Queensland University of Technology

School of Natural Resource Sciences

NUTRITION AND FEEDING BEHAVIOR IN TWO

SPECIES OF MUD CRABS

Scylla serrata and Scylla paramamosain

PHUONG HA TRUONG

M.Sc. Aquaculture

Submitted in fulfillment of the requirements for

the degree of Doctor of Philosophy

2008

ABSTRACT

Mud crabs of the genus Scylla are widely exploited for aquaculture in the Asia-

Pacific region. In the current study, a series of in vivo experiments were carried to assess

the protein requirement, protein sparing effects of starch and the capacity of Scylla

serrata to digest diets that contained different animal and plant-based feed meals and

different levels and types of starch.

Results from a protein requirement study indicated that juvenile S. serrata fed

diets containing 45% or 55% protein demonstrated significantly higher growth responses

than those fed the diet containing 25% protein. The subsequent study was carried out to

determine if responses to dietary protein could be influenced by using purified wheat,

potato, rice or corn starch to manipulate the gross energy level of fishmeal- based diets

(18 or 15.5 MJ kg-1), i.e., to see if starch had a protein sparing effect in these animals.

Overall, growth responses in this study appeared to be positively correlated with the level

of protein in the diet with the highest growth rates achieved using diets containing 45%

protein, regardless of the energy level of the diet. In addition, at a dietary protein level of

40% there was no evidence that the source of starch had any significant impact on growth

performance or feed utilisation suggesting it had no protein sparing effect. By contrast, it

was found that growth of juvenile S. serrata was strongly correlated with the intake of

digestible dietary protein.

The investigation of the capacity of sub-adult S. serrata to digest different animal

and plant- based feed meals showed that apparent dry matter digestibility (ADMD) and

apparent gross energy digestibility (AGED) values were not significant different for most

ii

selected feed meals (cotton seed, poultry, canola, fishmeal, soybean, and lupin meal).

Apparent crude protein digestibility (ACPD) for all test feed meals were relatively high

(86-96%). A subsequent study was carried out to determine if purified starch from

different sources influenced the digestibility of fishmeal based diets. Overall, most diets

containing starch were readily digested by mud crabs. In particular, there were no

negative impacts on the digestibility of major nutrients (e.g. protein) observed following

the inclusion of wheat, rice or corn starch in formulated feeds. Nevertheless, the apparent

starch digestibility (ASD) of wheat starch decreased significantly as the inclusion level

was increased from 15% to 60%, although there was no significant effect on ACPD

values. At a 30% inclusion level, the ASD of diets containing different starches decreased

in the order corn > wheat > potato = rice. Moreover, ACPD values were significantly

higher for diets containing corn or rice starch than for those containing wheat or potato

starch.

The capacity of another species of mud crab commonly exploited for aquaculture

in South East Asia, S. paramamosain, to digest the local plant-based ingredients

(defatted soybean meal, rice bran, cassava and corn flour) was also conducted in

Vietnam. Overall, the findings of this study showed that at a 30% inclusion level diets

containing soybean meal or rice bran were well digested by mud crabs. In particular, the

ACPD and AGED values for all diets containing soybean meal were not significantly

different from the fishmeal based reference diet. Likewise, all digestibility values for the

diet containing 30% rice bran were relatively high and not significantly different from the

reference diet. By contrast, diets containing cassava flour appeared to be poorly utilised

since their digestibility values for all parameters were lower than those from other test

iii

ingredients. In summary, the apparent digestibility of dry matter, protein and energy was

in the following order (from most to least digestible) soybean meal ~ rice bran > corn

flour > cassava flour.

In the next study the effects of attractants in diets (chicken meal, betaine, tuna oil

and bait enhancer), temperature (26.5oC, 28.5oC and 30.5oC), sex (female and male) and

size (small, medium and large) on feeding responses of S. serrata were investigated.

Significant differences were observed in the behavioral responses of mud crabs to diets

containing different attractants. Specifically, consumption of diets with chicken meal or

betaine was significantly higher than for other treatments. With the exception of betaine,

no significant difference in food consumption was observed when attractant inclusion

levels were raised from 2% to 5%. Overall, small crabs consumed significantly more of

the ration (as a percentage of body weight) than larger crabs. Temperature showed a

significant impact on most behaviour of mud crabs, excepting continuation response and

there was some evidence that females were significantly more active than males. Light

intensity was considered as a main factor effect to crab response since there were

extremely high percentage time of crab spent in half-shaded of the Y –maze which valued

at 95.6%, 93.8 and 94.4% (corresponded to small, medium and large size respectively) in

comparison to those of crabs spent in the unshaded side.

Overall, the findings from these studies demonstrated that mud crabs have a high

capacity to digest a range of plant based feed ingredients. In particular, soybean meal

appeared to be well digested by both species of mud crabs examined. It was also shown

that a range of purified starches were well digested by S. serrata although starch

iv

inclusion in diets did not appear to reduce the requirement for protein to promote growth.

Subsequent attractant studies demonstrated that chicken meal and betaine produced

significantly elevated feeding responses and food consumption when added to diets.

Based on these results we propose that these ingredients can be utilised to increase the

attractiveness and consumption of artificial mud crab feeds.

Keywords

Aquaculture, mud crab, Scylla serrata, Scylla paramamosain, starch, plant ingredients,

protein, energy, digestibility, video behaviour analysis, chemo-attractants

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SUBMITTED MANUCRIPTS

Truong, H.P., Anderson, A.J., Mather, P.B., Richardson, N.A. & Paterson, B.D. (2008).

Effect of selected feed meals and starches on diet digestibility in the mud crab, Scylla

serrata. Aquaculture Research (in press)

Truong, H.P., Anderson, A.J., Mather, P.B., Richardson, N.A. & Paterson, B.D. Effect of

dietary protein and starch on growth and feed utilisation in the juvenile mud crab, Scylla

serrata. (In review)

Truong, H.P., Anderson, A.J., Mather, P.B., Richardson, N.A. & Paterson, B.D. (2008).

Apparent digestibility of selected feed ingredients in diets formulated for the sub-adult

mud crab, Scylla paramamosain in Vietnam. Aquaculture Research (in press)

Truong, H.P., Paterson, B.D., Anderson, A.J., Richardson, N.A., Mather, P.B. The effect

of culture environments, sex, size and feed attractants on feeding responses in the mud

crab, Scylla serrata (In prep.)

vi

TABLE OF CONTENTS

ABSTRACT……………………..………………………………………………………...i

KEYWORDS……………………………………………………………………………..v

LIST OF SUBMITTED MANUCRIPTS………………………………………………vi

TABLE OF CONTENTS…………………………….………………………………...vii

LIST OF TABLES AND FIGURES…………………………………………………...xi

STAEMENT………………………………………………………………….………...xvi

ACKNOWLEDGEMENTS…………………………………………………….…….xvii

DEFINITION OF ABBREVIATIONS………………………………… ………….xviii

CHAPTER 1. GEENRAL INTRODICTION

1.1 Introduction……………………………………………………1

1.2 Scope and aims of study……………………………………..29

References………………………………………………………..32

CHAPTER 2. THE EFFECTS OF DIETARY PROTEIN AND STARCH ON

GROWTH AND FEED UTILISATION IN THE JUVENILE

MUD CRAB, SCYLLA SERRATA

2.1 Abstract………………………………………………………48

vii

2.2 Introduction…………………………………………………..50

2.3 Materials and methods……………………………………….53

2.4 Results………………………………………………………..57

2.5 Discussion and conclusion …………………………………..60

Acknowledgements………………………………………………64

References......................................................................................65

Tables and figures ……………………………………………….69

CHAPTER 3. THE EFFECTS OF SELECTED FEED MEALS AND

STARCHES ON DIET DIGESTIBILITY IN THE MUD

CRAB, SCYLLA SERRATA

3.1 Abstract………………………………………………………78

3.2 Introduction…………………………………………………..79

3.3 Materials and methods……………………………………….83

3.4 Results………………………………………………………..89

3.5 Discussion and conclusion …………………………………..91

Acknowledgements ……………………………………………...94

viii

References ……………………………………………………….95

Tables and figures ……………………………………………….99

CHAPTER 4. APPARENT DIGESTIBILITY OF SOME NUTRIENT

SOURCES BY JUVENILE MUD CRAB, SCYLLA

PARAMAMOSAIN

4.1 Abstract……………………………………………………..108

4.2 Introduction…………………………………………………109

4.3 Materials and methods……………………………………...111

4.4 Results………………………………………………………114

4.5 Discussion and conclusion …………………………………117

Acknowledgements …………………………………………….120

References ……………………………………………………...121

Tables and figures ……………………………………………...124

CHAPTER 5. THE EFFECTS OF ATTRACTANT DIETS ON RESPONSES

OF THE MUD CRAB, SCYLLA SERRATA

5.1 Abstract……………………………………………………..131

5.2 Introduction…………………………………………………133

ix

5.3 Materials and methods……………………………………...136

5.4 Results………………………………………………………142

5.5 Discussion and conclusion …………………………………146

Acknowledgements …………………………………………….151

References ……………………………………………………...152

Tables and figures ……………………………………………...158

CHAPTER 6. GENRAL DISCUSSIONS AND CONCLUSIONS

Discussions …………………………………………………….170

Conclusions……………………………………………………..179

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LIST OF TABLES AND FIGURES

Page

Chapter 1.

Table 1.1. Dry basis proximate composition and gross energy of selected aquafeed

ingredients………………………………………………………………..14

Table 1.2. Summary of parameters in several different types of cultured mud crab

systems in Southeast Asia ……………………………………………….21

Figure 1.1. World aquaculture production and value of major species groups in

2002………………………………………………………………………..4

Figure 1.2. Aquaculture and wild fish capture production of Vietnam during the

period of 1993-2003 ………………………………………………………6

Figure 1.3. Estimates of current and future fish meal and fish oil used in aquaculture

worldwide…………………………………………………………………9

Figure 1.4. Photographs of adult female Scylla serrata showing diagnostic feature...18

Figure 1.5. Photographs of adult female Scylla paramamosain showing diagnostic

features…...………………………………………………………………19

Chapter 2.

Table 2.1. Composition of diets formulated for Study 1……………….…………...69

xi

Table 2.2. Composition of diets formulated for Study 2………….………………...70

Table 2.3. Influence of dietary protein on final weight, specific growth rate (SGR)

food conversion ratio (FCR) and survival of S. serrata…………………71

Table 2.4. Weight gain (WG), specific growth rate (SGR), average moulting

frequency and survival rate (SR) of juvenile mud crabs fed experimental

diets during ten weeks of experimental culture………………………….72

Table 2.5. Feed conversion ratio (FCR) and protein efficiency ratio (PER) of

juveniles fed experimental mud crab diets ……………………..……….73

Figure 2.1. Correlation between weight gain (mean ± s.e.m.) and protein intake of

juvenile mud crabs over a ten week growing period ………….………...74

Chapter 3.

Table 3.1. Composition (% dry matter of the diet) of the formulated diets for the

digestibility trial using commercial feed ingredients………..…………...99

Table 3.2. Proximate nutrient composition (%) in dry matter of basal diet and test

ingredients used in the digestibility trial………………..………………100

Table 3.3. Composition (% dry matter of the diet) of the formulated diets for the

digestibility trial using different starches. ……………….……………..101

Table 3.4. Proximate nutrient composition (%) in dry matter of experimental diet

used in the starch digestibility trial……….…………………………….102

xii

Table 3.5. The apparent digestibility coefficients (%) of dry matter (ADMD), crude

protein (ACPD) and gross energy (AGED) for yeast and selected animal

feed meals……………………………………………………………..103

Table 3.6. Impact of starch on apparent digestibility coefficients (%) for dry matter

(ADMD), crude protein (ACPD), gross energy (AGED) and starch (ASD)

in fishmeal-based formulated mud crab diets…………………………104

Chapter 4.

Table 4.1. Composition of the nine formulated diets for the digestibility trial using

different local ingredients (as % dry weight basis)…………… ………124

Table 4.2. Dry matter (DM), gross energy (GE MJ kg-1), crude fat (CF), crude

protein (CP) and ash of experimental diets and test ingredients used in the

formulated diets (as % dry weight basis)……………………………….125

Table 4.3. Apparent digestibilities (%) for dry matter (ADMD), crude protein

(ACPD) and gross energy (AGED) in experimental diets.……………..126

Table 4.4. Apparent digestibility coefficients (%) for dry matter (ADMDI ADCDM),

crude protein (ACPDI ) and gross energy (AGEDI ) of the test ingredients

used in the formulated diets ………………………………….………...127

xiii

Chapter 5.

Table 5.1. Mud crab behaviour description and response value measurements.…..158

Table 5.2. Formulation of reference diet (as % dry matter basis)… ………………159

Table 5.3. Percentage of time crabs engaged in each arm of the maze under five

combinations between seawater (no food) and five test diets……..……160

Table 5.4. Percentage of time crabs engaged in each arm of the maze under four

combinations between reference and four test diets ……………...…...161

Table 5.5. Percentage of time crabs spent on specific behaviour under sea water and

five test diets……………………………………………...…………….162

Table 5.6. Time spent of medium-sized crabs on specific behaviours at 26.5oC,

28.5oC and 30.5oC using 2% chicken meal diet …………..……………163

Table 5.7. Time spent of crabs on specific behaviours testing with equal amounts of a

2% chicken meal diet placed in the two arms of the Y-maze under light

and dark environments ……………………………...………………….164

Figure 5.1. The Y-maze chamber with (a) from the top side (plan) and (b) from the

horizontal side (elevation)…………………………..…………………..165

Figure 5.2. Effect of size on crab response to diet containing 2% CM ………….…166

xiv

Figure 5.3. Initiation response of different sized crabs exposed to diets containing

different attractants ……………………..……………………………..167

Figure 5.4. Effect of sex on crab responses …………….……………………………..168

Figure 5.5. Effects of different attractants on food ………………………………..169

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STATEMENT

I, Phuong Ha Truong, hereby declare that the work described in this thesis has never been

previously submitted for a degree or diploma in any University. I also declare that to the

best of my knowledge and belief, this thesis contains no material previously published or

written by another person excepting where due reference is made in the thesis itself.

Signed ……………………………………………..

Phuong Ha Truong

Date 20th February 2008

xvi

ACKNOWLEDGEMENTS

I would like to thank my supervisors, Dr Alex Anderson, Dr Neil Richardson,

Associate Professor Peter Mather (QUT) and Dr Brian Paterson (BIARC) for their

advice, encouragement and technical support throughout this project and also their

valuable criticism during the preparation of this thesis and of the manuscripts of papers.

I would like to thank the Bribie Island Aquaculture Research Centre (Australia)

and Research Institute for Aquaculture No3 (Vietnam) for providing me the facilities to

conduct this project. I thank my co-workers at Bribie Island Aquaculture Research Centre

and Research Institute for Aquaculture No3, David Mann, Marko Pavasovic, Tom

Asakawa, Beverly Kelly, Nguyen Co Thach, Truong Quoc Thai, Nguyen Xuan Nam, Le

Van Chi, Nguyen T.X. Thu for their technical assistance, time and patience throughout

this project. I would also like to gratefully acknowledge the financial support provided by

the Ministry of Education and Training, Vietnam and ACIAR Mud Crab Project

FIS/2000/065, Australia. I would like to thank Mr. Vincent Chand of the Queensland

University of Technology for his assistance in the biochemical analysis of this project.

My gratitude is also extended to Dr Richard Smullen of Ridley Aqua-Feed (Australia)

and Tran Dinh Thanh of Seaprodex, Danang, Vietnam for supplying the fishmeal, John

Harsant of Radford Park Aquafeeds (Australia) and Dr Linda Browning of DSM

Nutritional Products (Australia) for supplying experimental materials.

Finally, I would especially like to thank my beloved wife, son, daughter and

parents for their ongoing encouragement and support throughout my studies. Without

their encouragement and support my studies would not have been possible.

xvii

DEFINITION OF ABBREVIATIONS ACIAR Australian Centre for International Agricultural Research

ACPD Apparent digestibility coefficient for crude protein

ADMD Apparent digestibility coefficient for dry matter

AGED Apparent digestibility coefficient for gross energy

ANOVA Analysis of variance

ASD Apparent starch digestibility

BIARC Bribie Island Aquaculture Research Centre

BSE Bovine spongiform encephalitis

CHO Carbohydrate

FCR Food conversion rate

G Gram

GE Gross energy

h Hour

kg Kilogram

L Liter

m Meter

MJ Megajoule

mL Milliliter

mm Millimeter

MOET Ministry of Education and Training

MOFI Ministry of Fishery

nm Nanometer

xviii

p The probability of the observed differences occurring due to

random variation

QUT Queensland University of Technology

® Registered brand name

RIA3 Research Institute for Aquaculture No 3

se Standard error

sem Standard error of the mean

SGR Species growth rate

xix

1

CHAPTER 1. GENERAL INTRODUCTION

2

1.1 Introduction

Traditionally, capture fisheries have provided a major source of animal aquatic

food for human consumption. Over recent decades, however, increased fishing

pressure has severely depleted this resource. Even with the pursuit of new species and

improved fishing technologies, many over exploited stocks are yielding catches that

have reached their maximum sustainable limits and possess little potential for further

growth (FAO 2000). As a consequence, there has been a dramatic expansion in

aquaculture that is broadly defined as the culture of aquatic plants and animals. In

particular, aquaculture has become increasingly important in many parts of the Asia-

Pacific region where it has helped to address a rapid rise in human consumption of

aquatic products and provided greater food security.

Aquaculture production

Over recent decades, aquaculture has been the fastest growing food production

sector worldwide, with production levels increasing at 12.8% year-1 (Tacon 1999).

This growth has been driven by increases in human populations, increased human

consumption of aquatic products, and declines in major capture fisheries (FAO 2003).

The contribution from aquaculture to total fisheries continues to grow, increasing from

5.3% in 1970 to 32% by weight in 2000 (Tacon 2003). Aquaculture has grown at an

average of 18.6% per year in period of 1997-2006, while capture fisheries has reduced

0.25% per year over the same period (FAO 2008). Moreover, production from

aquaculture has greatly outpaced human population growth, with the world average

per capita supply from aquaculture increasing from 0.7 kg in 1970 to 6.4 kg in 2002

(FAO 2003).

3

At present, Asia has the dominant position in global aquaculture. For example,

aquaculture output from Asia contributes 82% of molluscs, 84% of crustaceans, 89%

of finfish and almost all aquatic plants produced world wide (Hassanai 2003). In 1997,

combined production from 22 Asian countries and territories was 30.7 million metric

tones and was valued at 37.6 billion US dollars. This represents an increase of 11.2%

by weight and 10.6% by value from 1995. Aquaculture production in the region,

including from west Asia, has been growing more than four times faster than have

capture fisheries. Moreover, the contribution to total fisheries landings in the region

increased nearly two-fold from 20.7% in 1984 to 38% in 1995 (Hassanai 2003).

Development of aquaculture in Vietnam provides a clear example of the rising

importance of this important food production industry in Asia. The commercial culture

of aquatic species is recognised by the government of Vietnam as an industry with

great potential to generate income, to provide food security and to alleviate rural

poverty. Over the last two decades aquaculture production in Vietnam has increased

ten fold and in 2004 accounted for 37% of total aquatic product (MoFI 2005). As

shown in Figure 1.1, this is in direct contrast to growth in capture fisheries that has

been estimated at only 6.1% year-1 over the period 1993-2003 (FAO 2003).

Despite the recent rapid expansion of aquaculture in Asia, there is some

evidence that the future expansion of this industry may be constrained by physical,

genetic, social and/ or economic factors. In particular, overall rate of growth of the

industry in Asia appears to be declining. For example, in 1994 the rate of growth of

Asian aquaculture was 13.5% while in 1997 this growth had declined to only 6.3%. It

is noteworthy that this slowdown is happening in the face of continuing high

population growth and a rapidly growing demand for foodfish in much of the region

4

(Hassanai 2003). Improving the productivity and cost effectiveness of aquaculture

systems represents an important strategy that can guarantee future food security in this

region of the world.

Figure 1.1. Aquaculture and wild fish capture production in Vietnam during the period

of 1993-2003 (FAO 2003).

Animal species in aquaculture

In 2000, there were approximately 210 aquatic species cultured worldwide

(Tacon 2003). This diversity reflects the wide range of species available in different

countries and the wide variety of systems used. For an aquatic animal to be considered

as a commercially viable species however, it needs to meet a number of biological and

economic criteria. These criteria include:

• High growth and survival rates;

• High market price;

5

• Low protein requirement and low production costs;

• Acceptance of artificial diets;

• Low incidence of disease;

• Availability of broodstock or fry; and

• Ability to be domesticated.

Although few species currently meet all of these criteria, it is anticipated that

development of low costs diets and improved culture systems together with genetic

selection to improve survival and growth rates, increasing numbers of aquatic

organisms will become available for commercial farming.

Crustaceans are an important aquaculture commodity. Important crustaceans

produced in culture include marine and freshwater prawns, lobsters, crabs and

freshwater crayfish. Marine prawns (or shrimp) are the principal cultured species

exploited for aquaculture and in 2000 accounted for 66.0% of global crustacean

aquaculture production (Tacon 2003). In 2000, global aquaculture production of

crustaceans was estimated at about 1.65 million tonnes. This accounted for only 3.6%

by weight of global aquaculture production but comprised approximately 16.6% by

total value and was estimated to be worth about U$ 9.37 billion (Tacon 2003). Figure

1.2 shows that production of crustaceans was ranked fifth by weight but second by

value. It is important to note that there has been a particularly significant increase in

crab aquaculture in recent years. For example, global crab production increased about

46.2% by weight and 66.2% by value between 1988 and 1997 (FAO 2003).

6

Figure 1.2. World aquaculture production and value of major species groups in 2002

(FAO 2003).

Aquafeed production

While aquaculture production has increased rapidly in recent years and is

projected to continue to increase considerably, the natural productivity of aquaculture

systems is insufficient to meet all of the nutritional requirements of candidate species.

As a consequence, supplemental feeds must be provided to facilitate growth and

7

prevent excessive mortalities that result from starvation or cannibalism (New &

Wijkström 2002). On a global scale, 11.5 million tones of aquafeed were required to

produce 12.9 million tonnes of product during 1997 (Tacon 1999). In fact, production

of aquafeeds has been widely recognized as one of the fastest expanding agricultural

industries in the world, with growth rates in excess of 30% per year (Halwart 2003).

A major challenge currently facing the world aquaculture industry is the need

to develop feeding strategies for finfish and crustacean farming systems based on use

of low cost, locally available feed components. At present, nearly all farming

operations for carnivorous, diadromous finfish, marine finfish and crustaceans that

require supplemental aquafeeds are net consumers of fishery resources rather than net

producers. Input of dietary fishery resources in the form of such products as fishmeal,

fish oil or “trash fish” however, exceeds production outputs by a factor of two to three.

There is a general consensus that such aquaculture practices are unsustainable in the

long term and, therefore, increased attention has been placed on identifying potential

ingredients such as terrestrial animal by-product meals, plant oilseed and grain legume

meals, cereal by-product meals; and miscellaneous protein sources such as single-cell

proteins, leaf protein concentrates and invertebrate meals as potential ingredients for

aquafeeds (Halwart 2003).

Fishmeal and fish oil production

Fishmeal and fish oil are the primary protein and energy sources included in

most aquafeeds and account for up to 75% and 35% of total feed, respectively (Tacon

1999). However, the price of fishmeal and fish oil increased about 62.9% and 170%

from August 2005 to June 2008, respectively (Hammersmith Marketing Ltd, 2008),

8

therefore the inclusion of fishmeal and fish oil in aquafeeds has been reduced to 45%

and 33% and targeted to the levels of 16% and 10% respectively for some fish species

in Europe (Aquamax 2007). In 1989, 7.1 million tones of fishmeal were produced

globally of which 10% was used in aquafeeds (FAO 2000). This had increased to 37%

by 1997 (Tacon 1999) and 46% by 2002 (Pike 2005). The expected future expansion

of global aquaculture, particularly of carnivorous species, has the potential to utilize

about 70% of total global supplies of fishmeal by the year 2015 and to exceed the total

supplies of fish oil well before this date (New & Wijkström 2002).

High quality fishmeal and fish oil play a key role in modern intensive

aquaculture systems. Future availability of fish-based products for aquafeeds is

predicted to constrain further expansion of global aquaculture industries. Specifically,

by 2015 the global aquafeed industry is expected to have the potential to utilize nearly

4.6 million tones of fishmeal and nearly 1.9 million tones of fish oil (New &

Wijkström 2002). Thus, the global aquafeed industry has the potential to utilize 70%

of the average historical annual fishmeal supply by the year 2015. If supplies of

fishmeal do not increase, the ‘fishmeal trap’ will start to constrain producers of shrimp

and carnivorous fish as the world market price of fishmeal increases in response to

increasing demand. Furthermore, the global aquafeed industry has the potential to

exceed the average historical annual supplies of fish oil before the year 2010 and to

reach 145% by 2015. This means that if supplies of fish oil do not increase, the ‘fish

oil trap’ will become a very real constraint on producers of shrimp and carnivorous

fish well before 2010. Fishmeal and other sources of raw material (waste, offals) are

not likely to permit any significant and sustainable increases in the supplies of

fishmeal and fish oil between now and 2015 (New & Wijkström 2002).

9

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

1992 1995 1999 2000 2010 2015

Year

Fish

mea

l and

fish

oil u

ssag

e (x

1000

)

FishmealFish oil

Figure 1.3: Estimates of current and future fish meal and fish oil used in

aquaculture worldwide (New & Csavas 1995, Tacon 1998, Barlow 2000, New

& Wijkström 2002, Chamberlain 2000).

The potential for fish-based products to limit aquculture expansion is

particularly evident in Asia. For example, in Vietnam, there are two types of fishmeal

commonly used: (1) “fish powder” produced in a traditional way by sun- drying and

grinding, and (2) fishmeal product produced using an industrial process in which raw

materials are cooked before being dried. In a recent survey of trash fish and fishmeal

in Vietnam it was reported that locally manufactured fishmeal was generally of poor

quality and, therefore, unsuitable for inclusion in most aquafeeds (Edwards et al.

2004). Locally produced fishmeal contains approximately 60% protein and is mainly

used for feeding livestock and some freshwater fish species (Edwards et al. 2004).

Fishmeal of higher quality (minimum 70% protein) used for feeding fish fingerlings

and crustaceans is mainly imported from Japan, Taiwan and South America and

represents about 90% of the total fishmeal used in Vietnam. According to Ministry of

10

Fisheries of Vietnam (MOFI 2005), the feed manufacturing companies in Vietnam

produced only about 10,000mt year-1 that accounted for only 10-15% of the demand

from aquaculture recently. As a consequence, there is heavy reliance on imported,

expensive, high quality fishmeal for manufacture of formulated fish and crustacean

feeds. With future demand for fishmeal expected to increase as aquaculture production

expands, there is concern that only species of very high market value (e.g. lobster) will

be able to compete for this critical feed ingredient.

Alternative ingredients to fishmeal

Many plants and animal proteins have potential as replacements for fishmeal

protein in aquafeeds. A large number of possible fishmeal replacers are available

locally including; invertebrate animal by-products (e.g. silkworm pupae, earthworms,

zooplankton), vertebrate animal by-products (e.g. blood meal, liver meal, meat and

bone meal, poultry by-products), single-cell proteins (mainly from fungal and bacterial

sources), oilseeds (e.g. soybean, rapeseed, sunflower, cottonseed), legumes (e.g.

beans, peas, lupins) and miscellaneous plant protein products (e.g. corn gluten meal

and concentrates made from potatoes and leaves). The major constraints to the use of

alternative protein sources to replace fishmeal protein identified by Tacon (1994)

were: (1) Limited availability and cost of single-cell proteins; (2) Lack of palatability

and anti-nutritional factors in poorly processed plant oilseeds and legumes; (3)

Limited availability, erratic quality and microbial contamination in terrestrial animal

by-product meals; (4) Palatability problems, and limited availability and high cost of

miscellaneous plant proteins.

11

Traditionally, in common with feed ingredients from other animal sources

(mammalian, poultry), the raw materials used in production of aquafeeds have been

regarded as potential sources of agricultural chemical residues, microbial pathogens

and heavy metals. After processing, they can also become sources of mycotoxins and

microbial pathogens. Two potential problems have become particularly important

recently. The first is the presence of dioxin and PCB residues in human food products

of animal origin and the potential carry-over of these substances from animal feeds.

The increase in demand for fishmeal and fish oil, compared with supply, has led to

increased prices. Strict guidelines have also been set down by the European Union on

the maximum acceptable levels of dioxin and polychlorinated biphenyls in fishmeal

and fish oil used in animal feeds. This is likely to lead to an even higher demand and

global market prices for fishmeals in time (Stone 2002). The second is the relationship

between meat and bone meal and the incidence of bovine spongiform encephalopathy

(BSE) in ruminants, coupled with their link to Creutzfeld Jacob Disease (CJD) (New

& Wijkström 2002).

Terrestrial animal meals are usually higher in total protein content than plant

ingredients (Table 1.1) and have been shown to be a suitable replacement for fishmeal

in many species of fish (Tacon 1994; Allan et al. 2000b; Stone et al. 2000; Alexis &

Nengas 2001; Edwards et al. 2004). Global production of terrestrial animal meals (5.5

million tones year-1) is similar in volume to fishmeal and however they also suffer

from problems of availability (Stone 2002). The potential application of terrestrial

animal meals in aquafeeds has also been severely impacted by the advent of “mad

cow- disease” (bovine spongiform encephalopathy-BSE) (New & Wijkström 2002;

Stone 2002). Countries in the European Union have banned use of any terrestrial

12

animal meals in aquafeeds, while the USA is also considering prohibiting the

importation of any seafood that has been produced using terrestrial animal meals

(Alexis & Nengas 2001; New & Wijkström 2002; Stone 2002).

Theoretically, a ban on (or a deterrent to) the use of any high-protein

ingredients creates a large potential market for replacements. The ban on meat and

bone meal may increase the demand for fishmeal dramatically, and affect price as a

consequence. If this proves to be true, the aquaculture industry would experience

enhanced competition from other agriculture sectors for supplies of fishmeal in the

future.

Further research on potential total or partial replacement ingredients for both

fishmeal and fish oil is therefore essential. In the case of fish oil replacement, the need

is now urgent. Oilseeds, legumes and grains could provide a cheap source and huge

resource for essential nutrients for aquatic animals (Tacon 1994; Tacon 1999; Allan et

al. 2000b; Alexis & Nengas 2001; Stone 2002). Fish product substitution studies must

take into account not only the effect of marine resource replacers on standard farming

parameters, such as growth and survival rates and food conversion ratios, but also on

their impact on other factors including immune function and disease resistance. The

effect of potential marine resource replacers on the quality of farmed aquaculture

products also needs further study, it means that more knowledge about the impact of

such replacement on the nutritional, sensory, processing, and safety characteristics of

the farmed products will be required (New & Wijkström 2002).

The aquafeed and aquaculture producing sectors must be ready to apply the

results of such research as soon as economic and other factors dictate (New &

13

Wijkström 2002). Finally, alternative forms of aquaculture that require less utilization

of marine resources (e.g. the culture of omnivores and herbivores) require further

promotion.

According to New (2001), relatively poor digestibility, lower availability of

some essential amino acids, palatability problems, and, in some cases, the presence of

anti-nutritional factors, have limited replacement of fishmeal with plant proteins. To

some extent these factors have been ameliorated by inclusion of supplemental

(synthetic) amino acids and flavour enhancers. More recently, the use of enzymes to

enhance the nutritional value of diets based on plant proteins has been suggested, and

according to Gerin (1999) is ‘used on a confidential basis in aquafeeds’. New strains

of plants, with lower levels of phytates and anti-nutritional factors may also be

developed. Furthermore, if public opinion allows, plants may be genetically modified

to improve their fatty acid and amino acid profiles (Chamberlain 1993). Other

alternative protein sources, such as single cell proteins (SPC), have also been

considered (Tacon 1995; Åsgård et al. 1999). Brandsen et al. (2001) cautioned that

fish growth is not the only factor to be considered when assessing potential

replacement ingredients for fishmeal, indicating that their effects on disease resistance

and immune function need to be investigated but are seldom mentioned. Such effects

may be beneficial or detrimental. .

14

Table 1.1: Dry basis proximate composition and gross energy of selected aquafeed

ingredients.

Ingredientsa Protein

(g 100g-1)

Lipid

(g 100g-1)

CHOb

(g 100g-1)

Energy

(MJ kg-1)

Ash

(g 100g-1)

Fish meals

Danish fish meal

Peruvian fish meal

72.9

70.2

11.4

11.3

-

-

21.5

20.9

13.0

17.6

Terrestrial animal meals

Blood meal (spray dried)

Poultry meal

Meat and bone meal (lamb)

Meat and bone meal (beef)

94.9

60.0

54.3

49.2

-

18.2

7.2

9.2

-

-

-

-

23.9

22.7

16.2

16.1

3.1

15.0

34.5

36.0

Plant materials

Soy bean meal (solvent extracted)

Soy bean meal (whole expeller)

Soy bean meal (dehulled full-fat)

Canola meal (solvent)

Peanut meal

Wheat gluten

Wheat (Australia standard)

Corn gluten meal

47.8

45.0

35.8

36.6

41.2

76.9

12.2

62.0

3.7

6.4

19.5

2.6

1.3

-

1.9

-

40.5

42.3

39.2

53.4

52.3

-

84.1

-

17.0

20.9

23.3

19.9

19.7

23.1

18.3

24.1

8.0

6.3

5.5

7.4

5.2

-

1.8

1.1 a Data from Allan et al 2000b and Stone 2002; b Total CHO = Total carbohydrate

(including fibre) (g 100g-1) calculated by difference = 100 – (protein+ lipid + ash).

Grains, oilseeds, and legumes may provide a cheap source of essential

nutrients for fish and crustaceans. These ingredients contain a significant level of

protein and energy, and have the potential to replace quantities of fishmeal in

aquafeeds (Table 1.1) (Tacon 1994; Tacon 1999; Allan et al. 2000b; Allan et al.

2000c; Alexis & Nengas 2001; Catacutan et al. 2003; Stone 2002; Edwards et al.

2004; Pavasovic et al. 2004; Tuan et al. 2006)

15

Plant-based carbohydrates as potential feed ingredients

Carbohydrates form the most widespread organic compounds in the biosphere.

The term ‘carbohydrate’ covers a large number of molecules, mainly formed of

carbon, oxygen and hydrogen atoms. They form the primary component of grains,

legumes, oilseeds, and roots and tubers. Plant carbohydrates are polyalcohols with one

or several aldehyde or ketone functions. Simple sugars (monosaccharides) include a

single unit of hydroxyaldehyde or hydroxyketone. Oligosaccharides contain between

two and ten monosacchride molecules linked by glycoside bonds. Polysacchrides are

polymers with either straight or branched chains (Kaushik 2001). Plant carbohydrates

are generally classified into two categories, energy reserve polysaccharides and

structural polysaccharides (non-starch polysaccharides - NSPs). Starch is the

predominant energy reserve carbohydrate in most plant materials (Stone 2002).

Carbohydrates of starch origin are often referred to as digestible carbohydrate, while

NSPs are often referred to as indigestible carbohydrate. In general, NSPs are a

complex group, composed predominantly of linked monomers of hexoses and

pentoses such as glucose, galatose, mannose, arabinose, and xylose (Stone 1996; van

Barneveld 1999). Although many carbohydrates exist, only a few have nutritive value

in animal nutrition. These mainly involve hexoses (glucose), disaccharides (formed

from two hexose molecules), and some homopolysaccharides, including starch

(Kaushik 2001).

Plant carbohydrates have been identified as the least expensive potential source

of dietary energy for human and domestic animals (Wilson 1994; Stone 2002;

Edwards et al. 2004). Digestibility of starch varies however, with botanical origin,

inclusion content, and with how it is treated. Furthermore, inclusion of starch in

16

aquafeeds can affect the digestibility of protein and lipids but effects vary from one

species to another (Cousin et al. 1996; Sales & Britz 2002; Stone et al. 2003).

Inclusion of digestible dietary carbohydrate as an energy source in aquafeeds

can also reduce the feeding costs of culture, for example in mud crabs where it

accounts for about 40-50 % of the total operating costs (Quinitio 2004). Based on

previous studies, digestible carbohydrate has been recommended at an inclusion level

of 20% for many carnivorous species (salmonids, marine fish and crustaceans) and up

to 40% for warm water omnivorous species (Wilson 1994; Catacutan & Coloso 1997;

Kaushik 2001).

Mud crab investigations

Mud crabs are widely distributed across the Indo-Pacific region where they are

fished as a commercial commodity (Hill 1983). They are also an important source of

income and fresh food for many coastal communities and are viewed as a luxury item

in many places where they are appreciated for taste and texture, low fat content and

high levels of protein, vitamins and minerals. Currently, many mud crab fisheries are

suffering from the effects of overfishing. This has threatened the capacity of wild

fisheries to meet future demand for local and export markets.

Knowledge of the morphology and distribution of any species and its

population structure are important for developing sustainable culture systems and for

developing and implementating approriate fisheries management options (Keenan

1999b). In general, the poor understanding of the systematic and genetic relationships

in the genus Scylla has been a primary constraint on management of the wild fishery

and development of aquaculture (BOBP 1992).

17

Until 1998, one of the major constraints on development of mud crab culture

was problems associated with presence of cryptic species in crabs included in the

Scylla genus. Previously, it was generally assumed that the genus Scylla constituted

only a single species. Even after Estampador’s classification (1949), all forms

continued to be referred to as Scylla serrata for many years (Fortes 1999; Nguyen

2000). Before 1998, very little was known about the individual characteristics of each

species and their relative suitability for culture. Variation in colour, morphology and

biological characteristics in this genus however reported from Philippines, Vietnam,

India and Japan have established the existence of more than one species (Estampador

1949; BOBP 1992; Keenan et al. 1995; Fuseya & Watanabe 1996; Keenan et al. 1998;

Sugama & Hutapea 1999).

Recently, Keenan (1999b) revised the genus and reported the following re-

classification:

Estampador (1949)

S. serrata

S. oceanica

S. serrata var paramamosain

S. tranquebarica

Keenan (1999b)

S. serrata

S. olivacea

S. paramamosain

S. tranquebarica

Scylla serrata (Figure 1.4) is the most common species in samples collected

from the Gulf of Carpentaria, Moreton Bay, Northern Territory, and Western Australia

(Keenan 1999b) but countries in southeast Asia appear to have suitable environmental

conditions for most of the four species recognised in the genus Scylla. For example, S.

paramamosain (Figure 1.5), S. serrata and S. olivacea are found in Indonesia

(Sugama & Hutapea 1999); S. paramamosain, and S. olivacea are found in the

18

Mekong Delta Vietnam; S. olivacea, S. serrata and S. tranquebarica are found in the

Philippines and S. olivacea, and S. tranquebarica occur in Malaysia (Keenan 1999b;

Quinitio 2004).

Figure 1.4. Photographs of adult female Scylla serrata showing diagnostic features:

high, bluntly pointed frontal lobe spines; pairs of large spines obvious on carpus and

propodus; polygonal patterning clearly present on all appendages. Photo: Queensland

Museum, reproduced by Keenan 1999.

19

Figure 1.5. Photographs of adult female Scylla paramamosain showing diagnostic

features: moderately high, pointed and triangular frontal lobe spines usual; pairs of

large spines obvious on propodus, on carpus inner spine absent and outer spine

reduced; polygonal patterning clearly present on last two pairs of legs, weak or absent

on other appendages. Photo: Queensland Museum, reproduced by Keenan 1999.

Mud crab grow out

Fisheries exploitation and aquaculture production of the world’s portunid crabs

has increased seven fold over the past thirty years (Keenan 1999a). For example, the

finite capacity of wild fisheries to service growing demand for mud crabs in Vietnam

has promoted increased interest in aquaculture of this species. In 2006, the total area

devoted to mud crab aquaculture was 102,355 ha which yielded 22,082 tonnes

(Nguyen, 2007 pers. comm.) with the majority of production centred in South

Vietnam, especially the Mekong Delta. A major factor facilitating the recent growth of

mud crab aquaculture in Vietnam has been improvements made to hatchery

20

technologies. For example, in 2006 a total of 18 million crablets produced in

hatcheries were sold to farmers in the Mekong Delta (Tuan, 2007 pers. comm.).

As described above, mud crab farming is well established across southeast

Asia and there is an obvious unmet market demand for mud crab wherever crab farms

exist. The major food items for cultured mud crab include fish of various species, or

‘trash fish’, golden snails, clams, telescope snails, small bivalves, mussel, etc.,

(Keenan 2004; Nguyen 2004; Quinitio 2004).

In southeast Asia, mud crabs are grown in many different production systems

including monoculture, polyculture with fish and/or shrimp, integrated mangrove- crab

culture and fattening (Quinitio 2004), and pen culture in mangrove areas (Table 1.3)

(Genodepa 1999; William Chang & Ikhwanuddin 1999). In Vietnam two types of crab

farming are employed: extensive farming with a stocking density of 0.5 -1 crab 10m-2

and intensive farming with a stocking density of 0.5-1.5 crabs m-2 (Nguyen 2004;

Christensen et al. 2004) and cage culture with a stocking density of 3-5 crabs m-3

(Nguyen 2004).

The experience base for crab aquaculture in Australia is small and until recently, most

activity confined to R & D institutions (Mann & Paterson 2004b). Interests in

commercial farming have developed over the past two years in the Northern Territory

and Queensland. In the Northern Territory, a proposed mangrove pen, mud crab

operation run by indigenous communities is at an advanced planning stage (Fielder

2004). In addition intensive, recirculating, cellular systems are being used for both

grow-out and shedding. These systems have been designed to stop cannibalism while

21

holding large crabs at high densities under controlled conditions (Mann & Paterson

2004a).

Table 1.2: Summary of parameters in several different types of cultured mud crab

systems in Southeast Asia (Keenan 1999a).

22

Mud crab nutrition

While crab farming has been practiced for several decades in tropical Asian

countries, information about their nutritional requirement is however still limited

(Yalin & Quingsheng 1994; Keenan 1999a; Anderson et al. 2004; Quinitio 2004).

Crustaceans are known to possess many of the digestive enzymes required to break

down key nutrients in their diet including proteins, carbohydrates and lipids. Previous

studies of protein digestion by crustaceans have shown the presence of trypsin

(Zwilling & Neurath 1981; Vega-Villasante et al. 1995; Jones et al. 1997; Figueiredo

et al. 2001; Cordova-Murueta et al. 2003; Pedroza-Islas et al. 2004; Johnston et al.

2004; Von Elert et al. 2004), chymotrypsin (Jones et al. 1997; Figueiredo et al. 2001;

Cordova-Murueta et al. 2003; Pedroza-Islas et al. 2004; Johnston et al. 2004; Von

Elert et al. 2004; Vega-Villasante et al. 1995), and carboxypeptidases and

aminopeptidases (Vega-Villasante et al. 1995; Figueiredo et al. 2001). Carbohydrase

activity in crustacean digestive tissues has also been reported as have relatively high

levels of amylase activity in crabs (Brethes et al. 1994; Wilson 1994; Pavasovic et al.

2004), prawns (Jones et al. 1997; Cordova-Murueta et al. 2003), lobsters (Johnston et

al. 2004), and crayfish (Figueiredo et al. 2001, Pavasovic et al. 2006). In recent years,

dietary lipid levels for mud crab have been investigated and the optimum dietary level

of cholesterol was found to be 0.51% (5.1g kg-1 diet) (Sheen & Wu 1999; Sheen

2000).

Formulation of any artificial diet requires detailed information on the specific

nutritional requirements and digestive capabilities of the candidate species.

Traditionally, mud crabs have been viewed as carnivores that prefer natural diets

containing molluscs, crustaceans and fish (Hill 1979). Examination of foregut

23

contents, however, has frequently also revealed the presence of plant- based materials

(Hill 1976; Tacon & Akiyama 1997). In general, digestion of complex carbohydrates

such as starch is relatively uncommon in aquatic animals (Kaushik 2001). However,

previous studies on the digestibility of experimental mud crab feeds containing plant

materials have shown high digestibility for all nutrients including fibre and ash

(Catacutan et al. 2003). The dietary requirements of this species have also been

demonstrated to be not nearly as stringent as those of most penaeid prawns with good

growth occurring over a wide range of protein and lipid levels (Catacutan 2002). For

example, diets tested in the previous studies have shown a wide range of nutritional

requirements for mud crabs in which protein varied from 32-54%, fat 4.8-10.8%, fibre

2.1-4.3, 18.7-42.5 nitrogen free extract (NFE) and 0.6-22% ash (Anderson et al. 2004)

while many existing artificial prawn diets contain higher protein levels.

Aquatic animal feeding behaviour

As profit margins decline for many aquaculture species, there is a need to

optimize feed efficiency so that farmed animals consume the minimum amount of

nutrients required to maximize growth. In addition, it is important to minimize the

amount of time between feed application and ingestion and to reduce feed input and

any concomitant wastes. Crab aquafeeds should also contain highly digestible

nutrients and chemical stimulants to improve feed intake levels.

In the mud crab aquaculture industry, the greatest constraint to large scale

farming is cannibalism in juveniles. This signifies the importance of the interaction

between feeding and cannibalism. A better understanding of this interaction will

potentially influence culture management and feeding practices and thus potentially

24

reduce cannibalism rates. The key lies in developing a better understanding of crab

behaviour and understanding the triggers for specific behaviour (Mann & Paterson

2004b).

A hierarchical testing protocol for screening compounds or mixtures as

chemical stimuli for Penaeus spp was designed, utilizing: (1) a static chamber for an

initial rapid assay for behavioral excitation; (2) a novel flow-though chamber for

demonstrating chemotaxis; (3) a modified Y-maze choice chamber for more detailed

studies of chemotaxis and feeding stimulation; and (4) aquarium feeding trials in

which the most potent chemical attractants and/or feeding stimulants are incorporated

and trialled into experimental feeds (Pittet et al. 1996).

The hierarchical role for crustacean chemical stimuli as modulators of feeding

behaviour was also modified by Lee and Meyers (1996) and has been applied in most

of the recent studies (Zimmer-Faust 1989; Pittet et al. 1996). The model is based on

the idea that an animal’s response to chemical stimuli can be classified into five

phases:

(1) detection- when an animal becomes aware of the presence of a chemical stimulus

(excitant);

(2) orientation- when an animal prepares for movement (attractant, repellent or

arrestant);

(3) locomotion- when an movement occurs (attractant or repellent);

(4) initiation of feeding- when an animal begins to handle and consume the food

(incitant or suppressant); and

25

(5) continuation or termination of feeding- when an animal feeds until satiation

(stimulants or deterrent).

While there have been few studies of feeding and foraging behaviour in the

genus Scylla, there have however, been numerous observations made of foraging

behaviour in other portunid swimming crabs and their responses to odour plumes,

particularly Callinectes sapidus (Clark 1999, 2000; Diaz et al. 2003).

The importance of chemo-attractants and/or feeding stimulants in improving

both initial palatability and overall feeding rate as a means to reduce wasted feed is

now widely recognized (Lee & Meyers 1996). In a recently developed paradigm for

feeding behavior of crustaceans, chemical stimuli are postulated to play a key role in

mediating the various stages of feeding from initial excitation to sustained feeding

(Pittet et al. 1996).

Taste and smell are very important attributes in feeding behaviour. An animals’

capacity to detect and respond to chemical stimuli is controlled by peripheral and

central neural mechanisms (Zimmer-Faust 2004). It has been suggested that

chemosensory stimuli are much more important than visual stimuli in provoking a

feeding response (Hazlett 1997). Interestingly, it has also been noted in investigations

of chemoreceptive behaviour in marine animals that other environmental factors such

as temperature, light, and water current may all affect an organism’s behaviour and

chemo-sensitivity.

Many marine animals, especially those that are active at night, use chemical

cues to detect and locate food items (Prosser 1973). Experiments conducted with

freshwater (Pseudemys spp; Chelydra serpentine) indicate that feeding experiences

26

early in development can result in olfactory-mediated orientation and food preferences

that persist for days or even weeks (Owens et al. 1986).

In behavioural reviews, emphasis has been placed on the importance of visual

body patterning for social communication (Hanlon & Messenger 1996) and chemical

cues. A wide variety of odours including homogenized fish (Hazlett & Mclay 2000;

Kozlowski et al. 2003; Hazlett 2004; Budelmann 1994), squid (Zhou & Shirley 1997),

clam body odour (Zimmer-Faust et al. 1996), extract (Pittet et al. 1996; Salierno et al.

2003), oysters (Diaz et al. 2003), betaine, taurine, glycine (Hartati & Briggs 1993,

Circuna et al. 1995), and crushed conspecifics (Gherardi et al. 2002; Hazett 2004;

Wall 2005) have been trialled as potential feed attractants. In most molluscs, chemical

cues are important for social communication. Cues can be detected from both local

(‘touch’) and distant (‘smell’) sources (Audesirk 1977).

Compounds that can act as chemo-attractants include primary amino acids,

although quarternary ammonium bases, nucleotides and nucleosides as well as organic

acids are also thought to elicit a response (Zimmer-Faust 2004). Betaine has been the

most effective attractant identified by far for all species studied, either alone or in

combination with other substances (Pittet et al. 1996; Reig et al. 2003; Hartati &

Briggs 1993). L-aminoacids are present in tissue of vertebrates and invertebrates

(Mackie & Mitchell 1985). While almost all marine invertebrates have a large amount

of betaine, only a trace is present in teleost fish tissue (Love 1980). Cadena-Roa et al.

(1982) reported that the best performing sole diets (Solea solea) contained a dose of

4.4% of betaine, as part of a mixture of chemical feeding activators that provide 7% of

the biomass of the diet. It has been shown that the essential amino acid requirement of

organisms from various groups approximate their body tissue amino acid profile (De

27

Silva & Anderson 1995). The basis of this concept is that since dietary amino acids are

used for protein deposition, the concentration of amino acids in the lean tissues of an

animal should indicate the balance of amino acids required in its diet (Bautista-Teruel

1999). This raises the possibility that amino acids can be used as attractants for mud

crab species.

1.2 Scope and aims of the study

It is generally agreed that knowledge about mud crab nutritional requirements

will be the key consideration to allow further expansion of the mud crab aquaculture

industry. Apart from a lack of knowledge of nutritional requirements of mud crab,

shortages of fishmeal supply and limitations on use of terrestrial meal for aquafeeds

are also key issues that need to be addressed. Some plant-based products have been

viewed as having the potential to supplement or replace fishmeal because they have

the capacity to supply protein and/or energy. To test this hypothesis, studies need to be

carried out to test the capacity of mud crabs to digest candidate ingredients and to

determine if they can reduce requirements for high cost marine animal-based meals in

the formulated diets for Scylla species. Associated changes in relative palability,

chemo-attractants may need to be incorporated into feeds as stimulants in order to

increase the efficient utilization of artificial feeds and to improve the quality of water

used for culture.

The aims of this study therefore were to determine:

The effects of dietary protein and starch on growth and feed utilisation in the

juvenile mud crab, Scylla serrata (conducted in Australia)

28

The effects of selected feed meals and starches on diet digestibility in the mud

crab, Scylla serrata (conducted in Australia)

Apparent digestibility of selected feed ingredients in diets formulated for the

sub-adult mud crab, Scylla paramamosain (conducted in Vietnam)

The effects of attractant containing diets on feeding responses of the mud crab,

Scylla serrata (conducted in Australia)

Research findings addressing the four issues in S. serrata and S.

paramamosain are presented and discussed in Chapters 2 to 5 of this thesis.

In chapter 2, we conducted an experiment to determine the minimum

requirement for protein in diets formulated for juvenile mud crab (S. serrata). Also

the use of starch (wheat, potato, rice and corn starch) to manipulate energy levels

of diets (18 MJ kg-1 or 15.5 MJ kg-1) was investigated to see if inclusion of starch

in the diet had an effect on protein requirements.

In chapter 3, the capacity of the mud crab, S. serrata to digest experimental

diets that contained different animal and plant-based feed meals (fish meal, meat

and bone meal, poultry meal, lupin meal, soybean meal, cotton seed meal and

canola meal) and different levels and types of starch (wheat, potato, rice and corn

starch) was investigated. The ADC (apparent digestibility coefficient) values for

dry matter, protein and gross energy were determined from animal and plant-based

diets. Moreover, ADC values for starch were determined from diets that included

starches.

In chapter 4, an experiment was carried out to determine if similar sorts of

ingredients available in Vietnam have potential for inclusion in diets formulated

29

for the local mud crab species, S. paramamosian that is the species commonly

farmed in southeast Asia. In this study, four local potential ingredients were used

(rice bran, cassava flour, corn flour and defatted soybean meal). The ADC values

for dry matter, crude protein and energy were determined in order to see how this

species reacts to and digests local ingredients.

In chapter 5, the effects of diets containing combinations of attractants,

temperature, sex and size on behavioural responses in S. serrata were investigated

during feeding. Experiment 1 was conducted with three different groups of mud

crab (small, medium and large size) and tested with seawater only (without food),

reference diet, and 2% attractant diets including chicken meal, betaine, tuna oil and

bait enhancer. Experiment 2 investigated the responses of males and females crabs

at three temperature levels of 26.5oC, 28.5oC and 30.5oC using diets containing 2%

chicken meal. Experiment 3 was designed to measure food consumption using a

reference diet, and diets containing chicken meal 2 and 5%, betaine 2 and 5%, tuna

oil 2 and 5% and a commercial bait enhancer 2 and 5%.

30

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Statement of Joint Authorship

Manuscript 1

Phuong H. Truong, Alex J. Anderson, Peter B. Mather, Neil A. Richardson & Brian D.

Paterson. Effect of dietary protein and starch on growth and feed utilisation in the

juvenile mud crab, Scylla serrata (in review).

45

Phuong H. Truong (Candidate)

• Wrote the manuscript

• Designed and formulated sampling design and experimental protocols

• Undertook all laboratory work, analysis and interpretation of data

Alex J. Anderson

• Supervised the experimental design and experimental protocols

• Assisted in the interpretation of data

• Provided editing and contributed to the structure of the manuscript

Peter B. Mather

• Co-supervised the experimental design and experimental protocols

• Assisted in the interpretation of data

• Provided editing and contributed to the structure of the manuscript

Brian D. Paterson

• Co-supervised the experimental design and experimental protocols

• Assisted in the interpretation of data

• Provided editing and contributed to the structure of the manuscript

Neil A. Richardson

• Co-supervised the experimental design and experimental protocols

• Assisted in the interpretation of data

• Provided editing and contributed to the structure of the manuscript

46

• Acted as the corresponding author

47

Chapter 2.

EFFECT OF DIETARY PROTEIN AND STARCH LEVELS ON GROWTH

AND FEED UTILISATION IN THE JUVENILE MUD CRAB, Scylla serrata

Phuong H. Truonga,b, Alex J. Andersona, Peter B. Mathera, , Brian D. Patersonc and

Neil A. Richardsond

aSchool of Natural Resource Sciences, Queensland University of Technology, GPO

Box 2434, Brisbane QLD 4001, Australia

bPermanent address:Research Institute for Aquaculture No3 (RIA3), 33 Dang Tat

Street, Nhatrang, Vietnam

cBribie Island Aquaculture Research Centre (BIARC), PO Box 2066, Woorim, QLD

4507, Australia

dSchool of Life Sciences, Queensland University of Technology, GPO Box 2434,

Brisbane QLD 4001, Australia

Correspondence: N.A. Richardson, School of Life Sciences, Queensland University of

Technology, GPO Box 2434, Brisbane QLD 4001, Australia. Ph:+61 7 3138 1388;

Fax: +61 7 3138 1537, E-mail: n.richardson@qut.edu.au

48

2.1 Abstract

A major consideration in the formulation of any aquaculture diet is the

provision of adequate nutrients to support growth at the lowest possible cost. Plant-

derived carbohydrates, like starch, are relatively cheap sources of dietary energy that

have also been investigated for their potential to spare protein in formulated diets in

some aquatic species. The present study was conducted to determine; 1) the

requirement for protein in diets formulated for the juvenile mud crab Scylla serrata,

and 2) if this requirement was influenced by the use of starch to manipulate energy

level in diets. In the first study, the requirement for protein in juvenile mud crab diets

was determined in an experimental cellular culture system using fishmeal-based diets

containing 25%, 35%, 45% or 55% crude protein. After ten weeks of culture, crabs fed

diets containing 55% or 45% protein demonstrated significantly higher growth

responses than those fed the diet containing the lowest level of protein (25%). A

second study was then conducted to determine if responses to dietary protein were

influenced by using starch to manipulate the energy level of fishmeal-based diets.

Specifically, six diets were formulated containing 35%, 40% or 45% crude protein

with gross energy values adjusted to 18 MJ kg-1 or 15.5 MJ kg-1 by incorporation of

purified wheat starch. In addition, three more diets were formulated containing potato,

rice or corn starch to determine if starch type altered the response of juvenile mud

crabs to fishmeal-based diets. Overall, growth responses in this second study appeared

to be positively correlated with level of protein in the diet with highest growth rates

achieved using diets containing 45% protein, regardless of the energy level of the diet.

In addition, at a dietary protein level of 40%, there was no evidence that the source of

starch had any significant impact on growth performance or relative feed utilisation.

49

Based on the results of the current study we propose that; 1) the growth response of

mud crabs is positively correlated with level of crude protein in the diet, and 2) at all

protein levels tested, inclusion of dietary starch offered no significant improvement to

growth performance.

Key words: Mud crab, Scylla serrata, dietary protein, starch, feed utilisation

50

2.2 Introduction

Mud crabs of the genus Scylla are a valuable source of nutrition and provide

income for many coastal communities in the Asia-Pacific region. Aquaculture of mud

crabs, however, is hampered by unsustainable feeding practices such as the use of

“trash fish” for grow out (Fielder 2004). Specifically, the use of natural feeds can

deplete local marine communities, foul waterways and provide vectors for the

transmission of disease. There is also increased use of natural feed for other purposes

leading to reduced availability and increased price. It is widely considered that a major

constraint to large scale mud crab farming is the lack of low cost formulated diets

based on readily available ingredients.

Some studies have been conducted on mud crab nutrition during larval stages

of development such as those that have investigated the effects of Artemia enrichment

on megalop larva survival (Suprayudi et al. 2004) or assessment of a microbound

larval diet as an Artemia replacement (Genodepa et al. 2004). There are also reports

that artificial diets designed for other crustacean species can be used to support the

growth of juvenile mud crabs. For example, formulated feeds designed for penaeid

shrimps have been used successfully for mud crab grow out (Mann & Paterson 2004).

Many of these diets, however, are relatively expensive and it is unclear exactly how

effectively they meet the nutritional requirements of mud crabs. In particular, there is

evidence that the dietary requirements of mud crabs are not nearly as stringent as those

of many penaeid species. For example, good growth has been obtained using relatively

wide ranges of protein and lipid in formulated mud crab diets (Sheen & Wu 1999;

Catacutan 2002). Moreover, many formulated penaeid diets are based on marine

animal meals, such as fishmeal. Fishmeal is typically one of the most expensive

51

ingredients incorporated into aquaculture diets and reducing reliance on this ingredient

is widely recognised as a priority for lowering the costs of prospective mud crab diets.

In recent years, evidence has emerged that the mud crab digestive system has

the capacity to digest many relatively inexpensive plant-derived feed ingredients. For

example, enzymes such as amylase and cellulase, that are required to digest plant-

based carbohydrates, have been detected in the mud crab digestive system (Pavasovic

et al. 2004). Consistent with these findings, high digestibility coefficients have been

reported for various plant-based feed meals and flours incorporated into mud crab

diets (Catacutan et al. 2003; Tuan et al. 2006). Recently, we have also reported that

purified starches incorporated into fishmeal-based formulated diets are readily

digested by sub-adult mud crabs (Truong et al. 2008). Elsewhere, plant-based

carbohydrates such as starch have been investigated for their potential to be utilised in

aquafeeds. For example, Allan et al. (2003) demonstrated a protein sparing effect by

incorporating starch into diets formulated for two different fish species. Using

carbohydrates to supply energy or spare protein in diets formulated for mud crabs

could offer the potential to significantly reduce feed costs for this species. A key aim

of the current study was to determine the impact of dietary starch on growth and

protein utilisation by juvenile mud crabs. In the first part of this study, formulated

diets based on fishmeal were used to assess the protein requirements of juvenile mud

crabs in an experimental cellular culture system. The second part of the study was

conducted to determine if there were interactions between dietary wheat starch and

protein, specifically, if using starch to adjust the crude energy level of diets will

improve growth responses or relative feed utilisation. Also potato, rice and corn starch

52

were compared with wheat starch in order to see if different types of starch influence

the growth of crabs.

53

2. 3 Materials and methods

2.3.1 Experimental site

Growth trials were conducted at the Queensland Department of Primary

Industries and Fisheries (DPI & F), Bribie Island Aquaculture Research Centre

(BIARC).

2.3.2 Study 1: The effect of dietary protein on growth

Forty eight crablets with an average body weight of 0.65g ± 0.10g were

collected from the nursery ponds, and the average initial weight of crabs at the

commencement of the study did not differ significantly among treatments (F=0.907,

p= 0.535). At the commencement of the trial, crabs were assigned randomly into four

groups of 12 crabs and housed in individual plastic containers (10cm x 10cm x 13cm).

Crabs were then fed a commercial penaeid diet (Turbo®, 49.7% crude protein, 6.7%

crude lipid; CP Feeds, Thailand) to apparent satiation for a week to acclimate to the

culture environment. For all studies, containers were supplied with recirculated,

aerated seawater that was gravity fed through an electrically heated overhead tank.

Water temperature was maintained within the range 27.5 ± 0.5oC.

Four experimental diets (n=12 crabs/treatment) were used for this study. Power

analysis of published figures on crab growth (Catacutan 2002) showed that with n=12,

the experiment had a power of 80% to detect differences of 20% in mean crab weights

at p=0.05. Diets were formulated at BIARC and prepared to contain 25%, 35%, 45%

or 55% crude protein (CP) with 10% lipid, to a final gross energy (GE) value of 17.8

MJ kg-1 (Table 2.1). Dry matter, crude protein, lipid, ash and gross energy content of

54

individual dietary ingredients were determined at the Health and Nutritional

Biochemistry Laboratory (Yeerongpilly, Queensland, Australia) and have been

reported previously (Truong et al. 2008, Chapter 3). Analyses were performed

according to the methods of AOAC (1984). Dry dietary ingredients were mixed with

oil and water added until a crumbly dough consistency was achieved. The dough was

then extruded through a 3 mm die to obtain pellets 5 to 10 mm in length. Pellets were

cooked in a rice steamer for 10 min then dried at 60oC for 24 h. Dried pellets were

crushed and sifted to obtain particles with a width from 0.7 mm to 1 mm (weeks 1 to

5 of growth trial) and 1 mm to 2 mm (weeks 6 to 10 of growth trial). All diets were

stored at 4oC until required. Crabs were fed experimental diets twice daily at a feeding

rate of 5% body weight (BW) per day. This feeding rate was selected since previous

observations had indicated that it was equivalent to satiation level feeding for juvenile

mud crabs (data not shown). Animals were weighed individually at the start of the

feeding trial and once a week thereafter for ten weeks. A daily record was kept of

moults and mortalities in each test group.

2.3.3 Study 2: The effect of dietary starch on growth

As for study 1, twelve individual crabs were used in each treatment group with

crabs held in individual black containers (10 cm x 10 cm x 13 cm). As previously

(2.3.2), crabs were fed the Turbo® diet for one week to acclimate to the culture

environment prior to the start of the experiment. Crabs were weighed individually at

the start of the culture period and each week thereafter. At the commencement of the

study, crabs were fed one of the experimental diets listed in Table 2.2 at a rate of 5%

body weight per day. Six of the fishmeal-based diets used in this study were

formulated to contain 35%, 40% or 45% crude protein with gross energy values

55

adjusted to 15.5 MJ kg-1 or 18 MJ kg-1 by addition of purified wheat starch (Sigma-

Aldrich®). The range of dietary protein used in this study was selected on the basis of

the outcomes of study 1 which showed that 45% dietary protein, but not 35%,

stimulated specific growth rates that were significantly higher than those obtained

using diets containing only 25% protein (Table 2.3). The dietary energy levels of 15.5

MJ kg1 or 18 MJ kg-1 were selected since they were within the range previously found

appropriate for crustacean diets (Cuzon & Guillaume 1997; Catacutan 2002;

Pavasovic et al. 2004). Three other diets were also prepared containing 40% crude

protein with gross energy values adjusted to 18 MJ kg-1 by addition of corn, rice or

potato starch (Sigma-Aldrich®). Experimental diets were prepared for feeding trials as

described previously (2.3.2). To confirm the suitability of the experimental fishmeal-

based diets developed for this study, a control crustacean diet consisting of the

commercial Turbo® P.monodon feed (Diet 1) was also included in this growth trial.

Crabs were cultured for ten weeks with the amount of feed administered adjusted

weekly according to body weight. Uneaten food was removed by siphoning before

each feeding time. Crabs that had moulted or had died were recorded daily in order to

calculate survival rate and moulting frequency at the end of the trial.

2.4 Assessment of growth performance and feed utilisation

Biological parameters used to evaluate relative growth performances and

nutrient utilization in this study were calculated as follows:

Average moulting interval (MI, day) = T/Mt

Intermoult period = Number of days between first and second

moult

Survival rate (SR,%) = Nf x 100/Ni

56

Specific growth rate (SGR% day-1) = (ln Wf-lnWi) x 100/T

Food conversion ratio (FCR) = Df/WG

Protein efficiency ratio (PER) = WG/Dp

Where Mt is the number of each crab moult during culture period (T); Ni is the

number of initial crabs; Nf is the number of final crabs; Wf is the final wet weight (g);

Wi is the initial wet weight (g); T is the duration of trial in days; Df is the dry feed

intake (g). Dp is the dry protein intake (g).

2.5 Statistical analyses

The effects of protein level on growth performance were determined using

one-way ANOVA (SPSS version 13.0) and post hoc comparison by Tukey’s HSD.

The effects of the inclusion of starch at different protein levels was analysed by two-

way ANOVA, again followed by post hoc comparison by Tukey’s HSD. For all

analyses the significance level of p<0.05 was used as standard. Regression analysis

was used to test weight gain response to protein and energy intake.

57

2.4 Results

2.4.1 Study 1: The effect of dietary protein on growth

Progressively higher final weights and SGR values were observed as the level

of dietary protein was raised from 25% to 55% (Table 2.3). In particular, the average

SGR values for crabs fed diets containing 55% (Diet P55) or 45% (Diet P45) protein

were significantly higher (p<0.05) than those obtained from crabs fed diets containing

only 25% crude protein (Diet P25). No significant differences were observed in FCR

values obtained using the experimental diets while survival rates for all treatments

were generally high (83% to 92%) (Table 2.3). Power analysis revealed that variances

in this study were higher than reported by other workers (Catacutan 2002) thus

reducing the experimental power below the 80% level expected.

2.4.2 Study 2: The effect of dietary starch on growth

Final weigth, average moulting frequency, specific growth rate and survival

rate of crabs fed reference (Diet 1) and test diets are presented in Table 2.4. Survival

rates for all treatments were high, ranging from 82% to 100%. Juveniles fed the

commercial diet (Diet 1) had the highest weight gain and SGR values, however, these

values were not significantly different (p>0.05) from any experimental diets except

Diet 35L (35% CP, 15.5 MJ kg1). The lowest weight gain and SGR values were

obtained using the diet containing 35% crude protein and 15.5 MJ kg-1 (Diet 35L)

although these values were not significantly different (p>0.05) from those obtained for

crabs fed diets containing 40% protein (Diets 40H, 40L, 40R, 40P and 40C),

regardless of the starch or energy content. Interestingly, SGR and weight gain values

obtained from juveniles fed the experimental diet containing only 35% protein with 18

58

MJ kg1 were not significantly different (p>0.05) from those obtained from juveniles

fed diets containing 45% protein (Diets 45H and diet 45L). By contrast, if the energy

level of diets was lowered to 15.5 MJ kg-1, SGR obtained from juveniles fed the diet

containing only 35% protein (Diet 35L) was significantly less (p<0.05) than those of

juveniles fed diets containing 45% protein.

The average moulting frequency of crabs over the culture period is shown in

Table 2.4. While there was no significant difference in average moulting frequency in

response to different dietary treatments, the treatment group fed the diet with only

35% protein and 15.5 MJ kg-1 (Diet 35L) was the only group where no individual

crabs progressed to a fourth moult.

As shown in Table 2.5, there were no significant differences in protein

efficiency ratios obtained from crabs fed experimental or reference diets. FCR values

were generally similar except for the value obtained using Diet 35H (35% CP, 18 MJ

kg-1) that was significantly higher than values obtained from crabs fed diets containing

45% protein (Diets 45H and 45L).

A significant relationship was observed between total weight gain and total

protein intake for all crabs combined in each group (Figure 2.1). Two- way ANOVA

showed no interaction between protein and starch in the experiment diets. Regression

analysis showed a linear relationship and a correlation coefficient (r) of 0.98 between

weight gain and digestible protein intake (p<0.001), caculated using digestibility

coefficients reported in Chapter 3 of this thesis. Although there was a significant

relationship between weight gain and gross energy intake (r = 0.64, p< 0.05), no

relationship was observed between weight gain and either gross non-protein energy

59

intake or total starch intake, a result indicates that the relationship found between

weight gain and gross energy intake was in fact due to the protein component of the

diet.

60

2.5 Discussion

In the first part of this study, growth responses of mud crabs fed diets

containing 35%, 45% or 55% protein were relatively high and also broadly consistent

with those reported by other workers who have shown good growth occurs in response

to diets containing between 32% and 59% crude protein. For example, Catacutan

(2002) reported no significant difference in growth performance when juvenile mud

crabs were fed diets that contained 12% lipid with either 32% or 48% crude protein.

Triño and Rodriguez (2002) also demonstrated that S. serrata in mangrove pens

demonstrated good growth when fed diets containing between 32% and 47% crude

protein. Likewise, no significant difference in growth performance of S.

paramamosain or S. olivacea was found under feeding regimes containing 38% to

59% dietary protein (Christensen et al. 2004).

Traditionally, mud crabs have been viewed as carnivores that have a relatively

high requirement for protein. This assumption is supported by analysis of the digestive

enzyme profile of mud crabs. For example, Pavasovic et al. (2004) demonstrated that

protease activity levels in the mud crab midgut gland were approximately two fold or

one hundred fold higher than those detected for amylase or cellulase, respectively. In

addition, protease activity levels in extracts from the mud crab digestive system are

significantly higher than those of other crustacean species, such as the redclaw

crayfish Cherax quadricarinatus, a species with a preference for diets based on plant

material or detritus (Pavasovic et al. 2007). Although mud crabs have a relatively high

requirement for protein, this requirement does not appear to be as stringent as those

61

reported for some penaeid species. Typically, many artificial diets developed for

penaeid aquaculture contain between 50%-60% protein (Teshima & Kanazawa 1984).

Such diets have been used successfully to support mud crab grow out (Mann &

Paterson 2004), however, results of the current study show that good growth can be

obtained with a little as 45% crude protein, regardless of energy level in the diet.

Diets designed for crustaceans usually source a significant proportion of

protein from marine animal species. Marine animal-based protein sources included in

the diets in the current study were fish meal and dried squid. A selection of plant based

carbohydrates, specifically wheat, corn, rice, and potato starch were also tested for

their impact on the responses of mud crabs to a fishmeal-based diet since they are

known to be highly digestible by some crustacean species (Catacutan & Coloso 1997;

Catacutan 2002; Catacutan et al. 2003; Stone et al. 2003; Anderson et al. 2004;

Pavasovic et al. 2004; Truong et al. 2007; Chapter 3). A major finding of the current

study was that the amount or source of starch included in diets had no significant

impact on growth performance of juvenile mud crabs. A significant difference was

detected however, between groups fed diets containing 45% or 35% crude protein

when the energy level of the diet was lowered to 15.5 MJ kg-1. A possible

interpretation of this finding is that in diets with suboptimal dietary protein and energy

levels more protein is utilized for purposes other than promoting growth, such as

meeting for energy requirements.

In this study, lipid content was not used to manipulate the energy level of diets.

Elsewhere evidence has been presented that in other crustacean species such as P.

japonicus, carbohydrates are more effective at sparing protein than are lipids (Teshima

& Kanazawa 1984). Nevertheless, diets were formulated to contain 10% lipid to

62

maintain consistency with diets described in previous studies. For example, Catacutan

(2002) reported that growth of crabs fed a diet with 40% protein was not significantly

improved if lipid content was increased from 6% to 12%. Likewise, Sheen and Wu

(1999) suggested that between 5.3% to 13.8% dietary lipid promoted good growth of

juvenile mud crabs. On the basis of these findings we propose that the lipid content of

our experimental diets is appropriate for promoting optimal growth in mud crabs.

Evidence from the current study indicates that the level of protein, and not

starch, in the diet was the main factor that affected growth rate. This conclusion is

supported by the significant linear relationship between protein intake and weight gain

and by a lack of correlation between weight gain and both non-protein energy intake

and starch intake. An interpretation of this finding is that the usefulness of high starch

levels in formulated feeds for mud crabs may be limited. This trend is supported by a

study of the penaeid, L. vannamei by Rosas et al. (2002) who demonstrated that

dietary carbohydrate utilization was limited by saturation of α- amylase when dietary

carbohydrate exceeded 33%. By contrast, Pavasovic et al. (2004) reported that

amylase activity in extracts from mud crab midgut gland were highest when they were

fed diets containing 47% carbohydrate. Likewise, Lopez-Lopez et al. (2005)

demonstrated that diets for C. quadricarinatus that contained wheat starch produced

the highest amylase activity while digestive enzyme activity in L. vannamei was

enhanced in the presence of high levels of dietary starch (42.3%) (Gaxiola et al. 2005).

We suggest that further studies will be required to resolve the basis of these apparent

species related differences in the responses of crustaceans to dietary carbohydrate.

The moulting frequencies observed in the current study were similar to those

reported elsewhere. For example, Sheen and Wu (1999) reported that the average

63

molting frequency of juvenile crabs fed a diet containing 48% protein over 63 days

was 3.6. A significant finding of the current investigation was that juvenile mud crabs

fed the diet with the lowest energy (15.5 MJ kg-1) and protein level (35%) did not

progress to a 4th moult. This finding supports our previous suggestion that if crabs are

fed diets containing suboptimal protein and energy levels, more protein may be

utilized to support activities other than growth.

A key aim of this study was to determine if the source of dietary starch had an

impact on growth responses of crabs. Elsewhere, it has been shown that in some

crustacean species wheat starch is digested more efficiently than is corn starch (Davis

et al. 1990) and wheat starch is more digestible than potato starch (Truong et al. 2008;

Chapter 3). The results of the current study indicate however, that although there may

be differences in the digestibility of different starches in mud crab diets, there appear

to be no direct relation between starch digestibility and relative growth performance.

In a previous investigation we have shown that wheat, rice and corn starches

are all well digested by mud crabs (Truong et al. 2008; Chapter 3). In the current

study, however, it has been shown that for any selected level of dietary protein the

inclusion of starch in formulated mud crab diets has no significant protein-sparing

effect. Nevertheless, the current studies confirm that relatively high levels of starch

may be included in formulated diets without any apparent impact on relative growth

performance of juvenile mud crabs. Therefore we suggest that further investigations

are warranted to assess the potential of starch as a low cost energy source, binder or

bulk filler ingredient in formulated mud crab diets.

64

Acknowledgements

The authors would like to thank David Mann, Marko Pavasovic, Tom Asakawa

and Beverly Kelly at BIARC for their technical assistance. This study was supported

by Ministry of Education and Training, Vietnam and ACIAR Mud Crab Project

FIS/2000/065, Australia. Astaxanthin was kindly provided by Dr Linda Browning of

DSM Nutritional Products (Australia). Our gratitude is also extended to Dr Richard

Smullen of Ridley Aqua-Feed (Australia) for supplying the fishmeal.

65

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metabolism in silver perch and barramundi. Recent advances in Animal Nutrition in

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Anderson, A.J., Mather, P.B. & Richardson, N.A. (2004) Nutrition of the Mud Crab,

Scylla serrata (Forskål). In Allan, G. and Fielder, D. eds. Mud Crab Aquaculture in

Australia and Southeast Asia. Proceedings of the ACIAR Crab Aquaculture Scoping

Study and Workshop, pp. 57-61. ACIAR Working Paper No. 54.

AOAC (1984) Official methods of analysis, 14th edn. Association of Official

Analytical Chemists. Washington, DC, USA.

Catacutan, M.R. & Coloso, R.M. (1997) Growth of juvenile Asian seabass, Lates

calcarifer, fed varying carbohydrates and lipid levels. Aquaculture 149, 137-144.

Catacutan, M.R. (2002) Growth and body composition of juvenile mud crab, Scylla

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Aquaculture 208, 113-123.

Catacutan, M.R., Eusebio, P.S. & Teshima, S. (2003) Apparent digestibility of

selected feedstuffs by mud crab, Scylla serrata. Aquaculture 216, 253-261.

Christensen, S.M., Macintosh, D.J. & Nguyen, T.P. (2004) Pond production of the

mud crab Scylla paramamosain (Estampador) and Scylla olivacea (Herbst) in the

Mekong Delta, Vietnam, using two different supplementary diets. Aquaculture

Research 36, 1013-1024.

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Cuzon, G. & Guillaume, J. (1997) Energy and protein: energy ratio. In Crustacean

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carbohydrate and moult cycle on digestive carbohydrases and hexokinases in

Litopenaeus vannamei (Bonne, 1931). Comparative Biochemistry and Physiology,

Part A 140, 29-39.

Genodepa, G.J., Zeng, C. & Southgate, P.C. (2004) Preliminary assessment of a

microbound diet as an Artemia replacement for mud crab, Scylla serrata, megalopa.

Aquaculture 236, 497-509.

Lopez-lopez, S., Nolasco, H., Villarreal-Colmenares, H., Civera-Cerecedo, R. (2005)

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Pavasovic, A., Richardson, N.A., Anderson, A.J., Mann, D. & Mather, P.B. (2007)

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69

Table 2.1. Composition of diets formulated for Study 1

Diet Ingredients

(% dry matter basis) P25 P35 P45 P55

Fishmeal 1

Casein

Binder (Wheat gluten)

Wheat starch

Cod liver oil

Fullers earth

Common Ingredients2

Calculated3

Crude Protein (%)

Digestible Protein (%)

Lipid (%)

Ash (%)

Gross energy (MJkg-1)

22.3

-

5.0

52.4

5.3

1.9

13.1

25.0

22.1

10.0

8.9

17.8

36.7

-

5.0

39.0

4.1

2.1

13.1

34.9

30.8

10.0

12.2

17.8

49.1

0.9

5.0

25.7

3

3.2

13.1

44.9

39.7

10.0

15.4

17.8

56.2

6.1

5.0

12.6

2.3

4.7

13.1

55.1

48.7

10.0

18.0

17.81Ridley Aquafeed, Australia

2 Common ingredients (g/100g): Vitamin mineral premix, 3; CaHPO4, 3; astaxanthin/

Carophyll pink (DSM, Netherlands), 0.1; Minced dried squid, 5; Lecithin, 2

3Calculated using coefficients from Truong et al 2008 (Chapter 3).

70

Table 2.2. Composition of diets formulated for Study 2

Diet Ingredients

(% dry matter basis) 11 35H 35L 40H 40L 45H 45L 40R 40P 40C

Fishmeal

Binder (Wheat gluten)

Wheat starch

Rice starch

Potato starch

Corn starch

Cod liver oil

Fullers earth

Common Ingredients2

Calculated3

Crude Protein (%)

Digestible Protein (%)

Lipid (%)

Ash (%)

Gross energy (MJkg-1)

Digestible energy

(MJkg-1)

P/E ratio (mg protein

kj-1)

-

-

-

-

-

-

-

-

-

49.1

44.4

6.7

15.3

19.1

17.1

25.7

36.8

5.0

41.0

-

-

-

3.7

0.4

13.1

35.0

30.5

10.0

10.6

18.0

15.9

19.4

37.0

5.0

25.6

-

-

-

3.9

15.4

13.1

35.0

30.6

10.0

25.5

15.5

13.6

22.6

43.6

5.0

34.0

-

-

-

3.2

1.1

13.1

40

35.0

10.0

12.5

18.0

15.8

22.2

43.8

5.0

18.8

-

-

-

3.3

16.0

13.1

40

35.1

10.0

27.2

15.5

13.6

25.8

50.3

5.0

27.4

-

-

-

2.6

1.6

13.1

45

39.5

10.0

14

18.0

15.8

25.0

50.5

5.0

11.9

-

-

-

2.8

16.7

13.1

45

39.6

10.0

29

15.5

13.5

29.0

43.6

5.0

-

33.9

-

-

3.2

1.2

13.1

40

35.0

10.0

12.5

18.0

15.3

22.2

43.6

5.0

-

-

33.9

-

3.2

1.2

13.1

40

35.0

10.0

12.5

18.0

13.0

22.2

43.6

5.0

-

-

-

33.9

3.2

1.2

13.1

40

35.0

10.0

12.5

18.0

15.4

22.21Turbo: P.monodon prawn feed, CP Feeds, Thailand.

2Common ingredients (g/100g): dried squid, 5; astaxanthin, 0.1; CaHPO4 ,3; Lecithin, 2;

mineral and vitamin premix, 3; (kg –1 of total diet) - 4.68 g K2HPO4; 7.12 g MgSO4.7H2O;

1.84 g NaH2PO4.2H2O; vitamin premix (kg-1) - 100000 IU vitamin retinol; 500 mg thiamine;

1750 mg riboflavin; 1125 mg pyridoxine hydrochloride; 3750 mg cyanocobalamin; 25000 mg

ascorbic acid; 500 000 mg colecalciferol; 20 000 IU d-alpha-tocopheryl acid succinate; 50 mg

biotin.

3from Truong et al 2008 (Chapter 3).

71

Table 2.3. Influence of dietary protein on final weight, specific growth rate (SGR) food

conversion ratio (FCR) and survival of S. serrata.

Diet CP(%)

Final weight (g) SGR

(%day)

FCR Survival

(%)

P25 25 5.69 ± 0.31ab 3.09 ± 0.06a 1.9 ±0.12 92

P35 35 6.41±0.45abc 3.35 ± 0.10ab 1.99 ± 0.13 92

P45 45 9.22 ± 1.18bc 3.78 ± 0.17bc 1.85 ± 0.24 92

P55 55 9.92 ± 0.90c 3.9 ± 0.16bc 1.79 ± 0.18 83

Values are means ± s.e.m. (n = 10-12 crabs in each group) Data with different

superscripts within columns are significantly different from each other (p<0.05) as

determined by ANOVA.

72

Table 2.4. Specific growth rate (SGR), average moulting frequency and survival

rate (SR) of juvenile mud crabs fed experimental diets during ten weeks of

experimental culture

Diet Final weight

(g)

SGR

(%/day)

SR

(%)

Moulting

frequency

% juveniles

to 4th moult

1

35H

35L

40H

40L

45H

45L

40R

40P

40C

11.68±1.25b

7.53±0.66a

7.68±0.41ab

9.60±0.66ab

8.10±1.01ab

11.96±0.80b

12.04±1.30b

8.80±0.70ab

7.97±0.66ab

9.27±0.73ab

4.14±0.15b

3.60±0.10ab

3.50±0.05a

3.70±0.11ab

3.68±0.10ab

4.1±0.11b

4.07±0.18b

3.55±0.18ab

3.58±0.09ab

3.76±0.04ab

83

100

100

100

92

100

100

100

100

100

3.50±0.16

3.16±0.11

3.00±0.06

3.16±0.11

3.08±0.08

3.50±0.15

3.50±0.15

3.08±0.12

3.08±0.08

3.16±0.11

41.6

16.6

0

16.6

8.3

41.6

41.6

8.3

16.6

16.6

Values are means ± s.e.m. (n = 10-12 crabs in each group). Means in the same column

with different superscripts are significantly different (p<0.05) from each other as

determined by ANOVA

73

Table 2.5. Feed conversion ratio (FCR) and protein efficiency ratio (PER) of

juveniles fed experimental mud crab diets

Diet Feed intake1

(g)

Protein

intake2

(g)

FCR PER

1

35H

35L

40H

40L

45H

45L

40R

40P

40C

276

240

220

228

214

238

241

204

197

223

103.5

84.0

77.0

91.2

85.6

106.9

108.6

81.8

79.0

89.3

2.36 ± 0.29ab

3.09 ± 0.20b

2.70 ± 0.13ab

2.25 ± 0.15ab

2.55 ± 0.19ab

1.87 ± 0.15a

2.08 ± 0.26a

2.30 ± 0.26ab

2.40 ± 0.15ab

2.31 ± 0.06ab

1.75 ± 0.20

1.68 ± 0.15

1.66 ± 0.06

1.69 ± 0.16

1.62 ± 0.15

1.94 ± 0.16

1.98 ± 0.25

1.71 ± 0.14

1.74 ± 0.16

1.81 ± 0.05

Values are means ± s.e.m. (n = 10-12 crabs in each group). Means in the same column

with different superscripts are significantly different (p<0.05) from each other as

determined by ANOVA

1 Feed intake was recorded daily

2 Protein intakes for the total group were calculated based on protein contained in diets

on a dry weight basis.

74

100

120

140

160

180

200

220

240

60 65 70 75 80 85 90 95 100

Digestible protein intake (g)

Wei

ght g

ain

(g)

Figure 2.1: Correlation between weight gain (mean ± s.e.m.) and protein intake of

juvenile mud crabs over a ten week growing period. Least squares regression

line y = 3.09x - 89.43; r = 0.98, p < 0.001.

75

Statement of Joint Authorship

Manuscript 2

Phuong H. Truong, Alex J. Anderson, Peter B. Mather, Neil A. Richardson & Brian D.

Paterson. Effect of selected feed meals and starches on diet digestibility in the mud

crab, Scylla serrata (in review).

Phuong H. Truong (Candidate)

• Wrote the manuscript

• Designed and formulated sampling design and experimental protocols

• Undertook all laboratory work, analysis and interpretation of data

Alex J. Anderson

• Supervised the experimental design and experimental protocols

• Assisted in the interpretation of data

• Provided editing and contributed to the structure of the manuscript

Peter B. Mather

• Co-supervised the experimental design and experimental protocols

• Assisted in the interpretation of data

• Provided editing and contributed to the structure of the manuscript

Brian D. Paterson

76

• Co-supervised the experimental design and experimental protocols

• Assisted in the interpretation of data

• Provided editing and contributed to the structure of the manuscript

Neil A. Richardson

• Co-supervised the experimental design and experimental protocols

• Assisted in the interpretation of data

• Provided editing and contributed to the structure of the manuscript

• Acted as the corresponding author

77

Chapter 3.

EFFECT OF SELECTED FEED MEALS AND STARCHES ON DIET

DIGESTIBILITY IN THE MUD CRAB, Scylla serrata

Phuong H. Truonga,b, Alex J. Andersona, Peter B. Mathera, , Brian D. Patersonc and

Neil A. Richardsond

aSchool of Natural Resource Sciences, Queensland University of Technology, GPO

Box 2434, Brisbane QLD 4001, Australia

bPermanent address:Research Institute for Aquaculture No3 (RIA3), 33 Dang Tat

Street, Nhatrang, Vietnam

cBribie Island Aquaculture Research Centre (BIARC), PO Box 2066, Woorim, QLD

4507, Australia

dSchool of Life Sciences, Queensland University of Technology, GPO Box 2434,

Brisbane QLD 4001, Australia

Correspondence: N.A. Richardson, School of Life Sciences, Queensland University of

Technology, GPO Box 2434, Brisbane QLD 4001, Australia. Ph:+61 7 3138 1388;

Fax: +61 7 3138 1537, E-mail: n.richardson@qut.edu.au

78

3.1 Abstract

The present study examined the capacity of the mud crab, Scylla serrata to

digest experimental diets that contained different animal and plant-based feed meals

and different types and levels of starch. Overall, the apparent dry matter digestibility

(ADMD) coefficients for most selected feed meals tested in the first part of this study

were similar, ranging from 78% to 88%. Crude protein digestibility (ACPD)

coefficients for all feed meals tested were relatively high, with values ranging from

86% to 96%. Cotton seed meal, poultry meal, canola meal, fishmeal, soybean meal

and lupin meal had similar gross energy digestibility (AGED) values (p>0.05) ranging

from 84% to 89%. In the second part of this study, apparent digestibility coefficients

were determined for formulated diets containing purified starch. The apparent starch

digestibility (ASD) of wheat starch decreased significantly as the inclusion level was

increased from 15% to 60%. However, there was no significant effect on ACPD

values. At a 30% inclusion level, the ASD of diets containing different starches

decreased in the order corn > wheat > potato = rice. ACPD values were significantly

higher (p<0.05) in diets containing corn or rice starch than in those containing wheat

or potato starches.

Key words: Mud crab, Scylla serrata, digestibility, starch

79

3.2 Introduction

Mud crabs of the genus Scylla are a valuable source of nutrition and provide

income for many coastal communities in the Asia-Pacific region. In recent years,

however, many mud crab fisheries have experienced over harvesting that has

threatened capacity to meet future demand from local and export markets. Aquaculture

is now widely regarded as a key strategy for meeting increased demand for mud crab

product and to reduce pressure on wild stocks.

Typically, mud crabs kept in culture have been fed natural diets based on

marine animals such as “trash fish”. The collection and use of such natural feeds has

many disadvantages including depletion of local marine communities, fouling of

culture production systems, and variable feed availability and cost. It is now widely

recognised that the future viability and expansion of mud crab aquaculture will depend

on development of low cost but nutritionally adequate formulated diets (Fielder 2004).

Formulated feeds designed for penaeid prawns have been used with some

success for mud crab grow out (Mann & Paterson 2004). Many penaeid diets,

however, are relatively expensive and little data are available to indicate how

effectively they meet the specific nutritional requirements of mud crabs. In particular,

there is evidence that the nutritional requirements of mud crabs may not be as stringent

as those reported for penaeid prawns and that the levels of some relatively expensive

components in mud crab diets, such as protein, may be reduced. For example, many

artificial prawn diets contain between 50% - 60% protein (Teshima & Kanazawa

1984) yet good growth rates for mud crabs in culture have been reported using

substantially lower levels of dietary protein (Catacutan 2002; Tuan et al. 2006).

80

Another disadvantage of using formulated prawn diets for crab aquaculture is that

most are based on marine animal meals, such as fishmeal. Typically, fishmeal is one

of the most expensive ingredients incorporated into any aquaculture diets (Hardy &

Tacon 2002). It has been predicted that with the future demand for fishmeal expected

to increase significantly as aquaculture production expands, only species of very high

market value will be able to compete for this critical feed ingredient (Edwards et al.

2004 ). Reducing reliance on fishmeal is now recognised as a priority for reducing

aquaculture feed cost.

Identification of feed ingredients with potential to replace fishmeal in

formulated aquaculture diets requires data on the nutritional requirements and

digestive capacity of candidate species. Traditionally, mud crabs have been viewed as

carnivores that show a preference for natural diets containing molluscs, crustaceans,

and fish (Hill 1979). Interestingly, significant amounts of plant-based material has

been found in the mud crab digestive system (Hill 1976; Tacon & Akiyama 1997).

Moreover, Prasad and Neelakantan (1988) demonstrated that detritus was the main

food source for crabs with a carapace of less than 70mm width. In recent years,

evidence has emerged that the mud crab digestive system possesses a significant

capacity to digest plant-based materials. Investigations into the digestive physiology of

S. serrata have confirmed that this species possesses the necessary endogenous

enzymes to digest many plant-based carbohydrates. For example, Pavasovic et al.

(2004) detected significant amylase, cellulase and xylanase activity in soluble extracts

from the mid gut gland. Findings such as these provide a rationale for investigating the

use of relatively cheap plant-based ingredients in mud crab diets. Supplying nutrients

81

from plant-based sources also offers potential opportunities to reduce reliance on

fishmeal and therefore to lower diet costs.

One of the first steps in estimating the potential of a new ingredient for use in a

formulated aquaculture diet is to test its digestibility in candidate species. Recent

digestibility studies have confirmed that the mud crab has a significant capacity to

utilise feed ingredients from a variety of terrestrial animal or plant-based sources. For

example, Catacutan et al. (2003) showed that plant-based feed ingredients such as

soybean meal, corn meal and copra meal were all highly digestible to mud crabs, while

Tuan et al. (2006) reported that the protein digestibility of blood meal and soybean

meal were not significantly different to that of fishmeal.

In a recent study, Pavasovic et al. (2004) demonstrated that mud crabs have a

relatively high capacity to digest purified carbohydrates derived from plants.

Specifically, high digestibility coefficients were obtained for soluble cellulose

derivatives and starch in formulated feeds. The inclusion of digestible carbohydrate

has already been recommended for diets formulated for carnivorous fish and some

crustacean species (Wilson 1994; Catacutan & Coloso 1997; Kaushik 2001).

Nevertheless, the digestibility of plant based materials such as starch can vary with

botanical origin and treatment. Furthermore, the inclusion of starch in aquafeeds can

affect the digestibility of other dietary elements, such as protein, that are essential for

growth (Cousin et al. 1996; Sales & Britz 2002; Stone et al. 2003). As a consequence,

digestibility coefficients of plant-based carbohydrates should be determined before

they are included routinely in formulated diets for candidate species.

82

The objective of the current study was to evaluate the potential of a range of

feed ingredients for use in diets formulated for S. serrata. In the first part of this study,

we determined digestibility coefficients for selected commercial animal feed meals.

Feed meals tested were derived from either terrestrial animal (meat or poultry) plant

(soybean, lupin, canola or cotton seed) or single cell (yeast) sources. The aim of the

second part of the study was to examine digestibility of experimental diets containing

various plant-based starches. Specifically, digestibility coefficients were determined

for fishmeal-based formulated diets containing different levels (15%, 30%, 45% or

60%) or types (wheat, potato, rice or corn) of purified starch.

83

3.3 Materials and Methods

3.3.1 Animals

Experimental animals were supplied by Bribie Island Aquaculture Research

Centre Station (BIARC), Bribie Island, QLD Australia. For all experimental

treatments, crabs were supplied with recirculated, aerated seawater that was gravity

fed through an electrically heated overhead tank. Water temperature was maintained

within a range (27.5± 0.5oC) suggested for optimal growth of mud crabs.

3.3.2 Study 1: Digestibility of Selected Feed Meals

A digestibility trial was conducted to evaluate the digestibility coefficients of

Brewer’s yeast (BY) (Swift and Co, Australia) and selected animal feed meals: South

American fish meal (FM) (Ridley Aqua Feed, Australia), meat and bone meal (MBM)

(Southern Meats, Australia), poultry meal (PM) (AJ Bush, Australia), lupin meal (LM)

(MC Croker, Australia), soybean meal (SBM) and canola meal (CM) (Radford Park

Aquafeed, Australia). A reference diet was used as a control that consisted of a

commercial P.monodon diet (Turbo, Thailand). Experimental diets were formulated by

combining test ingredients and the reference diet in a 30%:70% ratio, on a dry weight

basis. A complete list of ingredients of all test diets is presented in Table 3.1. The

proximate nutrient content of the reference diet and test ingredients are presented in

Table 3.2. Proximate composition of diets and faecal material were determined at the

Animal Research Institute (Brisbane, Australia) according to AOAC protocols (1984).

Diets used in the experiment were prepared by thoroughly mixing dry

ingredients, followed by wet ingredients, until a crumbly dough consistency was

84

achieved. Diet mixture was pressure pelleted using a meat grinder with a 3mm die.

Pellets were steamed in a rice steamer in a microwave oven (Sanyo) for 10 min, prior

to drying at 50°C in a drying oven, overnight. All experimental diets were stored at -

20°C until required. All diets contained 0.5% Chromic oxide (Cr2O3) as an inert

indicator to allow calculation of digestibility coefficients for dry matter (ADMD),

crude protein (ACPD) and energy (AGED) using formula suggested by Jones and De

Silva (1997). Pilot studies determined that there was no significant loss of detectable

chromium (Cr) in feed pellets immersed in water at 26oC for 1h (data not shown).

At the commencement of this study, crabs were selected that had an average

body weight of 89g ± 18g. Crabs were then assigned randomly into ten groups and

housed individually in plastic containers (19.5cm x 28cm x 22cm). Nine dietary

treatments (n=12 crabs / treatment) were utilised in this study (Table 3.1). Crabs were

fed experimental diets twice daily at a feeding rate of 5% body weight (BW) per day

until approximately 1.5 to 2g of faecal material (dry weight) had been collected. A

daily record was kept of mortalities in each test group. Faecal material at the bottom of

the tank was collected by syphoning into a plastic sieve and removed individually

using forceps. To collect sufficient material for analysis, faecal material from three

crabs in each treatment was pooled (n=4 / treatment). All samples were lyophilized

and stored at -20°C until required for analysis.

The indirect method of Furukawa and Tsukahara (1966) was used to calculate

digestibility coefficients of all diets tested. Briefly, 0.5g of feed or faecal material was

added to 4.0mL of concentrated nitric acid (AnalaR grade, 16 M HNO3) and incubated

overnight at room temperature. Samples were then heated to 150oC for an additional

60 min. After cooling, samples were mixed with 5.0mL of concentrated perchloric

85

acid (AnalaR grade, 70% HClO4) then heated to 220oC for 30 min and 245oC for a

further 30 min. After cooling, absorbance of each sample was read at 346.5nm. For

calibration purposes, the above protocol was repeated using known quantities of

Cr2O3.

Coefficients for ADMD were determined using the formula:

ADMD = 100 – 100 (%Cr2O3 in feed / % Cr2O3 in faeces).

Coefficients for crude protein (ACPD) or gross energy (AGED) were

determined using the formula:

APD = 100 – 100 [(%Cr2O3 in feed / % Cr2O3 in faeces) x (% protein or Mj/kg

energy in faeces/ % protein or Mj/kg energy in feed)].

Apparent digestibility coefficients (ADC) of the test ingredients were

calculated using the following equations described by Bureau et al. (1999).

ADCI = ADCT + ((1 – s) DR / s DI) (ADCT – ADCR);

Where: ADCI = apparent digestibility coefficient of test ingredient; ADCT =

apparent digestibility coefficient of test diet; ADCR = apparent digestibility coefficient

of the reference diet; DR = % nutrient (or kJ/g gross energy) of the reference diet

mash; DI = % nutrient (or kJ/g gross energy) of the test ingredient; s = proportion of

test ingredient in test diet mash (i.e. 0.3 in this study); (1 – s) = proportion of reference

diet mash in test diet mash (i.e. 0.7 in this study).

3.3.3 Study 2: Impact of starch on diet digestibility

86

As described previously, other workers have recommended inclusion of plant-

based carbohydrates in formulated diets to supply energy for aquatic animal species. A

second digestibility trial was therefore conducted in this study to assess the impact of

starch included in formulated mud crab diets based on fishmeal. Adult sibling crabs

(164.3 ± 27.7g) from the population used in study 1 were included from a grow out

recirculating cellular system at Bribie Island Aquaculture Research Centre for this

trial. Each crab was weighed individually at the start and end of each experiment.

Crabs were assigned randomly into nine groups with twelve crabs in each group and

held in individual containers (19.5cm x 28cm x 22cm) that were covered with plastic

net lids. Crabs were kept in these containers for one week to acclimate to culture

environment conditions prior to the start of the experiment and fed a commercial

prawn diet as described previously (3.3.2).

To ensure dietary protein levels in all test diets were set above those reported

to promote good growth rates in culture (Catacutan 2002), a reference diet based on

high quality South American fishmeal was formulated (Diet 1; Table 3.3). Eight other

diets were also formulated where fishmeal in the reference diet was replaced with

different amounts (15%, 30%, 45% or 60%) or types (wheat, corn, rice and potato) of

purified starch (Sigma product). Diets used in the experiment were prepared as

described previously (3.2.2), with the exception of Diet 5 (45% wheat starch) that was

not steam cooked (uncooked) prior to drying. All diets contained 0.5% Cr2O3 as an

inert indicator to allow calculation of apparent nutrient digestibility coefficients.

As described previously, proximate composition and faecal material were

determined at the Animal Research Institute (Brisbane, Australia) according to AOAC

protocols (1984). Proximate compositions of diets are shown in Table 3.4. A total

87

starch assay (amyloglucosidase/ α-amylase method, AOAC method 996.11) was also

performed on all diets and faecal material collected. Briefly, 100mg of sample was

wetted with 0.2mL of aqueous ethanol (80%v/v) to aid dispersion, stirred on a vortex

mixer and then immediately 3mL of thermostable α-amylase (300Units) in MOPS

buffer (50mM, pH 7.0) was added; vigorously stirred on a vortex mixer. The tube was

then incubated in a boiling water bath for 6 minutes, placed in a bath at 50oC, then

sodium acetate buffer (4mL, 200mM, pH 4.5) was added. This was followed by

addition of amaloglucosidase (0.1mL, 20U), stirred on a vortex mixer and incubated at

50oC for 30 min. The test tube was mixed thoroughly and the volume adjusted to

10mL with distilled water, then samples centrifuged at 3000g for 10 minutes.

Duplicate aliquots (0.1mL) of this solution were transferred to the bottom of glass test

tubes and 3.0mL of glucose oxidase-peroxidase (GOPOD) reagent was added to each

tube (including the glucose controls and reagent blanks). The tube was then incubated

at 50oC for 20 min. A glucose control consisted of 0.1mL of glucose standard solution

(1gL-1) and 3.0mL of GOPOD reagent. A reagent blank solution consisted of 0.1mL

of distilled water and 3.0mL of GOPOD reagent. Absorbance of the supernatant

solution and the glucose control was read against the reagent blank at 510nm using a

Novospec spectrophotometer (LKB). Values of starch contained in diets and faecal

material were calculated according to the following formula:

Calculation of starch = Δ E x F x 1000 x 1/1000 x 100/W x 162/180

= Δ E x F/W x 90

Starch % (Dry weight basis) = Starch % (as is) x 100/ [100 – moisture content (%)]

Where:

Δ E = Absorbance (reaction) read against the reagent blank

88

F = 100 (μg of glucose)/ absorbance of 100μg of glucose

1000 = Volume correction (0.1mL taken from 100mL)

1/1000 = Conversion from micrograms to milligrams

100/W = Factor to express “starch” as a percentage of sample weight

W = Weight in milligrams (“as is” basis) of the sample

162/180 = Adjustment from free glucose to anhydro glucose (as occurs in

starch)

3.2.4 Statistical analyses

The significance of data were determined using one-way ANOVA (SPSS

version 13.0) and post hoc comparison by Tukey’s HSD. For all analyses the

significance level of p<0.05 was used as standard.

89

3.3 Results

3.3.1 Digestibility of Selected Feed Meals

Table 3.5 displays the apparent digestibility coefficients for dry matter, crude

protein and gross energy obtained using selected animal feed meals. Overall, ADMD

coefficients for most feed meals tested were not significantly different from the value

obtained for fishmeal and ranged from 79% to 88%. The ADMD value of meat meal,

however, was significantly lower (p<0.05) than those obtained for all other test

ingredients. ACPD coefficients for all feed meals tested were relatively high, with

values ranging from 86% to 97%. Interestingly, the ACPD value for yeast was

significantly higher (p<0.05) than those obtained for all other test ingredients. Cotton

seed meal, poultry meal, canola meal, fishmeal, soybean meal and lupin meal had

AGED values that were not significantly different (p>0.05) from one another that

ranged from 84% to 89%. The AGED coefficient for meat meal, however, was

significantly less (p<0.05) than values obtained for all other ingredients except cotton

seed meal and poultry meal. By contrast, the highest value for AGED was obtained for

yeast that was significantly higher (p<0.05) than those obtained for meat meal, cotton

seed meal and poultry meal. Survival rates for all treatments were high and ranged

from 92% to 100% (data not shown).

3.3.2 Impact of starch on diet digestibility

Apparent digestibility coefficients for dry matter, crude protein, gross energy

and starch for diets tested in this study are presented in Table 3.6. ADMD values

ranged from 72% to 85.3%. Inclusion of wheat, potato, rice and corn starch in cooked

diets had no significant impact on dry matter digestibility when compared to the

90

reference diet (Diet B). By contrast, the inclusion of 45% wheat starch in the uncooked

diet (Diet W45U) significantly increased dry matter digestibility. Apparent

digestibility of crude protein was relatively high for all treatments and ranged from 86

% to 92%. Interestingly, ACPD values for diets containing rice starch (Diet R30), corn

starch (Diet C30) or uncooked wheat starch (Diet W45U) were significantly higher

(p<0.05) than values obtained for all other experimental diets containing between 15%

and 60% starch. AGED values for test diets ranged from 73% to 92%. The highest

value obtained was for the uncooked diet containing wheat starch (Diet W45U) that

was significantly higher (p<0.05) than all other experimental diets, except diets B and

W30. By contrast, the AGED value obtained for the diet containing potato starch was

significantly lower (p<0.05) than values obtained for all other test diets. A consistent

and significant (p<0.05) decline in starch digestibility in cooked diets was recorded as

the level of wheat starch included was progressively raised to 60% (Diet 6). The type

of starch incorporated into test diets also appeared to impact significantly on ASD

values. Specifically, apparent digestibility of starch at a 30% inclusion level in cooked

diets was in the following order (from most to least digestible) corn > wheat > rice =

potato. Interestingly, for all parameters tested, significantly higher (p<0.05) ASD

values were obtained for the uncooked diet containing 45% wheat starch (Diet W45U)

compared with a cooked diet with the same amount of wheat starch (Diet W45).

91

3.4 Discussion

Many plants and animal-based feed ingredients have been reported to have

potential to replace fishmeal in formulated aquafeeds (Tacon 1994). In the present

study, we observed relatively high digestibility coefficients for a broad range of

animal, plant and single cell- based ingredients, reflecting the ability of mud crabs to

utilize a wide range of nutrient sources. These findings are consistent with other

studies that have reported high digestibility coefficients for a wide variety of animal

and plant-based ingredients in mud crab diets (Catacutan et al. 2003; Tuan et al. 2006).

In the current study, ADMD coefficients for most high protein feed meals tested were

similar, with the exception of meat meal. Similar results were reported by Catacutan et

al. (2003) and Tuan et al. (2006) using some terrestrial animal-based meals in diets for

mud crab. Reduced digestibility coefficients have also been associated with use of

meat meal in C. destructor (Jones & De Silva 1997) and P. setiferus (Brunson et al.

1997). A possible reason for the relatively poor digestibility of meat meal in the

current study was high ash content (20%).

Protein digestibility for all feed meals was relatively high, with values over

85%. This finding is in general agreement with the findings of Catacutan et al. (2003)

who observed high ACPD in adult mud crabs fed a range of animal and plant-based

ingredients. High capacity of mud crabs to digest protein is not surprising considering

their high expression levels of protease in the digestive system (Pavasovic et al. 2004).

Based on the findings of Pavasovic et al. (2004) and the current study we argue that

mud crabs have a high capacity to digest protein from a wide range of single cell,

terrestrial animal and plant-based ingredients. Further studies that attempt to exploit

92

the potential of these feed ingredients to replace fishmeal as a source of dietary protein

may help reduce feed costs for this species.

In the second part of this study, we examined the digestibility of different

types and amounts of starches in formulated mud crab diets. Overall, digestibility

coefficients for starch and the associated diets were high. This finding is in close

agreement with many other investigations that have reported that cereals containing

high levels of starch are readily digested by crustaceans (Shi-Yen & Chu-Yang 1992;

Cousin et al. 1996). The high digestibility of starch demonstrated in this study is not

surprising considering the detection of significant carbohydrase activity in the mud

crab digestive system (Pavasovic et al. 2004). Specifically, amylase, cellulase and

xylanase activity has been detected in extracts prepared from the mud crab midgut

gland suggesting a significant capacity to digest plant-based nutrients. Furthermore,

plant-based material and detritus has been reported from the digestive system of mud

crab juveniles sampled from the wild (Hill 1976; Prasad & Neelakantan 1988).

Another important finding of the current study was that diet digestibility was affected

significantly by the type of starch and the cooking process. These results are in close

agreement with other studies that have shown digestibility of starch varies with

botanical origin and treatment (Cousin et al. 1996; Sales & Britz 2002; Stone et al.

2003). In the current study the digestibility of corn starch however, was generally

higher than for wheat starch (at 30% inclusion level). This contradicts the findings of

Davis and Arnold (1993) who reported that in other crustacean species wheat was

digested more efficiently than corn. The reason for this apparent difference is unclear

but may reflect species specific differences in capacity to digest carbohydrates from

93

different sources or differences in the purity or preparation of carbohydrates

incorporated into the feeds.

Findings of the second part of this study suggest that starch should be

considered as a potential feed ingredient in formulated mud crab diets. Overall, most

diets that contained starch were readily digested. In particular, no negative impacts

were observed on digestibility of major nutrients (e.g. protein) following inclusion of

wheat, rice or corn starch in formulated feeds. These results argue that further studies

are warranted to investigate the potential of starch to supply energy in mud crab diets

and to reduce requirements for more expensive feed ingredients such as fishmeal.

94

Acknowledgements

The authors would like to thank David Mann, Marko Pavasovic, Tom Asakawa

and Beverly Kelly at BIARC for their technical assistance. This study was supported

by Ministry of Education and Training, Vietnam and ACIAR Mud Crab Project

FIS/2000/065, Australia. Astaxanthin was kindly provided by Dr Linda Browning of

DSM Nutritional Products (Australia). Our gratitude is also extended to Dr Richard

Smullen of Ridley Aqua-Feed (Australia) for supplying the fishmeal and John Harsant

of Radford Park Aquafeeds (Australia) for supplying the soybean, canola and cotton

seed meals. This manuscript is written in partial fulfillment of the Doctor of

Philosophy degree in aquaculture of Phuong Ha Truong, at the Queensland University

of Technology, Brisbane, Australia.

95

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99

Table 3.1. Composition (% dry matter of the diet) of the formulated diets for the

digestibility trial using commercial feed ingredients.

Diet Ingredient 1 2 3 4 5 6 7 8 9 Basal Diet (Turbo)

62.4 62.4 62.4 62.4 62.4 62.4 62.4 62.4 92.4

Fishmeal

30

Meat meal

30

Poultry meal

30

Soybean meal

30

Canola meal

30

Lupin meal

30

Cotton seed meal

30

Yeast

30

Binder (Wheat gluten)

5 5 5 5 5 5 5 5 5

Common ingredientsa

2.6 2.6 2.6 2.6 2.6 2.6 2.6 2.6 2.6

a Common ingredients (g/100g): mineral and vitamin premix (2%), (kg –1 of total diet

- 4.68 g K2HPO4; 7.12 g MgSO4.7H2O; 1.84 g NaH2PO4.2H2O; vitamin premix (kg-1)

- 100000 IU vitamin retinol; 500 mg thiamine; 1750 mg riboflavin; 1125 mg

pyridoxine hydrochloride; 3750 mg cyanocobalamin; 25000 mg ascorbic acid; 500

000 mg colecalciferol; 20 000 IU d-alpha-tocopheryl acid succinate; 50 mg biotin);

astaxanthin (0.1%); chromic oxide (Cr2O3) (0.5%).

100

Table 3.2. Proximate nutrient composition (%) in dry matter of basal diet and

test ingredients used in the digestibility trial.

_____________________________________________________________________

Ingredient

Dry Matter

Crude Protein (N x 6.25)

Crude Fat Ash Energy (Mj/kg)

Basal Diet (Turbo)

90.4 49.7 6.7 15.3 19.1

Fishmeal

91.7 75.5 8.7 17.2 15.9

Meat meal

97.7 59.6 13.4 20.4 16.9

Poultry meal

96.7 69.2 13.1 14.1 20.9

Soybean meal

88.3 53.2 1.9 7.2 20.6

Canola meal

90 44.1 3.8 7.4 21.5

Lupin meal

86.1 30.8 9.4 3.6 15.9

Cotton seed meal

89.2 48.4 2.4 7.3 19.3

Yeast

95.1 48.6 0.4 9.6 18.3

101

Table 3.3. Composition (% dry matter of the diet) of the formulated diets for the

digestibility trial using different starches.

Ingredient (%) Diet

B W15 W30 W45 W45U W60 P30 R30 C30

Fish meal 87.4 72.4 57.4 42.4 42.4 27.4 57.4 57.4 57.4

Wheat starch 0.0 15.0 30.0 45.0 45.0a 60.0

Potato starch 30.0

Rice starch 30.0

Corn starch 30.0

Binder

(Wheat

gluten)

5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0

Common

ingredients b 7.6 7.6 7.6 7.6 7.6 7.6 7.6 7.6 7.6

a Uncooked diet

b Common ingredients (g/100g): mineral and vitamin premix (2%), (kg –1 of total diet

- 4.68 g K2HPO4; 7.12 g MgSO4.7H2O; 1.84 g NaH2PO4.2H2O; vitamin premix (kg-1)

- 100000 IU vitamin retinol; 500 mg thiamine; 1750 mg riboflavin; 1125 mg

pyridoxine hydrochloride; 3750 mg cyanocobalamin; 25000 mg ascorbic acid; 500

000 mg colecalciferol; 20 000 IU d-alpha-tocopheryl acid succinate; 50 mg biotin);

dried squid (5%); astaxanthin (0.1%); chromic oxide (Cr2O3) (0.5%).

102

Table 3.4. Proximate nutrient composition (%) in dry matter of experimental diet

used in the starch digestibility trial.

Diet Dry

matter

Protein

(Nx6.25)

Crude

fat

Ash Starch

Gross

Energy

(MJkg-1)

P/E ratio

B

W15

W30

W45

W45U

W60

P30

R30

C30

93.7

93.3

92.8

90.8

92.3

87.6

89.0

93.4

93.8

70.5

63.6

53.2

41.8

41.9

33.9

49.1

53.3

53.6

8.7

7.5

6.3

5.1

5.1

3.9

6.3

6.3

6.3

15.4

13.0

10.6

8.2

8.2

5.8

10.6

10.6

10.6

1.3

14.9

31.2

48.5

47.6

60.0

33.2

28.6

29.8

20.6

20.6

19.9

19.2

19.3

19.0

19.7

19.9

19.9

3.42

3.09

2.67

2.18

2.17

1.78

2.49

2.68

2.69

103

Table 3.5. The apparent digestibility coefficients (%) of dry matter (ADMD),

crude protein (ACPD) and gross energy (AGED) for yeast and selected animal

feed meals

Ingredient ADMD ACPD AGED

Basal Diet (Turbo)

83.2 ± 0.5 b 90.4 ± 0.5 ab

89.3 ±0.9 cd

Fishmeal 85.4 ± 1.7b 88.3± 0.7ab 87.8 ± 1.3cd

Meat meal 67.0 ± 1.3a 86.3 ±0.9ab 78.2 ± 0.8ab

Poultry meal 78.9±3.0b 88.2±2.1ab 85.2 ±1.9bc

Soybean meal 80.4 ±1.2b 91.7 ±0.5bc 89.1 ± 0.9cd

Canola meal 83.5 ±4.7b 87.6 ± 2.7ab 87.5 ± 2.9cd

Lupin meal 88.1 ± 1.6b 89.1 ± 0.9ab 89.9 ± 1.4cd

Cotton seed meal 80.5 ± 0.8b 86.8 ± 0.6ab 83.9 ± 0.4abc

Yeast 85.7 ±3.2b 96.8 ± 1.6c 93.5 ± 1.7d

Values are means ± standard error (n = 4 replicates per treatment). Means in the same

column with the same superscript are not significantly different (p>0.05) from one

another

104

Table 3.6. Impact of starch on apparent digestibility coefficients (%) for dry

matter (ADMD), crude protein (ACPD), gross energy (AGED) and starch (ASD)

in fishmeal-based formulated mud crab diets

Diet ADMD ACPD AGED ASD

B

W15

W30

W45

W45U

W60

P30

R30

C30

75.4 ± 1.9ab

72.0 ± 2.8ab

75.2 ± 1.2ab

77.6 ± 0.3bc

85.3 ± 0.4d

80.7 ± 0.6bcd

76.4 ± 1.2abc

83.0 ± 2.5bcd

83.4 ± 0.9bcd

89.2 ± 0.8abcd

86.5. ± 1.1ab

88.0 ± 0.8abc

86.7 ± 0.1ab

91.9 ± 0.5d

86.9 ± 0.4ab

88.0 ± 0.9abc

91.6 ± 1.3d

91.6 ± 0.6d

86.0 ± 1.1def

83.7 ± 1.4de

87.7 ± 0.7ef

81.4 ± 0.7bcd

91.8 ± 2.0f

82.7 ± 0.9cde

73.1 ± 1.6a

84.8 ± 1.5de

85.2 ± 1.1de

97.0 ± 0.2h

92.8 ± 0.1f

90.0 ± 0.1de

87.2 ± 0.2b

91.1 ± 0.1e

84.4 ± 0.4a

88.5 ± 0.1c

88.5 ± 0.1c

92.5 ± 0.1f

Values are means ± standard error (n = 4 replicates per treatment). Means in the same

column with the same superscript are not significantly different (p>0.05) from one

another

105

Statement of Joint Authorship

Manuscript 3

Phuong H. Truong, Alex J. Anderson, Peter B. Mather, Neil A. Richardson & Brian D.

Paterson. Effect of selected feed meals and starches on diet digestibility in the mud

crab, Scylla serrata (in review).

Phuong H. Truong (Candidate)

• Wrote the manuscript

• Designed and formulated sampling design and experimental protocols

• Undertook all laboratory work, analysis and interpretation of data

Alex J. Anderson

• Supervised the experimental design and experimental protocols

• Assisted in the interpretation of data

• Provided editing and contributed to the structure of the manuscript

Peter B. Mather

• Co-supervised the experimental design and experimental protocols

• Assisted in the interpretation of data

• Provided editing and contributed to the structure of the manuscript

Brian D. Paterson

• Co-supervised the experimental design and experimental protocols

106

• Assisted in the interpretation of data

• Provided editing and contributed to the structure of the manuscript

Neil A. Richardson

• Co-supervised the experimental design and experimental protocols

• Assisted in the interpretation of data

• Provided editing and contributed to the structure of the manuscript

107

Chapter 4.

APPARENT DIGESTIBILITY OF SELECTED FEED INGREDIENTS IN

DIETS FORMULATED FOR SUB-ADULT MUD CRABS, Scylla paramamosain

IN VIETNAM

Phuong H. Truonga,b, Alex J. Andersona, Peter B. Mathera, , Brian D. Patersonc and

Neil A. Richardsond

aSchool of Natural Resource Sciences, Queensland University of Technology, GPO

Box 2434, Brisbane QLD 4001, Australia

bPermanent address:Research Institute for Aquaculture No3 (RIA3), 33 Dang Tat

Street, Nhatrang, Vietnam

cBribie Island Aquaculture Research Centre (BIARC), PO Box 2066, Woorim, QLD

4507, Australia

dSchool of Life Sciences, Queensland University of Technology, GPO Box 2434,

Brisbane QLD 4001, Australia

Correspondence: Truong HP, School of Natural Resource Sciences, Queensland

University of Technology, GPO Box 2434, Brisbane QLD 4001, Australia. Ph:+61 7

3138 1388; Fax: +61 7 3138 1537. E-mail: hp.truong@student.qut.edu.au or

phuongngaduy@yahoo.ca

108

4.1 Abstract

The present study was conducted to explore the potential to incorporate local

plant-based feed ingredients into diets formulated for the Asian mud crab, Scylla

paramamosain that is commonly exploited for aquaculture in southeast Asia. Four test

ingredients (defatted soybean meal, rice bran, cassava meal and corn flour) were

incorporated at 30% or 45% inclusion levels in a fishmeal based reference diet, and

compared in digestibility trials where apparent digestibility coefficients (ADCs) for

experimental diets and test ingredients were determined. Generally, high ADC values

were obtained using diets containing 30% soybean meal or rice bran. The diet with

30% soybean meal had the highest apparent digestibility for dry matter (ADMD)

(85.7%), crude protein (ACPD) (93.2%) and gross energy (AGED) (92.2%), while the

lowest values were obtained for the diet containing 45% cassava meal (70.9% ADMD;

77.1% ACPD; 80.2% AGED). Overall, ADC values were reduced when the level of

test ingredient incorporated into diets was increased from 30% to 45%. Similar trends

were observed when dry matter and crude protein digestibilities were compared for

specific feed ingredients. Specifically, highest ADC ingredient (I) values were

obtained for soybean meal when used at a 30% inclusion level (87.6% ADMDI;

98.4% ACPDI; 95.6% AGEDI) while lowest ADCI values were obtained using

cassava meal at a 45% inclusion level (53.8% ADMDI; 60.2% ACPDI; 67.3%

AGEDI). Overall, ADCI values for test ingredients reduced as ingredient inclusion

level in experimental diets was increased. Based on the current findings we propose

that soybean meal and rice bran could be considered as potential ingredients for

incorporation into formulated diets for S. paramamosain.

Key words: Mud crab, Scylla paramamosain, digestibility, feed ingredients

109

4.2 Introduction

Mud crabs in the genus Scylla are commercially important in several Indo-

Pacific countries and provide an important source of income and fresh food for many

coastal fishing communities (Hill 1983; Keenan 1999). Currently, mud crab farming is

well established across southeast Asia with most farmers using trash fish, bivalve

meats or animal by-products as feeds. This traditional feeding practice, however, is

now considered unsustainable and development of formulated low cost grow out diets

is widely viewed as a priority issue for development of the mud crab aquaculture

industry (William & Abdullah 1999; Edwards et al. 2004; Christensen et al. 2004;

Fielder 2004; Tuan et al. 2006).

Consideration of any feed ingredient for incorporation into aquafeeds requires

data on the target species capacity to digest and absorb the component. Recent studies

have confirmed that Scylla serrata has a significant capacity to utilise feed ingredients

from a variety of terrestrial animal or plant-based sources. In particular, many plant-

based ingredients have been evaluated for their potential to be incorporated into

aquafeeds for this species. For example, Catacutan et al. (2003) determined that

soybean meal, corn meal and copra meal were all highly digestible in diets formulated

for S. serrata. Tuan et al. (2006) also reported apparent digestibility values for

soybean meal that were not significantly different to those obtained using fishmeal in

diets formulated for S. serrata. Likewise, Truong et al. (2008) (Chapter 3) reported

high apparent digestibility coefficients for soybean, canola, lupin and cottonseed meals

incorporated into diets formulated for this species. The study described in Chapter 2

also demonstrated that inclusion of potato, wheat, corn or rice starches in diets

110

formulated for S. serrata had few negative effects on apparent digestibility values for

protein or energy

The present study was carried out to determine the capacity of the common

mud crab species cultured in Vietnam, Scylla paramamosain, to digest formulated

diets containing selected, locally available plant-based ingredients (defatted soybean

meal, rice bran, cassava meal and corn flour). Apparent digestibility coefficients for

dry matter, crude protein and gross energy were determined for test ingredients and

diets containing test ingredients. We also examined the effects of varying the inclusion

level of test ingredients on nutrient digestibilities in diets.

111

4.3 Materials and Methods

Experimental site and animals

The experiment was carried out at the Research Institute for Aquaculture No 3

(RIA3), Nha Trang, Vietnam from 20 November 2006 to 4 January 2007 with

hatchery reared sub-adult S. paramamosain (94.1±1.1g) collected from a culture pond

at RIA3. Crabs were fed a commercial diet (Turbo®, Thailand) twice daily at a

feeding rate of 3% body weight for a week to acclimate to experimental conditions.

Culture system

Crabs were assigned randomly to nine groups with twelve crabs in each group

and held in black individual containers (19cm x 28cm x 21cm) that were covered by

plastic net lids. For all experimental treatments, crabs were supplied with recirculated,

aerated seawater. During the experimental period, temperature, salinity, pH, and

dissolved oxygen of water temperature were maintained at 27.5 ± 0.5oC, 27.5 ± 1.5g

L-1, 7.67±0.09 and 4.26±0.18mg L-1, respectively.

Diet preparation

Diets used in the experiment were prepared by thoroughly mixing dry

ingredients, followed by wet ingredients, until a crumbly dough consistency was

achieved. Diet mixture was pressure pelleted using an electronic mincer with a 3mm

die. Pellets were then steamed in a rice steamer in a microwave oven (LG) for 5 min,

prior to drying overnight at 50 °C in a drying oven. After drying, diet strands were cut

into about 8-10mm lengths. All experimental diets were stored at -20°C until required.

112

A reference diet based on high quality South American fishmeal was

formulated (Diet 1; Table 4.1) ensuring that dietary protein levels in all test diets were

set above those reported to promote good growth rates in culture (Catacutan 2002).

Eight other diets were also formulated where fishmeal in the reference diet was

replaced with different amounts (30% and 45%) of rice bran, cassava flour, corn flour

or defatted soybean meal. All diets contained 0.5% Cr2O3 as an inert indicator to

allow calculation of apparent diet nutrient digestibilities (AD) and ingredient

digestibility coefficients (ADC) for dry matter (DM), crude protein (CP) and gross

energy (GE).

Feeding and faecal collection

Crabs were fed experimental diets twice daily at a feeding rate of 3% body

weight (BW) per day until approximately 1.5 to 2g of faecal material (dry weight) was

collected. A daily record was kept of mortalities in each group. Faecal material at the

bottom of each container was collected by syphoning into a plastic sieve and removed

individually using forceps. To collect sufficient material for analysis, faecal material

from three crabs in each treatment was pooled (n=4 / treatment). All samples were

lyophilized and stored at -20°C until required for analysis.

Chemical analysis and calculations

The proximate nutrient content of the experimental diets are presented in Table

4.2. Proximate composition of diets and faecal material were determined at Nha Trang

Fisheries University, Vietnam, following AOAC standards (1984). The indirect

method of Furukawa & Tsukahara (1966) was used to calculate the digestibilities of

all diets tested. ADMD was determined using the formula:

113

ADMD = 100 – 100 (%Cr2O3 in feed / % Cr2O3 in faeces).

Digestibilities of crude protein (ACPD) or gross energy (AGED) were

determined using the formula:

APD = 100 – 100 [(%Cr2O3 in feed / % Cr2O3 in faeces) x (% protein or MJ

kg-1 energy in faeces/ % protein or MJ kg-1 energy in feed)].

Apparent digestibility coefficients (ADC) of the test ingredients were calculated using

the following equations described by Bureau et al (1999).

ADCI = ADCT + ((1 – s) DR / s DI) (ADCT – ADCR)

Where: ADCI = apparent digestibility coefficient of test ingredient; ADCT =

apparent digestibility coefficient of test diet; ADCR = apparent digestibility coefficient

of the reference diet; DR = % nutrient (or kJ/g gross energy) of the reference diet

mash; DI = % nutrient (or kJ/g gross energy) of the test ingredient; s = proportion of

test ingredient in test diet mash (i.e. 0.3 and 0.45 in this study); (1 – s) = proportion of

reference diet mash in test diet mash (i.e. 0.7 and 0.55 in this study).

Statistical analyses

The significance of data were determined using one-way ANOVA (SPSS

version 13.0) and post hoc comparison by Tukey’s HSD. For all analyses a

significance level of p<0.05 was used as standard.

114

4.4 Results

4.4.1 Digestibility determinations: Experimental diets

Apparent digestibility coefficients for dry matter (ADMD), crude protein

(ACPD) and gross energy (AGED) obtained using experimental diets are presented in

Table 4.3. ADMD coefficients ranged from 70.9% to 85.7%. The highest ADMD

value was obtained using the diet containing 30% soybean meal (D8) that was

significantly higher (p<0.05) than those obtained using any other diet except for the

30% rice bran (D2) diet and the reference diet. By contrast, the lowest ADMD value

was obtained using the diet containing 45% cassava flour (D5) that was significantly

lower (P<0.05) than those obtained for any other experimental diet. An overall

reduction in ADMD values was observed as the level of test ingredient incorporated

into experimental diets was raised from 30% to 45%. In particular, incorporation of

more than 30% soybean or cassava meal into experimental diets resulted in a

significant reduction (p<0.05) in ADMD values.

ACPD coefficients obtained using experimental diets ranged from 77.1% to

93.2%. ACPD coefficients for diets containing soybean meal (D8, D9) or 30% rice

bran (D2) were either significantly higher (P<0.05) or equivalent to those obtained

using the fishmeal-based reference diet. By contrast, the lowest ACPD value was

obtained using the diet containing 45% cassava flour (D5) that was significantly lower

(p<0.05) than those obtained using any other experimental diet. Furthermore, it was

demonstrated that if the level of any test ingredient incorporated into experimental

diets was increased from 30% to 45%, a significant reduction (p<0.05) in the ACPD

value of the diet was observed.

115

AGED coefficients for experimental diets were generally high and ranged from

80.2% to 92.2%. The highest AGED value was obtained using the diet containing 30%

soybean meal (D8) that was significantly higher (p<0.05) than those obtained using

other experimental diets except the diet containing 30% rice bran (D2) or the reference

diet. By contrast, the lowest ACPD value was obtained using the diet containing 45%

cassava flour (D5) that was significantly lower (p<0.05) than that obtained with other

experimental diets. Additionally, if the level of rice bran, cassava meal or soybean

meal incorporated into diets was increased from 30% to 45% significant reductions

(p<0.05) in the AGED value of the diets were observed.

4.4.2 Digestibility determinations: Test ingredients

Apparent dry matter (ADMDI), crude protein (ACPDI) and gross energy

(AGEDI) digestibility coefficients calculated for specific feed ingredients are

presented in Table 4.4. The highest ADMDI value (87.6%) was obtained for soybean

meal (at 30% inclusion level) that was significantly higher (p<0.05) than values

obtained for all other test ingredients. By contrast, ADMDI values obtained for

cassava flour were significantly lower than those obtained for any other test

ingredient. The highest ACPDI value was obtained for soybean meal which was

significantly greater (p<0.05) than those obtained for any other test ingredient, with

the exception of rice bran (at 30% inclusion level). By contrast, the ACPDI value

obtained for cassava flour (at 45% inclusion level) was significantly lower than those

obtained for all other test ingredients. The ingredient with the highest AGEDI value

(95.6%) was soybean meal, when used at a 30% inclusion level, while values obtained

using cassava meal were significantly lower than those for any other test ingredient.

Overall, ADC coefficient values for test ingredients decreased as the level of

116

ingredient incorporated into diets was raised from 30% to 45%. In particular, with the

exception of soybean meal, the ACPDI value of test ingredients was significantly

reduced (p<0.05) if the test ingredient comprised more than 30% of the experimental

diet.

117

4.5 Discussion

In a previous study, we demonstrated that S. serrata has a high capacity to

digest a range of plant based feed ingredients (Truong et al. 2008). In the present study

we have extended these investigations and examined the potential to incorporate local

plant based feed ingredients into diets formulated for the mud crab species commonly

cultured in Southeast Asia (S. paramamosain). As was the case for S. serrata, S.

paramamosain also demonstrated a high capacity to digest soybean meal at inclusion

levels up to 45%. These results are also consistent with the findings of Catacutan et al.

(2003) who reported that the apparent digestibilities for dry matter and crude protein

in diets formulated for S. serrata were 90.9% an 95.5%, respectively. Likewise, Tuan

et al. (2006) also reported that ADC coefficients for dry matter, energy and crude

protein for soy bean meal were relatively high with values of 95.7%, 97.1% and

97.9%, respectively.

In this study, rice bran at a 30% inclusion level demonstrated generally high

ADC values. These findings are in close agreement with the observations of Catacutan

et al. (2003) who demonstrated that dry matter and crude protein digestibility

coefficients for rice bran for S. serrata were relatively high (89% and 94%

respectively). In a related study, Truong et al. (2008) also reported high ADC

coefficients for rice-based ingredients incorporated into diets formulated for S. serrata.

Furthermore, the incorporation of approximately 30% rice starch to fishmeal based

diets formulated for juvenile S. serrata has been shown to have no negative impacts on

growth (Chapter 2). Based on the above findings, and those of the current

investigation, we suggest that rice-based products, such as bran and starch, are

118

generally well digested and should be investigated further for their potential to be

incorporated into aquafeeds formulated for Scylla species.

A key finding of this study was that many of the ADC values for corn flour

were significantly less than those obtained for rice bran or soybean meal. This result is

surprising considering that Truong et al. (2007a) observed that S. serrata demonstrated

a high capacity to digest diets containing corn starch. Likewise, Catacutan et al.

(2003) reported high ACPD (96.4%) and ADMD (93.2%) coefficient values in diets

for S. serrata containing 30% corn flour. One interpretation of these data is that

significant differences may exist in the capacity of various Scylla species to digest

corn-based ingredients. Alternatively, the capacity of mud crabs to digest corn-based

ingredients may be influenced by the source of the corn-based ingredient or the nature

of its preparation. Further work will be needed to resolve these topics.

Relatively poor ADC values were obtained for sub-adult crabs fed diets

containing cassava meal. This result contrasts with other studies that examined the

potential of cassava meal for incorporation into crustacean aquafeeds. For example,

Gomes and Pena (1997) reported that the inclusion of 30% heated cassava meal in

diets formulated for Macrobrachium rosenbergii did not have a negative impact on

digestibility of protein and energy. The potential of cassava meal in diets formulated

for mud crabs may be limited as a consequence of the presence of anti-nutrional

factors in this ingredient. Specifically, Oboh and Akindahunsi (2005) investigated rats

fed with a diet containing 40% cassava meal and showed a significant rise in serum

glutamate pyruvate transaminase and serum glutamate oxaloacetate transaminase

activity indicating possible damage to the liver (hepatotoxic) and/or heart

(cardiotoxic). Further studies will be required to determine the factors that may inhibit

119

digestion of cassava meal in mud crabs and if the relatively poor digestibility of this

ingredient is consistent across all Scylla species.

In conclusion, here we show that at a 30% inclusion level, soybean meal and

rice bran had no negative impact on diet digestibility compared with a fishmeal-based

reference diet. Moreover, even at an inclusion level of 45%, crude protein and gross

energy digestibility were not reduced significantly by soybean meal. This suggests that

soybean meal and rice bran are ingredients with high potential for inclusion into

formulated diets for both S. serrata and S. paramamosain. By contrast, while corn

flour demonstrated some potential for inclusion into diets formulated for S.

paramamosian, overall, ADC values were significantly less than those observed in

related studies with S. serrata. Another important finding of the current study was that

cassava meal demonstrated poor potential for inclusion in diets formulated for S.

paramamosain, at least at the inclusion range described here.

120

Acknowledgements

The authors would like to thank Nguyen Co Thach, Truong Quoc Thai,

Nguyen T. X. Thu, and Nguyen Xuan Nam at Research Institute for Aquaculture No3

for their technical assistance. This study was supported by Ministry of Education and

Training, Vietnam and ACIAR Mud Crab Project FIS/2000/065, Australia. Our

gratitude is also extended to Tran Dinh Thanh of SEAPRODEX Company (Da Nang,

Viet Nam) for supplying the fishmeal. This manuscript is written in partial fulfillment

of the Doctor of Philosophy degree in aquaculture of Phuong Ha Truong, at the

Queensland University of Technology, Brisbane, Australia.

121

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Effect of pH, temperature and diet on digestive enzyme profiles in the mud crab,

Scylla serrata, Aquaculture 242, 641-654.

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digestibility of some nutrient sources by juvenile mud crab, Scylla serrata (Forskål

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124

Table 4.1. Composition of the nine formulated diets for the digestibility trial

using different local ingredients (as % dry weight basis).

Ingredient (%) Diet

g100g-1 RFa R30 R45 Ca30 Ca45 Co30 Co45 S30 S45

Fish mealb 81.5 51.5 36.5 51.5 36.5 51.5 36.5 51.5 36.5

Binder

(Wheat gluten) 5 5 5 5 5 5 5 5 5

Cod liver oild 3 3 3 3 3 3 3 3 3

CaHPO4 3 3 3 3 3 3 3 3 3

Common

Ingredientse 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5

Rice branc - 30 45 - - - - - -

Cassava mealc - - - 30 45 - - - -

Corn flourc - - - - - 30 45 - -

Soybean mealc - - - - - - - 30 45

a Reference diet without any test ingredients included

b Peruvian fish meal; c products of Vietnam

d Healthier of Australia (Healtheries of New Zealand, Auckland, New Zealand)

e Common ingredients (g100g-1): mineral and vitamin premix (2%), (kg –1 of total

diet - 4.68 g K2HPO4; 7.12 g MgSO4.7H2O; 1.84 g NaH2PO4.2H2O; vitamin premix

(kg-1) – 100000 IU vitamin retinol; 500 mg thiamine; 1750 mg riboflavin; 1125 mg

pyridoxine hydrochloride; 3750 mg cyanocobalamin; 25000 mg ascorbic acid;

500000 mg colecalciferol; 20 000 IU d-alpha-tocopheryl acid succinate; 50 mg

biotin); dried squid (5%); 0.5% chromic oxide (Cr2O3).

125

Table 4.2. Dry matter (DM), gross energy (GE MJ kg-1), crude fat (CF), crude

protein (CP) and ash of experimental diets and test ingredients used in the

formulated diets (as % dry weight basis)

Sources DM

(%)

CP

(%)

CF

(%)

Ash

(%)

GE

(MJ kg-1)

Experimental diets

RF

R30

R45

Ca30

Ca45

Co30

Co45

S30

S45

Test ingredients

Peruvian fish meal

Defatted soy bean

Rice bran

Cassava meal

Corn flour

93.6

91.2

90.1

91.1

92.4

89.0

90.9

93.4

93.8

93.6

91.2

90.2

93.6

92.2

65.3

48.5

40.1

46.5

37.1

46.6

37.2

56.0

51.4

71.2

40.1

15.1

8.2

8.6

11.2

11.7

11.4

10.8

10.0

11.2

10.6

10.2

9.4

8.7

1.4

6.8

3.6

4.9

16.4

11.7

9.3

11.7

9.3

14.4

13.3

13.8

12.4

16.8

7.4

8.1

9.2

4.6

20.4

18.9

18.6

18.6

18.0

18.8

18.4

19.6

19.6

21.0

20.1

18.8

17.4

18.2

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Table 4.3. Apparent digestibilities (%) for dry matter (ADMD), crude protein

(ACPD) and gross energy (AGED) in experimental diets

Diet ADMD ACPD AGED

RF

R30

R45

Ca30

Ca45

Co30

Co45

S30

S45

84.9 ± 0.5e

82.6 ± 0.2de

80.6 ± 0.4cd

75.8 ± 0.9b

70.9 ± 0.6a

80.2 ± 0.1cd

78.9 ± 0.3c

85.7 ± 0.3e

81.6 ± 0.2cd

90.9 ± 0.5d

91.8 ± 0.4d

87.8 ± 0.4c

84.7 ± 0.8b

77.1 ± 0.7a

87.8 ± 0.2c

83.6 ± 0.3b

93.2 ± 0.4e

92.1 ± 0.9d

90.7 ± 0.7de

90.6 ± 0.4de

87.3 ± 0.7c

83.8 ± 1.1b

80.2 ± 0.5a

88.7 ± 0.5cd

85.6 ± 0.37bc

92.2 ± 0.4e

88.6 ± 0.3cd

Values are means ± SE (n = 4 replicates per treatment). Means in the same column

with the same superscript are not significantly different (p>0.05) from one another

127

Table 4.4. Apparent digestibility coefficients (%) for dry matter (ADMDI

ADCDM), crude protein (ACPDI ) and gross energy (AGEDI ) of the test

ingredients used in the formulated diets

Sources Inclusion

(%)

ADMDI

(%)

ACPDI

(%)

AGEDI

(%)

Rice bran 30 77.3±1.7c 93.8±0.4d 90.2±1.8cd

45 75.3±1.2bc 84.0±0.7c 83.1±1.6bc

Cassava meal 30 54.8±0.9a 70.3±2.6b 67.8±0.9a

45 53.8±1.1a 60.2±1.3a 67.3±1.1a

Corn flour 30 69.2±1.2b 80.6±0.9c 83.9±1.9bc

45 71.5±0.8bc 74.7±0.5b 79.4±1.4b

Defatted soy bean 30 87.6±2.4d 98.4±0.4d 95.6±2.9d

45 77.5±0.5c 93.5±0.2d 86.1±0.7bc

Values are means ± SE (n = 4 replicates per treatment). Means in the same column

with the same superscript are not significantly different (p>0.05) from one another

128

Statement of Joint Authorship

Manuscript 4

Phuong H. Truong, Brian D. Paterson, Alex J. Anderson, Peter B. Mather, and Neil A.

Richardson. The effects of culture environments, sex, size and feed attractants on

feeding responses in the mud crab, Scylla serrata (in prep.)

Phuong H. Truong (Candidate)

• Wrote the manuscript

• Designed and formulated sampling design and experimental protocols

• Undertook all laboratory work, analysis and interpretation of data

Alex J. Anderson

• Supervised the experimental design and experimental protocols

• Assisted in the interpretation of data

• Provided editing and contributed to the structure of the manuscript

Peter B. Mather

• Co-supervised the experimental design and experimental protocols

• Assisted in the interpretation of data

• Provided editing and contributed to the structure of the manuscript

Neil A. Richardson

• Co-supervised the experimental design and experimental protocols

129

• Assisted in the interpretation of data

• Provided editing and contributed to the structure of the manuscript

Brian D. Paterson

• Co-supervised the experimental design and experimental protocols

• Assisted in the interpretation of data

• Provided editing and contributed to the structure of the manuscript

130

Chapter 5.

THE EFFECTS OF CULTURE ENVIRONMENTS, SEX, SIZE AND FEED

ATTRACTANTS ON FEEDING RESPONSES IN THE MUD CRAB, Scylla

serrata

Phuong H. Truonga,b, Brian D. Patersonc, Alex J. Andersona, Peter B. Mathera, and

Neil A. Richardsond

aSchool of Natural Resource Sciences, Queensland University of Technology, GPO

Box 2434, Brisbane QLD 4001, Australia

bPermanent address:Research Institute for Aquaculture No3 (RIA3), 33 Dang Tat

Street, Nhatrang, Vietnam

cBribie Island Aquaculture Research Centre (BIARC), PO Box 2066, Woorim, QLD

4507, Australia

dSchool of Life Sciences, Queensland University of Technology, GPO Box 2434,

Brisbane QLD 4001, Australia

Correspondence: Truong HP, Research Institute for Aquaculture No3 (RIA3), 33 Dang

Tat Street, Nhatrang, Vietnam E-mail: phuongngaduy@yahoo.ca

131

5.1 Abstract

The present study was conducted to investigate the effects of added dietary

attractants (betaine, chicken meal, tuna oil and commercial bait enhancer),

temperature, sex and size on behavioural responses (resting, searching, feeding

initiation, feeding continuation and food consumption) of mud crabs. Results showed

that behaviours of mud crabs were influenced significantly by different attractants in

diets. Specifically, chicken meal and betaine attractant treatments showed a significant

increase in consumption (p<0.05) compared with other treatments. No significant

difference in food consumption was found, however, when attractant level was

increased (rising from 2 to 5% inclusion) in pelleted diets, except for the 5% bait

enhancer treatment, that resulted in significantly higher consumption (p<0.05) than for

the 2% bait enhancer treatment. Small crabs (17g) consumed significantly more

(p<0.05) of the ration in percentage terms than did large (311g) crabs. A two-way

analysis of deviance also showed that proportion of time in resting, locomotion and

initiation was significantly affected by individual size (p<0.05). No interaction was

observed, however, between crab size and attractants in resting, locomotion and

continuation, but there was a significant interaction between size and initiation

response. Both sex and temperature significantly influenced the proportion of time

crabs spent in locomotion, but only temperature had a significant impact on the

proportion of time crabs spent in initiation and resting. No significant difference in

continuation response was found for either sex or temperature and no interaction was

observed between factors for all response classes. Females were significantly more

active than males, though the effect was small. Light intensity was found to be a

significant factor that affected crab responses since small, medium and large

individuals spent 95.6%, 93.8 and 94.4% of the time, respectively, in the half-shaded

132

side of the Y –maze, and consumed significantly more food than when not shaded.

Key words: Mud crab, video behaviour analysis, chemo-attractants

133

5.2 Introduction

Mud crabs (Scylla spp.) are general predators of benthic molluscs and

crustaceans, and use contact chemoreception to locate and manipulate food items (Hill

1979). Although small juveniles can survive on natural productivity in culture ponds,

Scylla in culture are typically fed with “trash fish” (Christensen et al. 2004). Aside

from the questionable sustainability of feeding trash fish and other coastal resources to

cultured crabs, carnivory has two other drawbacks for aquaculture of mangrove crabs.

Firstly, it pre-supposes manufactured diets must be attractive to predators by

incorporating a high (and hence expensive) marine protein content and secondly, it

means crabs will readily cannibalise other crabs if grown at high density (Williams &

Primavera 2001).

As profit margins decline for many aquaculture species, the need to optimize

feed efficiency is growing in economic importance. Important issues that need to be

optimised to improve crab culture include minimizing the amount of time between

feed application to the pond and ingestion by the crabs, reducing feed input and

concomitant wastes from uneaten food, and getting the crabs to consume the minimum

amount of nutrients required to maximise growth. This also means that the aquafeeds

used, should contain highly digestible nutrients and be palatable. Feed costs for

cultured crabs could probably be reduced if marine animal meals can be replaced with

lower cost terrestrial plant meals in formulated feeds. Such a change, however, may

reduce the palatability and attractiveness of feeds even though mud crabs have

enzymatic profiles that allow them to digest common terrestrial plant meals (Tuan et

al. 2006; Pavasovic et al. 2004). Thus, developing a basis understanding of what

stimulates crabs to feed can assist in design of better low cost diets in the future.

134

Although little has been published on feeding and foraging behaviour of mud

crabs, there have been numerous observations made of foraging behaviour in other

portunid swimming crabs and their responses to scent (or odour, olfactory stimulant)

plumes in the water, particularly in Callinectes sapidus (Weissberg & Zimmer-Faust

1994; Clark 1999, 2000; Diaz et al. 2003). The importance of chemo-attractants and/or

feeding stimulants to improve both initial palatability and overall feeding rate and as a

means to reduce feed wastage is now considered an important priority for crab

research in the future. In particular, a recently developed paradigm for feeding

behaviour of crustaceans postulated that chemical stimuli play a key role in mediating

the various stages of feeding from initial excitation to sustained feeding (Pittet et al.

1996).

Many marine animals, especially those active at night, use chemical cues to

detect and locate food items (Diaz et al. 2003). A wide variety of odours including

homogenized fish, crab, shrimp and brine shrimp (Lee 1992; Hazlett & Mclay 2000;

Diaz et al. 2003; Kozlowski et al. 2003; Hazlett 2004; Budelmann 1994), squid (Zhou

& Shirley 1997), clam body odour (Zimmer-Faust et al. 1996), extracts (Lee 1992;

Pittet et al. 1996; Salierno et al. 2003), oysters and clams (Clark 1999, 2000; Diaz et

al. 2003), betaine, taurine, glycine (Lee 1992; Hartati & Briggs 1993; Circuna et al.

1995), and crushed conspecifics (Gherardi et al. 2002; Hazett 2004; Wall 2005) have

been trialled as potential feed attractants for crabs, and other aquatic organisms. In

particular, betaine has proven the most effective attractant by far in all species studied

to date, either alone or in combination with other substances (Lee 1992; Pittet et al.

1996; Hartati & Briggs 1993; Reig et al. 2003). For example, Cadena-Roa et al. (1982)

reported that betaine provided the best results in sole diets (Solea solea) at a dose of

135

4.4% betaine, as part of a mixture of chemical feeding activators, that provided 7% of

the biomass of the diet.

Investigations of chemoreceptive behaviour in marine animals have noted that

other environmental factors such as temperature, light, and water current can affect an

organism’s behaviour and relative chemo-sensitivity (Atema 1987; Zimmer-Faust et

al. 1996). To improve growth and survival rates of mud crabs in culture, while

reducing feed costs, it is therefore important to understand the influence that such

factors play in directing feeding behaviours. The current study investigated the effects

of selected attractants added to formulated feeds as well as crab size and sex and

various environmental factors such as temperature and light levels on feeding

behaviours of the mud crab, S. serrata.

136

5.3 Materials and methods

5.3.1 Experimental animals

The research was conducted at the Bribie Island Aquaculture Research Centre

(BIARC), Queensland, Australia. Juvenile mud crabs were all siblings and were

reared from spawning of a single wild female. Juvenile crabs were classified into three

size-class groups, and eight crabs (4 female and 4 male individuals) were selected

randomly from each class to make the following test groups: (1) the small group had

an average weight of 17.6 ± 4.4 g (CW= 43.0 ± 4.5mm) (2) medium group had an

average weight of 114.8 ± 18.4g (CW= 79.8 ± 4.5mm), and (3) large group had an

average weight of 311.2 ± 32.0g (CW= 111.3 ± 4.9mm). Crabs were acclimatised to

culture conditions by feeding twice daily at a feeding rate of 3% of body weight, once

in the morning and once in the afternoon with a Ebistar® prawn feed (Higashimaru,

Japan).

Crabs were examined at the start of the culture period to ensure they were in

the inter-moult stage and had no visible injuries (e.g. limb loss). The definition of this

moult stage is described as; the shell is hard and there is no separation of the epidermis

from the cuticle (Drach & Tchernigovtzeff 1967).

5.3.2 Experimental design and procedures

Experimental system

137

A Y- shaped wooden aquarium was used for this study. It consisted of (a) a

base (60cm long x 36 cm wide x 50 cm high) and two adjoining arms (60 cm long x

36 cm wide x 50 cm high); (b) a removable screen allowed the crab to be acclimated

in the stem of the Y-maze and encouraged forward orientation at the beginning of a

trial; (c) The convergence of the two arms angle was 60o and the angles between the

arms and the base were 150o (Figure 5.1).

Test water was stored in an overhead tank equipped with a heater and air

supply. Temperature, salinity and dissolved oxygen in the overhead tank were

maintained as the same level as those in the recirculating cellular systems (27.5±0.5oC,

32.4 ± 1.5g L-1 and 4.4±0.2mg L-1 respectively). Water was then fed continuously into

each arm of the Y-maze. The water outlet pipe was placed at the end of the stem of

the Y (20cm high from the bottom, setting depth of water) to allow water to flow out

continuously. Water current was adjusted to 1 litre water min-1 down in each arm.

Crab behaviour was monitored using a black and white video camera (WV-

BP330) suspended directly over the raceways above the maze for later analysis. This

study was a modification of the method employed by Lee (1992) using videotape

(VHS using Teac MV-6000 and LG AC450W video recorders) to provide information

on the duration of time spent in each response behaviour and the amount of food used

by individual crabs after each test period. Numerical values used to assess animal

responses were calculated as follows: response value = 100 - (10 x time (min) for each

activity).

The criteria for the behaviour types recorded in Table 5.1 were based on

similar studies on other decapod crustaceans and included resting and locomotion

138

(Eales 1972; Lee 1992; Lee & Meyers 1996); grabbing and holding, pouncing on food

(Eales 1972; Lee & Meyers 1996; Clark et al. 1999), and feeding (Eales 1972; Lee &

Meyers 1996, Zhou & Shirley 1997; Hazlett 2004).

Experiment 1: Seawater (no food) versus test diets

In this experiment, eight crabs (121.8 ± 12.5g) were used to test responses to

five combinations between seawater (no food) and five test diets in order to investigate

the total time individual crabs spend in each arm of the maze (or diet). Attractant diets

included 2% betaine (BT, Sigma-Aldrich®) a known attractant,2% chicken meal (CM,

Tony Bush and Sons®, Australia) a new product which showed promise as an

attractant during digestibility trials, 2% tuna oil (TN, Product of Australia) or 2% bait

enhancer (BE, Product of New Zealand) incorporated into the reference diet (RF).

Both TN and BE were commercial products reputed to improve attractability of baits,

but the composition of BE was not given. Water in this experiment was replaced

completely after each test. Each crab was selected randomly and tested with only one

pair of test diets each day and crabs were held in individual containers until required

for the trial to allow the response of individual crabs to be determined. Individual

crabs were first placed in the Y-maze for approximately 5 minutes in order to

acclimate in a standard manner to test environment conditions. For each test, a pair of

the test feeds were randomized and placed at the far end of each of the two arms of the

Y maze. After acclimation, the camera was switched on to record behaviour when a

test individual had moved into one of either two arms of the maze. Experiments were

conducted in a dark room during the day and at night under red light illumination in

order to minimise disturbance and to ensure adequate quality of video recordings (cf.

Wall 2005). Each test was terminated after 10 minutes and was conducted before the

first daily feed had been given. In this experiment, total time of individual crab spent

139

in each arm (or each test diet) of the Y-maze was recorded and displayed in time

percentage (%).

Experiment 2: Reference versus test diets

Crabs and attractant diets used in this experiment were as described in

experiment 1. The four test diets were tested against the reference diet, using an

attractant dose rate of 2%. Other procedures were as described for experiment 1.

Experiment 3: Influence of size and attractant diets on crab behaviours

In this experiment, three size classes were tested (small, medium and large).

Diets containing attractants included 2% chicken meal (CM), betaine (BT), tuna oil

(TN) and bait enhancer (BE), seawater (SW: no food) and a reference diet (RF) were

tested independently (one diet was placed in each of the two arms of the Y-maze) in

order to see how behaviour of individual crab respond to that diet using more detailed

criteria as described in Table 5.1 (percentage of time individual crabs spent on specific

behaviours) and how size of crab respond to attractant diets tested. Feed left after each

test was removed and water in the Y-maze system was drained after 3-4 tests. In this

experiment, the camera and videotape recorder were switched on immediately after the

screen had been removed. All other procedures were used as described in experiment

1.

Experiment 4: Effects of sex and temperature on mud crab response

The chicken meal diet (2%) was used for this experiment since it was

determined in previous experiments to be one of the most attractive diets. Twenty four

medium sized crabs (W=114.8 ± 18.4g; 12 female and 12 male) were tested at three

140

different temperature levels, 26.5oC, 28.5oC and 30.5oC. All other procedures were

applied as described in experiment 1.

Experiment 5: Effects of crab size and attractant diets on food consumption

Two groups of crab were investigated (small= 17.6 ± 4.4 g, n=36; large=114.8

± 18.4g, n=36). Nine test diets were used for this test (reference, CM 2% and 5%, BT

2% and 5%, TN 2% and 5%, BE 2% and 5%). All other procedures were applied as

described in experiment 1. Food consumption value was calculated as FC = [100 – (%

amount of food left)].

Experiment 6: Influence of light on crab choice of food

This experiment was conducted to determine how light intensity affects mud

crab behaviour. In this study, a diet containing 2% chicken meal (the most effective

attractant identified in the previous experiment) was used to test crab behaviour for

three different size classes (small, medium and large) at 27.5oC. Equal number of the

same food pellets were placed in each arm of the Y-maze (2g for medium and large

groups, 0.4g for the small group) and each individual’s response to light and dark

environments was examined. Half of the Y-maze was shaded so that light did not

penetrate onto this side and the experiment was conducted under normal light (not red

light as in previous experiments).

5.3.3 Test diet preparation

An important issue when assessing relative efficiency of chemo-attractants in

aquafeeds is the methodology used to present the chemical stimuli. There are several

methods available: (1) inclusion in the feed before processing; (2) coating the feed

141

immediately following processing; (3) coating the feed at pond side prior to feeding;

and (4) adding the chemicals as a separate mixture to the pond at the time of feeding

(Lee & Meyers 1996). In this trial, attractants were added directly to the feed before

processing.

A diet based on 58% fishmeal and 30% corn starch that had been tested

successfully on mud crabs by the authors previously was used as the basal diet because

it had provided good growth response (Table 5.2). Nine test diets were made for this

trial: a basal diet that did not include attractant and eight other diets that included an

attractant of either tuna oil (TN), bait enhancer (BE), betaine (BT), and chicken meal

(CM) at concentrations of either 2% or 5%.

After drying, pelleted diets were cut to similar size (2cm long) for testing

medium and large size of crab groups. Pellets (~2g) per comparison were placed in

each arm of the maze and then counted again at the end of the test. For small crabs,

diets were ground and sieved to ensure all particles passed through a 2mm screen but

remained in a 1mm screen (1mm < particle size < 2mm). Forty particles (~0.4g) of

similar size of each diet were used for each test.

5.3.4 Statistical analyses

Response to each activity class estimated from the videotape recorders in time

were converted to percentage time as described in Table 5.1. Data sets were plotted

and tabulated as means ± standard error. For the behavioural studies, a binomial

Generalized Linear Model (GLM) using a logit link function on untransformed data

was employed to analyse proportional time data, using factors relevant to each

experiment. Paired sample T tests were used to analyse differences between pairs of

142

test diet combinations. Food consumption was examined using two-way ANOVA that

assessed effects of attractant type, crab size and their interactions. Post-hoc LSD

analysis was conducted on means of factors where significant differences in response

rates were detected (p<0.05) following initial accumulated analysis of deviance

(GLM) or variance (ANOVA) tests. Interaction tables were employed to explore any

significant interactions between factors. All analytical operations were performed

using Genstat (Genstat 2007).

5.4 Results

Experiment 1: Seawater (no food) versus test diets

The paired samples T tests showed a highly significant difference in

percentage of time spent (p<0.01) in the arm of the maze containing food when crabs

were tested for combinations between seawater and RF, CM, BT, TN and BE diets

(9.1% vs 90.9%, 7.4% vs 92.6%, 9.1% vs 90.9%, 13.6% vs 86.4% and 14.0% vs

86.0%, respectively, Table 5.3 )

Experiment 2: Reference versus test diets

Paired samples T tests showed that the proportion of time spent by crabs

assessing diets that included CM and BT was significantly higher than that for the

reference diet (p<0.05). However no significant difference was found in a comparison

between RF versus either TN or BE diets (p>0.05), although response rates were

slightly lower (48.2% vs 51.8% and 46.9% vs 53.1% respectively, Table 5.4).

Experiment 3: Influence of crab size and attractant diets on crab behaviours

143

Two-way analysis of deviance showed that proportion of time in locomotion

was significantly affected by both crab size (Deviance ratio = 11.21, DF = 2,126,

approximate p<0.001) and the attractant type included in the diet (Deviance ratio =

66.35, DF = 5,126, approximate p<0.001). No interaction was detected however,

between factors (Deviance ratio = 0.71, DF = 10,126, approximate p=0.715). Small

crabs showed significantly more locomotion than did either medium or large crabs

(Figure 5.2). All crabs spent the bulk of time resting during the trials. Because periods

of rest were inversely proportional to periods in locomotion, not surprisingly time

spent resting was also significantly affected by both crab size (Deviance ratio = 33.98,

DF = 2,126, approximate p<0.001, Figure 5.2) and the attractant used (Deviance ratio

= 298.22, DF = 5,126, approximate p<0.001, Table 5.5).

A two-way analysis of deviance was carried out on initiation and continuation

data after first excluding results for the seawater blank. This was done because in this

particular treatment no food was available for crabs to approach or eat. This analysis

showed that the proportion of time spent in initiation was significantly affected by

both crab size (Deviance ratio = 40.36, DF = 2,105, approximate p<0.001, Figure 5.2)

and the type of attractant used (Deviance ratio = 4.71, DF = 4,105, approximate

p=0.002, Table 5.5). At first glance, the lower F value for the attractant factor may

reflect removal of the seawater blank from the analysis, this time there was however,

also a significant interaction between individual size and type of attractant (Deviance

ratio = 4.76, DF = 8,105, approximate p<.001). The data indicate that small crabs

devoted significantly more time to initiation when presented with the BE and TN

attractants (Figure 5.3).

144

Ranking of attractants for continuation responses showed that attractant

treatment had a significant effect on proportion of time spent in continuation

(Deviance Ratio = 7.96 DF = 4,105, approximate p<0.001, Table 5.5) while individual

size played no significant role (Deviance Ratio = 1.31, DF = 2,105, approximate

p=0.275, Figure 5.2). There were also no interactions between factors (Deviance Ratio

= 0.26, DF = 8,105, approximate p= 0.978).

Experiment 4: Effect of sex and temperature on mud crab response

Both sex (Deviance Ratio = 7.56, DF = 1,18, approximate p=0.013, Figure 5.4)

and temperature (Deviance Ratio = 125.72, DF = 2,18, approximate p<0.001, Table

5.6) significantly influenced the proportion of time crabs spent in locomotion. Female

crabs were significantly more active than males, although the difference was not large

(Figure 5.4). Crabs tested at 26.5°C also showed significantly less locomotion than did

crabs tested at two higher temperatures (Table 5.6). No significant interaction was

observed between sex and temperature (Deviance Ratio = 0.72, DF = 2,18,

approximate p= 0.501).

Temperature affected the proportion of time spent resting (Deviance Ratio =

165.44, DF = 2,18, p<0.001, Table 5.6) but sex did not (Deviance Ratio = 2.20, DF =

1,18, approximate p= 0.155, Figure 5.4) and there was no significant interaction

between sex and temperature (Deviance Ratio = 1.72, DF = 2,18, approximate p=

0.207).

The proportion of time spent in initiation was only slightly influenced by

temperature, the two way analysis of deviance showed a significant effect of

temperature (Deviance Ratio = 4.53, DF = 2,18, approximate p= 0.025, Table 5.6)

but not sex (Deviance Ratio = 1.59, DF = 1,18, approximate p=0.224, Figure 5.4).

145

The lowest temperature treatment showed the lowest average proportion of time spent

in initiation. No significant interaction was observed between sex and temperature

(Deviance Ratio = 1.52, DF = 2,18, p= 0.246).

Continuation was not significantly influenced by temperature (Deviance Ratio

= 2.63, D = 2,18, approximate p=0.100) or sex (Deviance Ratio = 0.01, DF = 1,18,

approximate p=0.909) and there was no significant interaction between the two factors

(Deviance Ratio = 0.52 DF = 2,18, approximate p=0.602).

Experiment 5: Effect of crab size and attractants on food consumption

Two-way analysis of variance showed that the amount of feed consumed was

significantly influenced by the type of attractant added to the feed (F8,54 = 33.31,

p<0.001) and the size of crab (F1,54 = 17.44, p<0.001), but there was no significant

interaction between the two factors (F8,54 = 0.97, p=0.467).

Significantly more (p<0.05) of the CM and BT diet treatments were consumed

than other diet treatments, and adding more attractant (increasing from 2 to 5%

inclusion level) did not increase in the amount of feed consumed. The only exception

was for the 5% BE treatment, that showed significantly higher consumption (p<0.05)

than in the 2% BE treatment (Figure 5.5). Small crabs consumed significantly more

(p<0.05) ration in percentage terms (50.0±12.9%, mean ± SD, n=36) than did large

crabs (43.6±15.2%, mean ± SD, n=36).

Experiment 6: Influence of light on crab choice of food

In general, result from this study showed that all crabs spent the majority of

time in the half-shaded side of the Y –maze, estimated at 95.6%, 93.8%, and 94.4%

146

for small, medium and large sized individuals respectively (Table 5.7). Behavioural

responses of crabs tested under the shaded side of the Y-maze could not be recorded.

However, an indirect indicator of continuation behaviour in the dark environment, is

the percentages of feed consumed by individual crabs in either of the two arms (shade

vs no shade side). These values were highly significantly different with more

consumption occurring in the shaded arm of the maze.

5.5 Discussion

Results presented here show clearly that mud crab behaviours changes

significantly when they are provided with diets containing different attractants and

with seawater alone. Two attractants; chicken meal and betaine, produced significantly

elevated feeding and food consumption rates compared with a reference diet, and with

diets containing less favoured types of attractant (tuna oil and commercial bait

enhancer). The high attractability associated with chicken meal in this study was

consistent with results of Middleton et al. (2000) who demonstrated that poultry

caracasses performed well as an alternative bait for harvesting blue crabs (Callinectes

sapidus). The effective attractant of betaine in this study was similar with some

previous findings that have proven betaine was the most attractive substance by far in

all species studied to date, either alone or in combination with other components.

Betaine has been trialled in the past as an attractant for fish (Love 1980; Mackie &

Mitchell 1985) and other crustacean species (Harpaz 1997; Felix & Sudharsan 2004).

Macrobrachium rosenbergii fed a diet contained 0.5% glycine betaine achieved the

147

highest weight gain over a 60 day of culture period in comparison with prawns fed

diets containing 1% and 1.5% glycine betaine (Felix & Sudharsan 2004). For the same

species, the growth rate of prawns fed a diet containing 0.6% betaine-HCl increased

17% compared with prawns fed diets without betaine (Harpaz 1997). In contrast, the

investigations by Middleton et al. (2000) revealed that the baits which did not contain

betaine (anhydrous betaine) were significantly more attractive to crabs than were bait

products containing betaine supplementation. This raises an opportunity that the use of

betaine as an attractant should be applied carefully for specific species.

Increasing the dose of favourable attractants from 2% to 5% in diets did not

increase, however the amount of feed consumed significantly, except for the 5% BE

diet. This suggests that an inclusion of 2% chicken meal or betaine is sufficient as an

attractant in crab diets. This indicates that if betaine proves to be a good attractant in

crabs, only small quantities are required in the diet. An important question therefore, is

what the optimum level of attractant should be added to diets. Specifically, it will be

necessary to trial different levels of attractants for any new species. Lee and Meyers

(1996) observed that the high relative cost of potential feed attractants and stimulants

means that cost effective diets will require minimum inclusion levels to be determined

first.

Small crabs displayed less resting time (i.e. more activity) and more time in

locomotion (e.g. walking, running, swimming), initiation (e.g. grabbing, lunging,

pouncing) in comparison with larger crab size classes. Smaller individuals may be

driven by physiological processes to be more active or possibly the dimensions of the

test maze (360 mm wide) inhibited locomotion by larger crabs (average CW of 111.3±

4.9mm). Further studies need to address this issue. Small crabs also showed an

148

unexpected initiation response when presented with BE and TN attractants that

overrode the normal response initiated by chicken meal and betaine. This unusual

initiation response was not expressed however, in continuation data or food

consumption data. An initiation response without continuation may mean that the feed

attracts crabs but does not act as a feeding stimulant. Attractants for nursery/juvenile

crabs should therefore probably be examined separately from those developed for sub-

adults and larger crabs.

Water temperature was shown to influence intensity of behaviour by crabs in

the present study. Crabs showed significantly lower response for all non-resting

behaviours at 26.5oC in comparison with crabs tested at 28.5oC and 30.5oC, with the

exception of the continuation response. The studies described above were conducted at

temperatures normally experienced by this species in the wild including; in the Gulf of

Carpentaria, Moreton Bay, Northern Territory, and Western Australia and in southeast

Asian countries (Keenan 1999b; Sugama & Hutapea 1999). An experimental growth

trial showed that the highest survival and growth rate of juvenile mud crabs was

achieved at 30oC (Ruscoe et al. 2004).

Female crabs showed slightly more locomotion activity than did males in the

temperature experiment. This observation is similar to results achieved by Wall (2005)

who reported that females displayed significantly more tactile investigation than did

males. Middleton et al. (2000) also observed that blue female crabs were more

attracted to the poultry bait than were male crabs. Hayden et al. (2007) reported that

female shore crabs (Carcinus maenas) exhibit stronger feeding responses than males

across the year with a significantly reduced feeding response in males during the

summer reproductive season. Females in general may be a “hungrier” than males due

149

to physiological requirement and/ or because medium and large female crabs are

becoming sexually mature at this stage. A review of cannibalism has noted that

females were more cannibalistic in 86% of trials conducted (Polis 1981). This suggests

that where female monosex culture is applied to crabs, potent feed attractants like

chicken meal or betaine could be useful for their ability to potentially reduce

cannibalism of soft post-moult female crabs by hard shell females (Hayden et al.

2007).

Light intensity was also an important factor that affected crab behaviour. A

recent study showed that crabs tested under light and dark environments spent much

more time in darker environments. Unfortunately, data on behavioural responses of

crabs in the dark side of the maze were not available here, but an indicator of their

relative activity level comes from is the relative amount of feed individuals consumed.

Much higher feed consumption occurred in the dark side of the maze relative to the

light side, however this does not mean that shade enhances appetite. Since mud crabs

are a cannibalistic species, darker environments could also improve their relative

chances of avoiding cannibalism. A recent study on the effects of tank colour on

larval survival and development rates of mud crab showed significantly higher larval

survival in dark-coloured backgrounds. In particular, larvae reared in dark

backgrounds generally had shorter development times and more synchronized

moulting (Rabbani & Zeng, 2005). This finding may be applied to culture of mud

crabs where shelters may assist in reducing cannibalism since crabs tend to hide when

given the opportunity.

The system used to test behavioural response can be critical to the

effectiveness of the study. The Y-maze system used here was constructed initially for

150

testing behavioural responses in shrimps and so had relatively high sides (60cm), a

long arm length (120cm) and a relatively large convergence angle of the two arms

(60o). This caused some difficulties when observing crab behaviour (due to the camera

needing to be placed relatively high above the system). Turbulence also occurred at

the point of confluence of the liquids from the two arms of the Y-maze as it entered

the base (big angle) (distortion or reflection from the water surface). Pettet et al.

(1996) suggested that a convergence angle of 10o would reduce turbulence in the water

when it enters the base.

In conclusion, chicken meal and betaine diets produced significantly better

feeding responses and food consumption rate in mud crabs. Thus, they can be trialled

in diets formulated for crabs diets as potential feed attractants. Temperature was also

recognised as an important factor that affected mud crab behavioural responses.

Results suggest that temperature in the range of 28.5oC to 30.5oC is most suited for

this species. Monosex culture of mud crabs should also be considered in further

studies, since results revealed that sex can affect their behavioural responses. Further

studies should pay more attention to the size of container and identify ones that are

better suitable for individual mud crab observations. In order to reduce water

turbulence at the point between the arms and base of the Y-maze and to observe and

manipulate the system more easily, the Y-maze should probably be adjusted to include

a shorter arm length (40-50cm), with lower walls (25-30cm) and a reduced

convergence angle of the two arms angle 10o between the two arms and 175o between

the arms and the base is suggested.

151

Acknowledgements

The authors would like to thank David Mann, Tom Asakawa and Beverly

Kelly at BIARC for their technical assistance. This study was supported by the

Ministry of Education and Training, Vietnam and we gratefully acknowledge

cooperation from the Australian Centre of International Agriculture Research

(ACIAR) Mud Crab Project FIS/2000/065, Australia.

152

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158

Table 5.1. Mud crab behaviour description and response value measurements Behaviours Explanation of behaviour Response values measured

(as % time spent in each

activity)

Resting

Locomotion (e.g.

walk, run, swim,

search)

Initiation of

feeding (e.g. grab,

lunge, pounce)

Continuation of

feeding (e.g.

ingest or reject)

Not interacting or display any other

observable behaviour

Animal walks, runs or swims from one

to another and searches a large area in

deliberate manner

Animal grabs source or grabs at source

with chelipeds or dactyls. Springs

downward touching the source or

springs forward toward source.

Source of chemical signal is moved to

mouth and ingested wholly or partially

= 100 – [10 x time (min) for

this activity].

=100 – [10 x time (min)

spent for all these activities]

= 100 – [10 x time (min)

spent for all these activities]

= 100 – [10 x time (min)

spent for feeding]

159

Table 5.2. Formulation of reference diet (as % dry matter basis)

Ingredient Fishmeal Gluten Dried squid Corn starch Vitamins & minerals1

Amount in diet %

57.9 5.0 5.0 30.0 2.0

Composition Dry matter %

Protein % Lipid % Energy MJ/kg

93.8 53.6 6.4 19.9

1Multi vitamins & minerals (per tablet) , Blackmores, Australia: Vitamin A, 2500IU;

B1, 30mg; B2, 30mg; Nicotinamide 50mg; B5, 31.5mg; B6, 30mg; B12 30mcg; C,

250mg; D3, 200IU; E, 50IU; H, 100mcg; K1, 5mcg; Folic acid, 400mcg;

Bioflavonoids, 50mg; Calcium phosphate, 40mg; Magnesium phosphate, 30mg; Zin

sulfate, 25mg; Potassium sulfate, 20mg; Ferrous fumarate, 15.3mg; Manganese

sulfate, 15.4mg; Cupric sulfate, 750mcg; Potassium iodide, 65mcg .

160

Table 5.3. Percentage of time crabs engaged in each arm of the maze under five

combinations between seawater (no food) and five test diets

Combination SW Ref. CM BT TN BE

1

2

3

4

5

9.1±1.6a

7.4±2.0a

9.1±2.3a

13.6±1.2a

14.0±2.2a

90.9±1.6b

92.6±2.0b

90.9±2.3b

86.4±1.2b

86.0±2.2b Values are means ± standard error (n=8 medium sized crabs). Means in the same row

with the same superscript are not significantly different (p>0.05).

161

Table 5.4. Percentage of time crabs engaged in each arm of the maze under four

combinations between reference (RF) and four test diets: bait enhancer (BE 2%),

betaine ( BT 2%), chicken meal (CM 2%) and Tuna oil (TN 2%)

Combination RF CM BT TN BE

1

2

3

4

38.8±2.4a

43.6±1.7c

48.2±1.7e

46.9±1.5f

61.2b

56.4±2.4d

51.8±1.7e

53.1±1.5f Values are means ± standard error (n=8 medium sized crabs). Means in the same row

with the same superscript are not significantly different (p>0.05).

162

Table 5.5. Percentage of time crabs spent on specific behaviour under sea water

(SW) and five test diets; reference diet (RF), bait enhancer (BE 2%), betaine ( BT

2%), chicken meal (CM 2%) and Tuna oil (TN 2%)

Attractant diets Response

SW RF BE BT CM TN

Resting 97.1±0.4a 48.5±1.3c 52.9±1.3b 45.4±1.3c 43.5±1.3c 52.1±1.3b

Locomotion 2.9±0.4a 19.5±1.0bc 17.3±0.1b 20.6±1.1c 22.1±1.1c 16.9±0.9b

Initiation 15.5±0.5b 18.8±0.6a 16.3±0.5b 16.7±0.6b 17.5±0.6ab

Continuation 16.5±1.1ab 11.0±0.9a 17.7±1.1b 17.7±1.1b 13.5±1.0a

Values are means ± standard error (n=8 medium sized crabs). Means in the same row

with the same superscript are not significantly different (p>0.05).

163

Table 5.6. Time spent of medium-sized crabs on specific behaviours at 26.5oC,

28.5oC and 30.5oC using 2% chicken meal diet

Temperature levels (oC) Behavioural

Response 26.5 28.5 30.5

Resting 61.6±0.6a 48.3±0.6b 47.0±0.6b

Locomotion 10.0±0.5a 19.4±0.6b 21.9±0.6c

Initiation 13.1±0.3a 14.3±0.3b 14.3±0.3b

Continuation 15.3±0.9 18.1±0.9 15.8±0.9

Values are means ± standard error (n=8 medium sized crabs). Means in the same row

with the same superscript are not significantly different (p>0.05).

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Table 5.7. Time spent of crabs on specific behaviours testing with equal amounts

of a 2% chicken meal diet placed in the two arms of the Y-maze under light and

dark environments

Time spent each half of the

Y-maze (%)

Food consumed (%) Test

crab

Shaded side Unshaded side Shaded side Unshaded side

Small 95.7±1.2a 4.3±0.4b 69.0±0.4a 10.0±0.4b

Medium 93.6±3.1a 6.4±1.1b 65.0±0.4a 13.8±0.3b

Large 93.7±2.2a 6.3±0.8b 68±0.3a 12.5±0.4b

Values are means ± standard error (n=4 medium sized female or male crabs in each

group). Means in the same column with the same superscript are not significantly

different (Tukey HSD, two - factors ANOVA, p>0.05).

165

Figure 5.1. The Y-maze chamber with (a) from the top side (plan) and (b) from

the horizontal side (elevation)

60o

150o

150o

1.2m

0.9m 0.36m

0.6m

0.6m

Removable Screen

Water Outlet

Water Inlet

Water Inlet

Tested Crab

0.5m 0.2m

Outlet pipe Drain line

The arm Inlet pipe

Removable screen

THE BASE SECTION THE ARM SECTION

166

ec

a

ec

a

fd

b

0

10

20

30

40

50

60

70

Resting Locomotion Initiation Continuation

Crab response

Tim

e sp

ent (

%)

LargeMediumSmall

Figure 5.2. Effect of size on crab response to diet containing 2% CM (mean ± SE,

n=8). For each specific behaviour, bars with different superscripts are

significantly different (p<0.05) from one another.

167

c

a

db db

0

5

10

15

20

25

30

RF BE BT CM TN

Test diets

Initi

atio

n re

spon

se (%

)

Small crabs

Medium crabs

Large crabs

Figure 5.3. Initiation response of different sized crabs exposed to diets containing

different attractants (2% inclusion level) (mean ± SE, n=8). Within specific diet

treatments bars with different superscripts are significantly different from one

another (p<0.05)

168

a b

0

10

20

30

40

50

60

Resting Locomotion Initiation Continuation

Crab response

Tim

e sp

ent (

%)

FemaleMale

Figure 5.4. Effect of sex on crab responses (mean ± SE, n=8 medium sized crabs).

For specific behavioural criteria, bars with different superscripts are

significantly different from one another (p<0.05)

169

bc

cc c

b

aaaa

0

10

20

30

40

50

60

70

80

RF BE2 BE5 BT2 BT5 CM2 CM5 TN2 TN5

Test diets

Food

cum

sum

ed (%

)

Figure 5.5. Effects of different attractants on food consumption using reference,

bait enhancer 2% (BE2), bait enhancer 5% (BE5), betaine 2% (BT2), betaine

5% (BT5), chicken meal 2% (CM2), chicken meal 5% (CM5), tuna oil 2% (TN2),

tuna oil 5% (TN5) (mean ± SD, n = 8 medium sized crabs). Bars with different

superscripts are significantly different from one another (p<0.05).

170

Chapter 6.

GENERAL DISCUSSION AND CONCLUSIONS

Discussions

As described in the introduction, mud crabs in the genus Scylla are highly

desired by humans as a food source and provide income for many coastal communities

in the Asia-Pacific region. With only limited potential for further expansion of wild

fisheries, aquaculture is now widely regarded as a key strategy to meet future demand

for mud crab product while also providing an opportunity to reduce pressure on wild

stocks. The viability of any mud crab aquaculture industry, however, will require the

availability of cost-effective feeds. In particular, if intensive culture systems are to be

utilised for mud crab production, provision of supplemental feeding using nutritionally

adequate diets will be essential (Bautista 1986). Traditional feeding practices

employed in mud crab aquaculture, such as the supply of trash fish, are now

considered unsustainable. In particular, the use of such inappropriate feeding practices

may significantly impact on maximum culture output by affecting the viability of

production due to unpredictable feed costs and availability. It is now widely

recognised that the future viability and expansion of mud crab aquaculture will depend

on development of low cost, but nutritionally adequate artificial diets (Fielder 2004).

For this reason, a central focus of the current thesis has been to examine the potential

of a broad range of readily available, low-cost feed ingredients for incorporation into

aquafeeds designed for Scylla species.

A key step in developing an appropriate supplemental feed for mud crabs is to

determine the feeding habits of the candidate animal and the required physical

171

characteristics of preferred feeds. Traditionally, mud crabs have been viewed as

carnivores that show a preference for natural diets that contain molluscs, crustaceans,

and fish (Hill 1979). Reports of significant amounts of plant-based material in the mud

crab digestive system (Hill 1976; Tacon & Akiyama 1997) led us to hypothesise that

plant-based feed ingredients may also have potential for inclusion in supplemental

diets formulated for this genus. Evidence to support this hypothesis has been obtained

by investigations into the digestive physiology of S. serrata. In particular, significant

activity for amylase and cellulase enzymes was reported from extracts prepared from

the mid gut gland (Pavasovic et al. 2004). Such findings raise the possibility that plant

based materials ingested by mud crabs may provide a source of essential nutrients

capable of supporting growth or metabolism. Based on this assumption, many of the

feed ingredients tested in the current thesis were derived from terrestrial plant sources

(e.g. soybean meal, wheat starch).

Macronutrients such as protein, lipid and carbohydrate are all potential sources

of dietary energy for aquatic organisms (Cho & Kaushik 1990; Wilson 1994). Of

these, carbohydrates are generally the cheapest sources and in some instances may

also offer the potential to spare some of the more expensive dietary protein (Stone

2003). The utilisation of carbohydrates as an energy source, however, varies

significantly among species (Wilson 1994). As a consequence, a key element of the

current investigations was to assess the capacity of two different Scylla species to

utilise carbohydrate-rich plant-based ingredients such as wheat starch.

Providing cost-effective diets to achieve optimum growth in crustaceans will

rely on developing an understanding of the nutritional requirements of each candidate

species and the nutrient specifications that must exist in diets designed to meet these

172

nutritional requirements. The nutritional requirements of crustaceans vary

considerably among species. One of the most obvious differences is that carnivorous

crustaceans such as shrimps and lobsters typically require more protein (>40%) in

their diets than do omnivores such as many freshwater crayfish. Mud crabs have been

viewed traditionally as carnivores and therefore assumed to have a relatively high

dietary protein requirement. This assumption is supported by analysis of the digestive

enzyme profile of mud crabs (Pavasovic et al. 2004). Specifically, protease activity

levels in extracts from the mud crab digestive system are significantly higher than that

of other omnivorous crustacean species, including the redclaw crayfish Cherax

quadricarinatus (Pavasovic et al. 2006). Nevertheless, the requirement for protein in

mud crab diets does not appear to be as stringent as those reported for some penaeid

species. Typically, many artificial diets developed for penaeid aquaculture contain

between 50%-60% protein (Teshima & Kanazawa 1984) and such diets have been

used successfully to support mud crab grow out (Mann & Paterson 2004). Elsewhere,

it has been shown however, that mud crabs can grow well when fed diets containing

only 33%-40% protein (Catacutan 2002). This thesis has confirmed a requirement for

protein that is substantially less than that commonly included in many previous

feeding strategies used in mud crab aquaculture. In particular, we demonstrated that

good growth can be obtained with 45% crude protein, regardless of the energy level of

the diet (Chapter 2). We therefore suggest that a significant potential exists to lower

mud crab aquaculture production costs through the development of artificial diets that

incorporate substantially less protein than is currently contained in many of the

penaeid feeds used by this culture industry.

173

Ingredients based on terrestrial animal or plant sources that contain high levels

of nutrients and are digestible to target species are often targeted for their potential to

replace higher cost ingredients in aquafeeds, such as fishmeal. An important finding in

this thesis was that S. serrata demonstrated the capacity to digest a wide range of feed

meals derived from terrestrial animal, plant or single cell-based sources (Chapter 3).

This capacity is not surprising considering that other workers have demonstrated

significant levels of protease amylase, cellulase and xylanase activity in soluble

extracts from the mid gut gland of mud crabs (Pavasovic et al. 2004). High

digestibility coefficients for a wide variety of animal and plant-based ingredients in

mud crab diets were also obtained in studies by Catacutan et al. (2003) and Tuan et al.

(2006). Based on such data, we predict that significant economic gains may be

achieved by inclusion of cheap plant-based materials in artificial mud crab diets. In

particular, digestible ingredients containing high levels of protein, such as soybean

meal, may decrease dependence on more expensive marine animal meals such as

fishmeal.

In chapter 2, experiments were also conducted where purified starches from a

variety of plant sources (wheat, corn, rice, and potato starch) were used to manipulate

the energy level of experimental crab diets. A major finding was that the amount or

source of starch included in diets had no significant impact on relative growth

performance of juvenile mud crabs and that the level of dietary protein, and not starch,

was the main factor that affected growth. This is despite the fact that S. serrata

demonstrated a high capacity to digest starch from a variety of sources (e.g. wheat,

rice and corn). These results strongly suggest that an increase in digestible energy

from carbohydrates does not translate into increased growth performance. This

174

interpretation is supported by the linear correlation between protein intake and weight

gain and by a lack of correlation between weight gain and both non-protein energy

intake and starch intake. Based on these findings we propose that the use of plant-

based ingredients to promote growth in mud crab aquafeeds is likely to be restricted to

those ingredients with a relatively high protein, and not necessarily carbohydrate,

content.

As discussed previously, the findings of the current thesis confirmed that the

inclusion of starch in formulated mud crab diets did not promote increased growth.

Nevertheless, for any given level of dietary protein, the inclusion of relatively high

levels of starch did not appear to impact negatively on overall culture performance. As

a consequence, we propose that further consideration be given to the potential of

starch as a low cost energy source, binder or bulk filler ingredient for formulated mud

crab diets.

A surprising finding of the current studies was that the uncooked diet with

45% wheat starch was digested more efficiently than was a cooked diet (Chapter 3).

This contradicts the result of Stone et al. (2003) who reported that gelatinised wheat

starch was digested more efficiently than was raw wheat starch by silver perch. This

apparent discrepancy may be due differences in the digestible enzyme profiles of these

two species and requires further analysis to be resolved. While digestibility studies

play an important role in investigating the initial phase of carbohydrate utilisation by

fish, it must be emphasised that efficient digestibility of an ingredient does not

necessarily equate to efficient utilisation of the carbohydrate for growth (Stone 2003).

Hutchins et al. (1998), for example, observed a tendency for a reduction in weight

gain, feed efficiency and protein efficiency in sunshine bass that corresponded with a

175

reduction in carbohydrate complexity. Stone et al. (2003) also observed a similar trend

for silver perch. Based on these findings, we suggest that further studies should be

conducted to determine to what extent the capacity of mud crabs to digest and to

utilise carbohydrates is dependant on carbohydrate type incorporated into feed

formulations or the diet manufacture process.

As discussed previously, a key aim of this thesis was to determine the capacity

of mud crabs to digest a range of feed ingredient derived from terrestrial plant sources.

Such data is essential before any plant-based ingredient can be considered for

incorporation into an aquafeed. In particular, it has been shown that the presence of

non-starch polysaccharides in some plant-based feeds may reduce ingredient

digestibility. For example, Olli et al. (1994) reported that plant meals can contain anti-

nutritional factors such as protease inhibitors which limit their potential use in

aquafeeds. Moreover, the capacity to digest plant-based ingredients may vary among

species within a genus and the origin of ingredients. For this reason, the capacity of a

second mud crab species, S. paramamosain, to digest several plant-based ingredients

readily available in Vietnam was tested (Chapter 4). It was shown that, as for S.

serrata, this species generally showed a high capacity to plant-based products such as

soybean meal and rice bran. Similar results were achieved in the current project

(Chapter 3) and in other studies using S. serrata (Catacutan et al. 2003; Tuan et al.

2006). An unexpected result of the current investigations was that corn flour-based

diets for S. paramamosain yielded lower ADC values for dry matter and protein than

for other plant-based ingredients (Chapter 4). This finding appears to contradict

studies that have reported high ADC coefficients for corn-based ingredients in diets

formulated for S. serrata (Catacutan et al. 2003; Chapter 3). The basis for these

176

differences are currently unclear, although they may reflect differences in the digestive

capacities of different mud crab species or the type of corn-based ingredient included

in the diet. Nevertheless, the findings presented here indicate that care must be taken

not to assume that data relating to optimal diet specification obtained from examining

a particular species of mud crab can be automatically applied to all species.

Findings presented in this thesis provided a significant contribution to the

overall understanding of the relationships between diet compositions, environmental

conditions and feeding efficiency in Scylla species. Optimising feeding efficiency is

essential for promoting consumption of the least amounts of nutrients in the minimum

time while also reducing wastes. By this definition aquafeeds should not only contain

highly digestible nutrients but they should be attractive to the target species. The

importance of chemo-attractants and/or feeding stimulants in improving both initial

palatability and overall feeding rate as a means to reduce wasted feed is now fully

recognized (Lee & Meyers 1996). Information on use of attractants in diets for Scylla

crabs is limited, but there have been a number of studies completed on foraging

behaviour in other portunid swimming crabs and their responses to odour plumes,

particularly Callinectes sapidus (Weissberg & Zimmer-Faust 1994; Clark et al. 2000;

Diaz et al. 2003). The findings from the current study showed that behaviour of mud

crabs significantly changed towards diets containing different attractants (Chapter 5).

In particular, chicken meal and betaine showed good potential as attractants for

inclusion in mud crab diets. The potential for poultry-based materials as attractants in

crab diets has also been reported by Middleton et al. (2000) who demonstrated that

poultry caracasses performed well as bait for harvesting blue crabs (Callinectes

sapidus). An important question raised in application of attractants to diet is what the

177

best concentration that should be included in a particular diet, since some attractants

are very expensive or may occupy other important nutrient components in the

formulated diet. In the current study, an increase in dose of the best attractant from 2%

to 5% into diets did not promote any increase in feed consumption. Based on these

findings we suggest that the inclusion of relatively small amounts of attractants in mud

crab diets may be adequate to yield significant benefits in terms of improved feeding

efficiency and lower cost.

It has been reported that environmental factors such as temperature, light

intensity may also influence chemosensitivity as does background noise and odour

complexity (Atema 1987; Zimmer-Faust et al. 1996). In the current study, temperature

was investigated and shown to be a key factor that affecting crab behaviour. In

particular, significantly reduced responses in most non-resting behaviours were

observed at lower temperature in comparison with crabs tested at higher temperature.

These findings are not surprising, because crabs are found commonly in tropical

environments (Keenan 1999; Sugama & Hutapea 1999). Perhaps most importantly,

data presented here lead to the conclusion that any benefits gained from optimising

diet formulation may be severely reduced if feeding behaviours are negatively

impacted by use of sub-optimal temperatures in culture systems.

Crab size is also a factor that appears to affect crab behaviours. The current

study showed a difference in behaviors between small and large crabs, since small

crabs displayed less resting time and more time in locomotion (e.g. walking, running,

swimming), and initiation (e.g. grabbing, lunging, pouncing) comparison with medium

and large crabs. Smaller individuals may be driven by physiological mechanisms to be

more active or perhaps the dimensions of the maze used here inhibited locomotion by

178

larger individuals. Wall (2005) studied sexual behaviour in crabs and reported that the

females displayed significantly more tactile investigation than did males. Hayden et

al. (2007) demonstrated that female of shore crabs (Carcinus maenas) exhibit stronger

feeding responses than do males throughout the year. Here we reported that female

mud crabs performed slightly more locomotion activity than do males in the

temperature experiment (27.5oC). This may be due to physiological mechanisms and/

or because medium and large sized females crabs are becoming sexually mature. Polis

(1981) noted that females were in general more cannibalistic than males. This suggests

that where female monosex culture is applied to crabs, potent feed attractants like

chicken meal or betaine could be trialled to reduce cannibalism of soft post-moult

female crabs (Hayden et al. 2007). Overall, the above findings emphasise that

developing a clear understanding of the behavioural responses of mud crabs of

different ages and sexes in specific culture environments will be essential to fully

exploit the potential of improved cost-effective artificial feed that accurately meet the

dietary demands of Scylla species.

179

Conclusions

This thesis has confirmed that mud crabs are efficient at digesting a wide range

of plant-based ingredients. The botanical origin of feed ingredients and starch, feed

ingredient and starch inclusion, and processing of starch, all had significant effects on

starch digestibility and should be considered when formulating diets with ingredients

rich in plant-based carbohydrates. Ultimately, inclusion of cheap plant-based

ingredients should reduce diet costs and in some instances may also mitigate reliance

on expensive protein sources such as fish meal thereby aiding the future sustainable

culture of Scylla species. Understanding the interaction between diet, feeding

efficiency and culture environment is also essential in developing mud crab culture

practises that maximise productivity.

In conclusion, the major findings of this study were:

1. Mud crabs grew well on diets containing 45% crude protein, regardless of the

energy level of the diet (15.5 MJ kg-1 or 18 MJ kg-1).

2. There is no evidence that the source or level of starch had any significant impact on

relative growth performance or feed utilisation (at an inclusion of 34% wheat, rice,

corn and potato starch in diets; protein 40%).

3. Scylla serrata digested well a wide range of plant-based feed meals or single cells

including soy bean meal, cotton meal, lupin meal, canola meal, rice bran and yeast.

4. Scylla serrata is efficient at digesting carbohydrates from corn, wheat, rice, and

potato when included in the diet at 30%. The ASD of diets containing different

starches decreased in the order corn > wheat > potato = rice.

180

5. Wheat starch in an uncooked diet was digested more efficiently than an equivalent

cooked diet.

6. Scylla paramamosain could digest diets containing soybean meal and rice bran

obtained in Vietnam effectively. By contrast, cassava meal appeared inappropriate as a

feed ingredient for this species.

7. Feeding behaviours of mud crabs were significantly changed in response to

different attractants added to diets. In particular, chicken meal and betaine showed

promise as potential attractants for formulated mud crab diets. Age, sex, light and

culture temperature were also shown to have major impacts on crab feeding behaviour.

181

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