 1.64 research Crear et al. 1.29-7.95 112 103 16.9 me,pj 2.13 87 1.62 (unpublished) Sheppard 0.8-11.2 180 118 18 pc na 65 1.46 (2001) James eta!. 1.3-6.8 118 200 18 me na 89 1.39 (2001) Ward (1999) 3.60-10.28 84 48 18 me na 80 1.25 Crear et al. 2.31-10.09 112 90 16 me 1.33 98 1.2 (2000) Thomas eta!. 5.24-17.09 119 42 18.8 me 1.82 91 0.97 (2003) Crear et al. 2.36-6.8 112 90 16 fd 2.23 68 0.95 (2000) Present 0.83-1.755 80 na 18 sq 4.67 50 0.92 research (morning) Hollings 3.2-8.2 106 na 18 pc,me, na 100 0.89 (1988) a Tolomei ct al. 13.05- 120 10 19 pj na 100 0.83 (2003) 35.31 Ward (1999) 3.58-7.11 84 48 18 pj na 85 0.82 Thomas et al. 5.25-14.3 119 42 18.8 pj 1.29 75 0.82 (2003) Present 0.8-1.43 80 na 18 sq 4.06 25 0.76 research (night) Crear et al. 7.72-19.92 134 69 18 me 1.03 80 0.71 (2002) Manuel 3.8-35.9 336 59 18 pc na 56 0.67 (1991) Crear et at. 7.75-17.5 134 69 18 pj 0.77 73 0.62 (2002) Rayns (1991) 0.8-58 750 37 na cs na 100 0.57 Rayns (1991) 0.8-42 750 183 na cs na 50 0.53 Manuel 3.2-13.5 366 23 10 pc na 89 0.43 (1991) Hookeretal. 60-117.6 365 57 13-23 pc na na 0.18 (1997) Hooker et al. 115.7-180 365 39 13-23 pc na na 0.12 1997 Adapted from Thomas et al. (2003). Feed: cs, chione stutchburyi (cockles); me, Mytilus edulis (blue lip mussel); fd, fonnulated dry feed; pj, Penaeus japonicus feed; pc, Perna canaliculus (green lip mussel); a, Haliotis sp. (abalone); f, fish (albacore tuna); na, infonnation not available; mg, Mytilus galloprovincalis (blue mussel); sq, squid. Chapter Five: Growth Experiments 122

High digestive gland lipid levels are needed in J. edwardsii during the moult cycle in order for them to maintain energy during the non-eating stage while their shell is hardening after the moult (Hartnoll, 2001). In the present research, digestive gland lipid levels were reasonable high for both treatments during moult stage C and did not vary from the control. The overall relatively high digestive gland lipid levels in lobsters fed squid indicate that energy reserves were being efficiently replenished during the pre-moult energy accumulation period (Cockcroft, 1997; Ward et al., 2003). Therefore, it was unlikely that the squid diet resulted in the moult death syndrome and following low survival. Lipid and proteins are considered to be the major energy reserves in decapod crustaceans (Cockcroft, 1997). In starved Panulirus longipes, both the lipids in the digestive gland and protein in the muscle tissue were utilised, with the latter being a more important source of energy (Dall, 1974). The high protein levels observed in the abdomen of juvenile J. edwardsii in the present research were a source of energy in adverse conditions and suggest that they were not being used for energy metabolism. High protein levels were also desirable for the aquaculture of lobsters because the market desires lobster tails high in protein and low in fat (lipid).

According to many researchers, the digestive gland is the principal storage organ of glycogen, it contains more than 80 % of reserves and is the site for gluconeogenesis (Gibson and Barker, 1979; Oliveira and De Silva, 1997; Cuzon et al., 2001). In the present research, the percentage of glycogen in the digestive gland in juvenile J. edwardsii did not differ despite different feeding times. However, the squid diet significantly affected the percentage of glycogen, with increased levels compared to controls. Therefore, the squid diet provided enough glucose to build up the stores of glycogen. The glycogen levels found in this experiment were comparable to other experiments on prawns. Abel-Rahman et al. (1979) obtained glycogen levels of 0.022-0.053 % in the digestive gland of Penaeus japonicus.

In crustaceans, growth is a discontinuous process achieved by a succession of moults (or ecdyses) over time (Hartnoll, 2001). It consists of two discrete components, the moult increment (increased size at moult) and the intermoult period (duration between each successive moult). These two processes exhibit different responses to both intrinsic and extrinsic factors (Hartnoll, 2001; Thomas et al., 2003). Generally, with increasing size, the duration between moults increases and the moult increment is reduced. In the present research (experiment one) the Chapter Five: Growth Experiments 123 intermoult period increased for lobsters fed in the morning and at night. The moult increment however, increased from the first moult to the second moult for morning fed lobsters. It was unclear why this occurred as night fed lobsters displayed the typical behaviour and did not respond this way. The differences in growth observed in the present research were attributed to differences in moult increment and not the moult interval (which was unaffected by feeding regime).

5.4.2 Experiment Two

Feed type is a major factor in influencing the growth of J. edwardsii, with mussels consistently producing higher growth than formulated feeds (Ward, 1999; Crear et al., 2000; Sheppard, 2001; Crear et al., 2002; Thomas et al., 2003; this study). In the present research, growth rates of the artificial diets were similar. Therefore, the type of carbohydrate did not affect growth rates among the artificial diet treatments. Lobsters fed the artificial treatments however, did perform better than the squid treatment, with SGR and %WG being superior. After mussels, agar was the best carbohydrate diet in terms of survival, and because none of the other growth factors differed significantly, agar was deemed the best performing diet out of all the carbohydrate treatments. When nutritional intake is inadequate cultured rock lobsters are susceptible to moult death syndrome (Bowser and Rosemark, 1981 ). The poor survival rates of lobsters fed squid, carrageenans or alginate were a result of lobsters being susceptible to moult death syndrome. It was postulated that carrageenans and alginate inhibit the uptake of nutrients from the mussel incorporated into the diet but this needs to be researched more fully.

The results obtained in the present research for lobsters fed mussels were comparable to other research on J. edwardsii under the same conditions (Table 5.17). The superior biomass yield from mussel fed treatments was achieved through a combination of survival and faster growth. This highlights the need to further understand the nutritional requirements of rock lobsters to aid in the development of specific formulated diets.

Intermoult period for all treatments in the present research were similar to other reports and literature. Moult increment decreases and intermoult period increases as lobsters grow (Hartnoll, 2001; James and Tong, 1997; Hazell et al., 2001; Thomas et al., 2003). However, the moult 124 increment did not respond this way for lobsters fed the carrageenan treatment. The carrageenan treatment increased from WI1 to WI2, which was the opposite of the other treatments and the literature (Hartnoll, 2001; James and Tong, 1997; Thomas et a/., 2003). The differences in growth observed in the present research were attributed to differences in moult increment, and not the intermoult period. To date, the effects of nutritional factors on the components of growth of rock lobsters have been contradictory. In agreement with the present research, Thomas et a/. (2003) observed differences in the growth of J. edwardsii fed on either a low or high ration diet, one, two or four times a day. This was attributed to larger moult increments. However, James and Tong (1997) noted differences in growth of J. edwardsii fed with various mussel species and this was attributed to effects of both moult increment and intermoult period, depending on whether the mussels were fresh or frozen. This contrasts with the results of Chittleborough (1975) who found that food shortage lengthened the intermoult period of Panulirus cygnus without affecting the moult increment. These contradictory results indicate considerable inter­ specific differences in the growth response of rock lobsters to different food types (Hartnoll, 2001; Thomas et al., 2003) and further highlights the need for more research.

There was a direct relationship between food consumption and growth in juvenile J. edwardsii. Consumption in the present research increased steadily in relation to lobster size, except for lobsters fed squid (Figure 5.7). It was unclear why lobsters in the squid treatment reacted this way. It was postulated that the high mortality rates could have affected the results. In all the carbohydrate treatments, growth rates levelled off from 40 to 60 days along with food consumption. In the mussel treatment, there was increased weight gain every measuring period and this was associated with increased food consumption. Consumption was similar between mussel, carrageenan and agar treatments (Table 5.12) so therefore the type of carbohydrate in the diet did not affect palatability. The reduced growth rates were therefore most likely the result of the type of carbohydrate in the diet.

The DGI represents the overall physiological state of lobsters and is an indicator of their condition. The DGI of juvenile rock lobsters fed a diet of blue mussels in the present research was lower than that reported by Ward et al. (2003) but higher than that reported by Crear et al. (2002). The DGI was also lower than that reported by Cockcroft (1997) for wild caught adult Cape rock lobsters, J. lalandii. All the other treatments had a significantly lower DGI than Chapter Five: Growth Experiments 125 mussel or control treatments, suggesting that lobsters fed squid, agar, carrageenans and alginate were in relatively poor condition. This could explain the high mortalities observed in these treatments. The growth results however, suggest that this was not the case and the exact reason for the low DGI is unclear.

Cockcroft (1997) found that the lipid content of the digestive gland appeared to be a good indicator of growth in adult male Cape rock lobsters. Higher lipid levels in the digestive gland resulted in greater weight gain at moult. This did not occur in the present research where lobsters fed the mussel treatment had one of the lowest lipid levels but the greatest weight gain at moult. Lobsters fed the agar treatment also had low digestive gland lipid levels. This suggests that lobsters fed mussel and agar treatments were in poor nutritional condition. However, the growth and survival rates of mussel fed lobsters were the highest of any of the treatments used in the present research, which was in agreement with Crear et al. (2002). They also observed that J. edwardsii fed mussels had lower lipid levels but the highest growth rates and survival. Lobsters fed the alginate diet had the highest lipid levels yet the poorest survival and growth rates. Therefore, there are species specific differences and further research is needed before any clear cut interpretations of digestive gland lipid levels can be made.

It is expected that crustaceans with high growth rates would have high protein deposition in their abdomens (Calvert, 2000). This was the case for lobsters fed the mussel and carrageenan treatments, where the percentage of protein in the abdomen muscle increased in comparison with the rest of the carbohydrate treatments, including the control lobsters. This suggests that lobsters fed both the mussel and carrageenan treatments promote the highest growth than any of the other treatments. It was postulated that the carrageenan in the treatment partially spared the protein for growth and did not completely utilise it for energy metabolism. This did not occur in the agar and the alginate treatments which would explain the lower growth rate and protein deposition. However, because mussel fed lobsters demonstrated higher survival rates over carrageenan fed lobsters, this further supports the suggestion that mussels were the most successful diet.

The digestive gland is the storage organ for glycogen. The glycogen levels in the digestive gland reflected the amount that was contained within the diet. Lobsters fed squid, agar, carrageenans and alginate had significant increased glycogen levels in the digestive gland than control lobsters. Chapter Five: Growth Experiments 126

It was unclear whether juvenile J. edwardsii were using the additional carbohydrates added to the mussel base or utilising the mussel itself to build up glycogen stores. Glycogen levels in the digestive gland increased even more significantly in the lobsters fed the mussel treatment. This was probably because mussel tissue has an extremely high glycogen content (Isani et al., 1995; Dayton et al., 1996; Seramo et al., 1998).

5.4.3 Conclusion

In experiment one, juvenile J. edwardsii grew faster when they were fed in the morning. They out performed night fed lobsters in all aspects of growth, such as % WG and SGR. Morning fed lobsters also had greater survival rates. Digestive gland lipids, glycogen levels and protein levels in the abdomen muscle were similar between the two treatments. Therefore the different feeding times of the lobsters did not affect the biochemical composition. Feeding times had no affect on the intermoult period but did have an affect on weight gain at moult. Therefore, weight gain at moult resulted in the difference in growth rates between morning fed lobsters and night fed lobsters. The present research suggests that an aquaculture facility farming J. edwardsii should feed the juveniles in the morning rather than at night because faster growth rates were observed.

In experiment two, the mussel treatment showed significantly higher growth rates than any of the other treatments (squid, agar, carrageenan and alginate). It has been observed in all growth experiments using J. edwardsii that a mussel treatment always gives the greatest and fastest growth rates. Along with the greater growth rates, mussel fed lobsters also had the highest survival rates. There were no differences in growth rates amongst the algal carbohydrate treatments however agar fed lobsters did have the highest survival rates. However, all the algal carbohydrate treatments performed better than the squid treatment. The differences in growth rates among all treatments were attributed to differences in weight gain at moult with the intermoult period having no effect on growth rates. All treatments except for the alginate diet had increased protein levels of the abdomen muscle compared to the control. Therefore, out of the algal carbohydrates, alginate is inferior to agar and carrageenans. Glycogen stores in the digestive gland increased in all treatments compared to the control while lipid levels in the digestive gland remained relatively similar with the exception of the alginate diet. The DGI indicated that lobsters fed squid, agar, carrageenans and alginate were in poor nutritional Chapter Five: Growth Experiments 127 condition and this coincides with the low survival rates. However, lobster growth rates indicated otherwise and it was unclear why the low DGis were observed. With respect to using carbohydrates in formulated diets, the present research suggests that agar be the algal binding agent used. Although it did not have the highest growth rates, it did have the highest survival. Carbohydrates should be included in artificial diets because they are the cheapest form of energy and have the potential to maximise protein utilisation for growth. Finding the carbohydrate that can be digested and metabolised the most efficiently by J. edwardsii is crucial to the development of a nutritional, cost effective diet and their successful aquaculture. Chapter Six: General Discussion 128

CHAPTERS/X

GENERAL DISCUSSION

The international market demand for spiny lobsters is unsatisfied and increasing. The high value of lobsters and the growing pressure placed on wild fisheries has generated worldwide interest in the possibility of lobster aquaculture, which presents a means of supplying the market with reliable yields of quality produce. With improving larval rearing techniques and further development of pueruli collection technology, there is potential for full-scale intensive captive lobster rearing to become a commercial reality in New Zealand.

The present research investigated the specific dynamic action (SDA) of juvenile Jasus edwardsii in relation to feeding time. This is of interest to aquaculturists who need to know whether particular feeding times can optimise growth rates. The present research also investigated diet and nutrition, two of the most impm1ant issues involved in ensuring successful on-growing of rock lobsters in aquaculture to marketable size. To maintain a productive, cost-effective aquaculture operation, a consistent, reliable and economical feed must be found which is palatable, reduces lobsters' intermoult period, maximises moult increment and improves survival. So far, only natural marine feeds, in particular mussels, have been successful at fulfilling these criteria. However, natural feeds can be constrained by seasonality and expense in terms of acquisition, storage and labour costs.

Feeding time had a significant effect on the SDA in juvenile J. edwardsii, particularly the duration of the response and the SDA coefficient. When lobsters were fed at night, the duration of the SDA was longer than when fed in the morning. This demonstrates that digestion is quicker when lobsters are fed in the morning. The SDA coefficient is the percentage of the energy content of the meal used metabolically in the SDA response. Lobsters fed a meal in the morning had a significantly smaller SDA coefficient than fed at night. Energy loss as a result of 129

digestion was therefore less when lobsters were fed in the morning. These effects suggest that juvenile J. edwardsii could use more energy from their meal for somatic growth.

The common management practice for feeding lobsters is to feed them 25 % of their meal in the morning and the remaining 75% at night (Johnston et al., 2003; Ward et al., 2003). The present research suggests that it would be more efficient to provide lobsters with the entire portion of the meal in the morning. This should promote faster growth rates ofjuvenile lobsters.

There is growing evidence linking post-prandial oxygen consumption of crustaceans with growth (Robertson et al., 2001a; 2001b), suggesting that measurements of metabolic rate could be used as a research tool to investigate nutritional value of different dietary formulations (Jobling, 1981). In the present research the SDA was used to determine ifjuvenile J. edwardsii were able to digest certain ingredients (carbohydrates) that would prove useful in the development of an artificial diet. If the carbohydrate is digested by juvenile J. edwardsii then there should be a rise in oxygen consumption to a peak followed by a slow decline back to pre-feeding levels (disaccharides and polysaccharides only). This is because disaccharides and polysaccharides need to be hydrolysed to glucose before they can be absorbed. The rise in oxygen consumption should theoretically be greater than that observed for unfed lobsters. The results generally suggested that the more complex the carbohydrate, the greater the oxygen consumption profile. An exception was found with lobsters fed the algal carbohydrates. Despite the algal carbohydrates being complex, they did not produce an oxygen consumption profile that was significantly different from unfed lobsters. A more sensitive measure of digestion was to use the magnitude of the SDA response. Using this measure lobsters fed agar showed significantly higher oxygen consumption magnitudes compared to unfed lobsters but carrageenans and alginate did not.

An alternative method of determining if lobsters were able to digest a particular carbohydrate was to test for a rise in haemolymph glucose concentrations following feeding. The more complex the carbohydrates, the smaller the haemolymph glucose concentration profile. This held true for both general and algal carbohydrates. When fed a meal of sucrose, maltose, glycogen and agar, lobsters' haemolymph glucose concentrations were similar. However, those same haemolymph glucose concentrations were significantly lower than glucose and fructose fed lobsters and significantly higher than carrageenans, alginate and unfed lobsters. Lobsters fed glucose and 130

fructose (monosaccharides) had higher haemolymph glucose concentrations because they can be readily absorbed across the digestive tract. All carbohydrates with the exception of carrageenans and alginate can be digested by juvenile J. edwardsii.

Lobsters fed glycogen, sucrose and maltose diets all had a medium rise in haemolymph glucose concentration and high oxygen consumption. The SDA, along with the magnitude of the response was a good indicator of carbohydrate digestion in juvenile J. edwardsii. It was concluded that juvenile J. edwardsii have the ability to digest glycogen, sucrose, maltose, glucose, fructose and agar out of the carbohydrates tested in the present research. After feeding on a diet of glucose and fructose, juvenile J. edwardsii showed no rise in oxygen consumption but a significant rise in haemolymph glucose concentration. Glucose and fructose are absorbed straight across the gut wall, flooding into the haemolymph and cannot be metabolised quickly. This was consistent with Abdel-Rahman et al. (1979) who observed results for prawns fed glucose and fructose. Therefore it is postulated that glucose and fructose resulted in a negative physiological affect in juvenile lobsters. Complex carbohydrates need to be hydrolysed to glucose before they can be absorbed across the gut wall. There is a gradual release of glucose, the haemolymph does not flood and the glucose can be used as it enters the haemolymph. Therefore, glycogen, sucrose, maltose and agar are the carbohydrates digested and metabolised the most efficiently by juvenile J. edwardsii.

In a growth experiment run over 80 days at l8°C algal carbohydrates were compared to a fresh blue mussel (Mytilus galloprovincialis) control. Blue mussels were used as a control because they are commonly used as experimental controls in lobster nutritional studies. Although producing inferior growth rates compared to the blue mussels, the algal carbohydrate diets were palatable to the lobsters, resulting in high consumption rates (6.57±0.48 %bw d" 1 for agar; 6.94±0.36 %bw d" 1 for carrageenans; 5.12±0.85 %bw d"1 for alginate). Finding a selection of ingredients which are acceptable and are able to be metabolised by lobsters, is a vital part in the development of an artificial diet. The more palatable the diet, the greater the consumption rates and resulting growth. Unexpectedly, high consumption rates did not translate into wet weight gain when lobsters were fed algal carbohydrates. Overall, the artificial diets performed well in terms of SGR and %WG and there were no differences in growth between the three different algal carbohydrate diets. However, lobsters fed agar had higher survival rates (50 %). Chapter Six: General Discussion 131

Subsequently, agar should be the algal binder that should be incorporated into artificial diet formulas for J. edwardsii.

E 25 ..~ 20 ~ c ...... 15 .... 10 ...0 I!... 5 0:..' 0 .*=! (1.1 0 VI c: 0 ·c: ~"' () ~ ::J 0 C5 ~ 0

Figure 6.1: Mean carapace length of lobsters over an 70 day period. Results of interest to the present research were malt (maltose), glucose, fructose, sucrose and glycogen. Graph obtained with the permission of Jeffs and Devey.

Dr Andrew Jeffs and Michelle Devey (NIWA) tested a diet of general carbohydrates in a growth experiment at 18 °C. There were a few differences in protocols between the two studies. The present research was conducted over 80 days (18 °C) and used the blue mussel (M galloprovincialis) as a dietary control. In contrast.Dr Jeffs' study was conducted over 70 days and used the green lipped mussel (Perna canaliculis) as a dietary control. The results from Dr Jeffs study suggest that there was no significant difference in growth (F=0.9, P>0.05) (Figure 6.1 ). However, the lobsters fed on the glucose diet tended to be larger than the controls and other treatments. Survival of lobsters fed the general carbohydrates was significantly higher than lobsters fed the algal carbohydrates. Only 12 out of the 216 lobsters fed the general carbohydrates died compared 24 deaths out of the 48 lobsters fed the algal carbohydrates. Therefore, lobsters fed the general carbohydrates had higher survival than lobsters fed algal carbohydrates. From the present research it was postulated that the oxygen consumption and haemolymph glucose results of lobsters fed the simple monosaccharides (glucose and fructose) resulted in a negative physiological effect in juvenile J. edwardsii. However, the results obtained in Dr Jeffs' growth experiment for the general carbohydrates suggest otherwise because lobsters fed on the glucose diet tended to be larger than any of the other treatments. Therefore taking this Chapter Six: General Discussion 132

into account, it can be suggested that glucose be the carbohydrate diet to be added in the formulation of a nutritionally formulated diet for the aquaculture of J. edwardsii because it resulted in the highest growth rates.

Issues Highlighted For Further Study

The present research suggests that juvenile J. edwardsii fed a diet of squid grow faster and assimilate their food more efficiently when fed in the morning than at night. These results might not be replicated if different feed types and temperature was changed. Rock lobsters are poikilotherms, therefore environmental temperature affects the speed of their metabolism. Consequently if the temperature was changed, it could either speed up or slow down the SDA. Different feed types have different amino acid profiles, carbohydrates and lipid contents and thus could effect the duration of the response because it may take longer or shorter to digest the various constituents. The size of the meal may also alter the response, either by making it longer with larger sized meals or shorter with smaller sized meals. Therefore, more research needs to be conducted in this area. This research also needs to be repeated on adult J. edwardsii. Adult oxygen consumption rates are approximately 10 times smaller than juveniles, subsequently the magnitude and SDA coefficients could be smaller compared to juveniles. This could affect management practices because oxygen levels in the water would not need to be as high for adults compared to juveniles.

The SDA and the SDA magnitude of juvenile J. edwardsii is an excellent indicator of carbohydrate digestion. In order to determine if the SDA represents digestion for other specific ingredients of artificial diets, such as proteins and lipids, additional research needs to be conducted. It is postulated that proteins and lipids would exhibit a similar response to carbohydrates, that is, if they can be digested there would be a rise in oxygen consumption that would be significantly different to unfed lobsters. With proteins in particular, there should also be a significant increase in ammonia excretion.

The results of the growth experiment and haemolymph glucose concentrations indicate that agar is the algal carbohydrate that juvenile J. edwardsii can utilise most efficiently. Alginate is the binder most widely used in the development of artificial diets and agar is recommended as the Cht2DtE~r Six: General Discussion 133 binding agent in the development of an artificial diet for J. edwardsii. This could reduce the amount of extra carbohydrate added in the diet because agar can be utilised by the lobster. The overall digestibility of agar when included in an artificial diet should also be tested. This can be accomplished by adding an inert marker to the diet (e.g. Cr203). Agar should be tested in an artificial diet to determine the level required to produce a protein sparing effect. This would maximise the use of protein for tissue growth and prevent it from be partitioned into energy metabolism.

Results from the growth experiment carbohydrate experiments indicate that glucose is the carbohydrate that juvenile J. edwardsii utilise the most efficiently. Therefore, further research needs to be conducted on the digestibility of glucose, glycogen, maltose and sucrose in a complete artificial diet. The optimal quantity that lobsters require these carbohydrates in a nutritional diet also requires additional research. This can be achieved by adding varying quantities (e.g. 5, 10, 20, 25 and 50%) in a diet. Glucose, glycogen, sucrose and maltose should also be tested in a diet with agar as the binder. This would indicate whether glucose, glycogen, sucrose, maltose or agars utilisation by the lobsters is affected by one another. This could hold the key for carbohydrate utilisation for the formulation of a nutritional diet for the aquaculture of J. edwardsii. If an accurate formulation can be obtained from these carbohydrates, then a protein would be used for growth and not for the production of energy. This would make the diet more cost effective because the amount of protein can be reduced (most expensive component of a diet) and the amount of carbohydrate can be increased (the cheapest form of energy in a diet).

The present research investigated feeding energetics and lobster nutrition. These results, combined with previous and future research will aid in the success of the rock lobster aquaculture industry. References 134

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