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Queensland University of Technology
School of Natural Resource Sciences
FACTORS AFFECTING REPRODUCTIVE PERFORMANCE OF THE PRAWN,
Penaeus monodon
Gay Marsden
Submitted in fulfilment of the requirements for
the degree of Doctor of Philosophy
2008
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Statement of original authorship
The work contained in this thesis has not been previously submitted to meet the
requirement for an award at this or any other higher education institution. To the best of
my knowledge and belief, the thesis contains no material previously published or written
by another person except where due reference is made.
Signature…………………………………….. Date…………………......................................
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Acknowledgments
In terms of facilities I would like to acknowledge the extensive support of the Bribie
Island Aquaculture Research Centre (BIARC), Queensland DPI&F. Funding for the
research was gratefully received from FRDC and QUT. For valued friendship and
technical support I am indebted to the BIARC staff and in particular Michael Burke.
Valued statistical advice was given by David Mayer (DPI&F) and biochemical analysis
was carried out by Ian Brock (DPI&F). Thanks also to: fellow student Phil Brady for his
encouragement throughout all phases of the research and for his passion and willingness
to partake in lengthy discussions on prawn reproduction; Peter Duncan for his kindness
and patience while I made use of his kitchen table during the final stages; and to my three
supervisors Dr Neil Richardson, Associate Professor Peter Mather and Dr Wayne Knibb
for their unique contributions. Neils’ efforts to keep me on track deserve a medal. Lastly,
thanks to my family for their understanding and financial support, particularly Ian
Neilsen who in many ways provided the window of opportunity I needed to undertake
this challenge.
Keywords
Penaeus monodon, prawn reproduction, ovary, eggs, hepatopancreas, mating, methyl
farnesoate, ablation, captivity, sinus gland hormones, fatty acids, lipid, protein.
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TABLE OF CONTENTS
Chapter 1.
INTRODUCTION...........................................................................................................10
Chapter 2.
LITERATURE REVIEW
2.1 Aquaculture…………………………………………………………………………14
2.1.1 History of aquaculture systems…………………………………………...15
2.1.2 Animal species cultured in aquaculture…………………………...17
2.1.2.1 Prawn aquaculture……………………………………………….18
2. 2 Penaeus monodon………………………………………………………………21
2.2.1 Global production of Penaeus monodon…………………………………..21
2.2.2 Penaeus monodon farming in Australia……………………...……………22
2.2.3 Domestication of P. monodon……………………………………………..23
2.2.4 Life cycle and reproductive biology of P. monodon ………………………25
2.3 Ovary development and endocrine regulation……………………………………….27
2.3.1 Accumulation of nutrient reserves in the oocytes of penaeid prawns……..27
2.3.1.1 The process of vitellogenesis…………………………………….28
2.3.1.2 Cortical Rod formation…………………………………………..31
2.3.1.3 Patterns of nutrient fluctuation in hepatopancreas and ovaries….33
2.3.2 Endocrine regulation of reproduction in crustaceans………………………35
2.3.2.1 The CHH family of hormones…………………………………...37
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2.3.2.2 The Roles of Methyl Farnesoate (MF) in prawn reproduction….40
2.3.3 Endocrine manipulation strategies employed in prawn aquaculture……….42
2.3.3.1 Eyestalk ablation…………………………………………………43
2.4 Mating Behaviour of Penaeid Species……………………………………………….45
2.4.1 Mating strategies of crustaceans……………………………………………….46
2.4.2 Mating strategies of closed and open thelycum species of penaeids…………..51
2.4.3 Mating behaviour of P. monodon ....... ..........................................................52
2.4.4 Mating in captive-bred prawns including P. monodon ………………… .... …52
2.5. Summary…………………………………………………………………………….56
2.6. Project hypothesis and aims…………………………………………………………59
Chapter 3.
GENERAL METHODS
3.1 Prawns………………………………………………………………………………..62
3.1.1 Location……………………………………………………………………62
3.1.2 Capture method…………………………………………………………….62
3.1.3 Transport method………………………………………………………..…63
3.1.4 Arrival and acclimation…………………………………………………….64
3.1.5 Holding tanks………………………………………………………………64
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3.1.6 Ablation…………………………………………………………………….65
3.1.7 Feeding……………………………………………………………………..65
3.2 Tissue, egg and larval collection, classification and biochemical analysis………….66
3.2.1 Tissue collection……………………………………………………………66
3.2.2 Classification of tissues using gonad somatic index (GSI) and hepatopancreas
somatic index (HSI)………………………………………………………………66
3.2.3 Classification of ovary developmental stage using histology……………….67
3.2.4 Biochemical analysis………………………………………………………..67
Chapter 4.
THE EFFECTS OF CAPTIVITY AND ABLATION ON PROTEIN, LIPID AND
DRY MATTER CONTENT OF OVARY AND HEPATOPANCREAS TISSUES IN
THE PRAWN PENAEUS MONODON.
4.0 Abstract……………………………………………………………………………….69
4.1 Introduction……………………………………………………………………………70
4.2 Methods……………………………………………………………………………….73
4.2.1 Prawns……………………………………………………………………...73
4.2.2 Holding Conditions for Captive Prawns……………………………………73
4.2.3 Statistical analysis…………………………………………………………..75
4.3 Results..………………………………………………………………………….........76
4.3.1 GSI and Biochemical Analysis……………………………………………..76
4.3.2 Histology…………………………………………………………………...78
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4.4 Discussion………………………………………………………………………….…83
Chapter 5.
THE EFFECTS OF ABLATION AND STARVATION OF THE PRAWN PENAEUS
MONODON ON PROTEIN AND LIPID CONTENT IN OVARY AND
HEPATOPANCREAS TISSUES.
5.0 Abstract……………………………………………………………………………….89
5.1 Introduction…………………………………………………………………………...91
5.2 Methods……………………………………………………………………………….93
5.2.1 Prawns………………………………………………………………………93
5.2.2 Holding conditions and experimental design……………………………….93
5.2.3 Statistical Analysis…………………………………………………………94
5.3 Results………………………………………………………………………………..95
5.4 Discussion…………………………………………………………………………...98
Chapter 6.
METHYL FARNESOATE AS A POTENTIAL HORMONE FOR STIMULATING
OVARY DEVELOPMENT AND INCREASING EGG HATCH RATE IN THE
BLACK TIGER PRAWN, PENAEUS MONODON
6.0 Abstract..……………………………………………………………………………102
6.1 Introduction…………………………………………………………………………104
6.2 Methods……………………………………………………………………………..107
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6.2.1. Prawns and holding conditions…………………………………………..107
6.2.2 Diets………………………………………………………………………108
6.2.3. Statistical analysis………………………………………………………..109
6.3 Results……………………………………………………………………..……… 110
6.4 Discussion……………………………………………………………...…………..115
Chapter 7.
THE IMPACT OF CAPTIVITY AND ABLATION ON LIPID AND FATTY ACID
PROFILES OF PENAEUS MONODON EGGS AND EARLY LARVAL STAGES
7.0 Abstract………………………………………………………………….………….121
7.1 Introduction……………………………………………………………….……..….123
7.2 Materials and methods…………………………………………………………..….126
7.2.1 Prawns……………………………………………………………….……126
7.2.2 Egg and larval collection and processing………………………………...126
7.2.3 Biochemical analysis……………………………………………………..127
7.2.4 Statistical analysis…………………………………………………….…..129
7.3 Results………………………………………………………………………………130
7.4 Discussion…………………………………………………………………………..138
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Chapter 8.
REPRODUCTIVE BEHAVIOURAL DIFFERENCES BETWEEN WILD
CAUGHT AND POND REARED PENAEUS MONODON PRAWN
BROODSTOCK.
8.0 Abstract…………………………………………………………………………….144
8.1 Introduction………………………………………………………………………..145
8.2 Methods……………………………………………………………………………148
8.2.1 Experimental prawns……………………………………………………..148
8.2.2 Holding facilities…………………………………………………………149
8.2.3 Observation tanks……………………………………………………… 149
8.2.4 Observations……………………………………………………………...150
8.2.4.1 Behaviour classification………………………………………………...150
8.2.5 Statistical analysis………………………………………………………...150
8.3 Results………………………………………………………………………………152
8.4 Discussion…………………………………………………………………………..159
Chapter 9.
GENERAL DISCUSSION AND CONCLUSIONS…………………………………164
Chapter 10.
REFERENCES..............................................................................................................171
PUBLICATIONS……………………………………………………………………...211
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Chapter 1
INTRODUCTION
Prawn farming is now one of the largest aquaculture sectors by volume in Australia after
Tuna and Salmon. The main species cultured is the black (or giant) tiger prawn Penaeus
monodon which until recently also dominated world prawn aquaculture production
(ABARE 2007). The recent decline in production has been dramatic. For example, in
2003 P. monodon accounted for 50% of farmed prawns in Thailand but by 2007 this had
dropped to only 5%. While in much of South East Asia it has been replaced by an
imported species (P. vannamei), P. monodon continues to demand relatively high market
prices and remains the aquaculture species of choice in many countries, including
Australia (FAO 2007).
Much of the decline in global production of P. monodon can be attributed to disease
outbreaks, including viruses originating from the wild-caught spawners. Similar viruses
are already limiting the expansion of the Australian industry (Cowley 2005, Lobegeiger
and Winfield 2008). Thus the industry reliance, both in Australia and overseas, on
broodstock captured from the wild is seen as a major impediment to the continued large-
scale production of P. monodon. In Australia there is also evidence that relying on
broodstock from the wild has limited industry expansion because of the variability in
quality and quantity of its supply (Hansford and Marsden 1995, Marsden et al 1997). For
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example, in 2000 a major shortage of good-quality wild broodstock severely reduced
annual production of this species in Australia (Lobegeiger et al 2005).
Proposed solutions to the above problems include the domestication of P. monodon
enabling the rearing of successive generations in captivity with known reproductive
performance and specific pathogen free (SPF) status. It has been shown with other
species that controlled breeding programs can allow supply to be scheduled to meet
demand and that the risk of viral infections can be reduced (Argue et al 2002, Fjalestad et
al 1993). As a consequence, a major goal of the prawn aquaculture industry, both in
Australia and overseas, is to close the life cycle of this species on a commercial scale and
supply high quality, genetically improved, specific pathogen-free (SPF) larvae for
commercial growout (FAO 2007).
Despite the dedication of considerable resources to reach this goal by both industry and
research organisations there is still limited commercial availability and use of
domesticated broodstock (Coman 2007). To date, hatchery trials using domesticated
stocks indicate that these stocks are less responsive to induced spawing and egg hatch
rates are low compared to their wild counterparts (Kenway pers com 2007). Both
domesticated and wild-caught females held in captivity require unilateral eyestalk
ablation (a crude method of hormonal manipulation) to induce ovary development and
spawning. This indicates that the captive environment is in some way preventing
spontaneous reproduction. While ablation enables some control over larval production
this industry practise is not always successful and it can also result in a decrease in egg
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and larvae quality if multiple spawnings from ablated females are required (Har 1991,
Marsden et al 1997). Thus there is a need to further understand and control factors
affecting egg quality and spawning in P. monodon if the Australian prawn farming
industry, which heavily dependent on this species, is to enjoy stability of broodstock
supply and industry growth.
To this end, the research carried out and presented in this thesis was aimed at better
understanding key physiological processes in P. monodon broodstock that relate to both
quality and quantity of eggs produced. After reviewing the extensive body of work
related to prawn (shrimp) reproduction, research in the thesis was directed towards (i)
determining the levels of specific nutrients accumulated during ovary development and
the impact of industry protocols (including ablation) on this process. (ii) further
understanding the hormones involved in ovary development and spawning, and (iii)
determining if abnormal mating behaviour is a factor contributing to the low hatch rate of
eggs from captive-bred broodstock.
The research investigating patterns of nutrient accumulation associated with ovary
development was considered particularly important as these nutrients must meet all the
needs of the developing eggs and early lecitotrophic larvae (nauplii). Most significantly
there is an increasing body of evidence indicating that nutrients accumulated at various
stages of ovary development play specific roles during egg and larval development (for
example Yamano et al 2003, 2004). Thus, the patterns of accumulation could be
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significant to egg quality and changes to these patterns due to existing industry protocols
need to be identified.
It was also proposed that the pattern of nutrient accumulation would provide an insight
into how the endocrine system, which may be species specific, is operating in P.
monodon. In particular, assessing the effect of ablation on nutrient levels in the ovary and
hepatopancreas (an important storage tissue involved in ovary development) could help
clarify the involvement of inhibitory (most notably the sinus gland peptides) and the
existence of any stimulatory hormones. This is important if improved methods of
inducing ovary development, particularly in domesticated females, are to be developed
and spawning frequency, and therefore egg production, improved.
The final experimental chapter then considered the extent to which unsuccessful mating
was contributing to the poor egg quality in terms of low fertility and hence hatch rates,
typifying naturally mated domesticated P. monodon broodstock. Hatch rates have been
shown to improve when artificial insemination (AI) is used instead of natural matings.
This suggests that there are factors, other than egg integrity and sperm quality,
contributing to the low hatch rates of domesticated stock. Thus, the aims of this section of
the project were to determine if the mating behaviour of the domesticated broodstock
deviates substantially from that of the wild-caught broodstock and, if so, to determine at
what stage this occurs and whether it is due to the male and/or female. This study was
intended to confirm whether mating is contributing to low egg hatch rates and give an
indication of underlying causes.
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Chapter 2.
LITERATURE REVIEW
Traditionally, capture fisheries have been a major source of food for the human
population. Over recent decades, however, increased fishing pressure has severely
depleted this limited resource. Even with the pursuit of new species for exploitation,
global production from wild capture fisheries has over the ten years between 1996 and
2006 decreased from 93.5 million tonnes to 92.0 (FAO 2007). As a consequence, the
farming of aquatic organisms, or aquaculture, has become increasingly important in
many parts of the world to guarantee food security for expanding populations.
Thus aquaculture is expanding due to declines in wild catches of certain stocks (even
though total volume is stable) and increased human populations. It is also increasing in
terms of per capita consumption in response to increased wealth and increased valuing of
seafood as health food.
2.1 Aquaculture
Aquaculture can be defined as the farming of aquatic organisms including fish,
molluscs, aquatic plants and crustaceans (FAO 2003). Most product is for human or
animal consumption with some exceptions such as pearl oysters and aquarium fish. As a
food industry there is a general consensus that aquaculture is of importance, not only for
its increasing production of high value species, but also for its capacity to supply an
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affordable protein source in developing nations. Additionally, it has the potential to
relieve pressure on the natural environment by reducing reliance on wild fishery stocks.
On a global scale it is an industry that can offer a long-term capacity to meet an
increasing demand for aquatic product.
Currently, aquaculture makes a significant contribution to the total tonnes and value of
seafood consumed worldwide, including both fresh water, brackish and marine produce.
Worth US$56.5 thousand million in 2001, it is one of the fastest growing food producing
industries in the world with an average growth rate of 9.2% since the early 1970s (Talcon
2003). Significantly, by 2006 aquaculture was contributing 36.0% by weight of the total
seafood produced from aquaculture and capture fisheries (FAO 2009). Approximately
91% of global aquaculture production comes from Asia and Pacific with China estimated
to produce 70%.
2.1.1 History of aquaculture systems
Farming involves some form of intervention in the growing, reproduction, rearing or
fattening of cultivated species. Currently, the farming of aquatic organisms is extremely
diverse in terms of the species cultured, the systems used and its geographic distribution.
The first recorded evidence of aquaculture dates back to over 4000 years ago in China
with the trapping of carp in rice paddy fields. Methods were developed largely by trial
and error and passed down between generations of farmers. It is only since the 1930’s
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that rigorous scientific investigations seeking to develop production technologies and
candidate species has been applied to aquaculture. As a consequence, when compared to
many terrestrial types of primary production, aquaculture can be viewed as a relatively
young industry in terms of technical advancement.
Broadly speaking the systems used in aquaculture fall into three groups based on the
degree of control over the processes involved:
• Group 1 represents systems where there is control of the animal’s movement but
no control over the water flow and is seen in practices which utilise cage culture
and netted tidal areas;
• Group 2 has some additional control over water flow such as occurs with pond and
raceway culture; and
• Group 3 has complete control of water flow and quality as observed with the use
of recirculation systems (AQUAVETPLAN 2001)
Each of these systems can be operated at levels ranging from low maintenance-low input
to high maintenance-high input. Typically with high input systems there is a high density
of animals and subsequent need for additional aeration and artificial feed to meet the total
requirements of the animal. Economics and the biology of the animals dictate which
system and intensity of farming is appropriate for a particular species.
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Biology and economics also determines the structure of each species-specific industry.
For a number of species the production cycle begins with broodstock and a hatchery
phase followed by a growout phase. This structure offers a high level of control over
production. If broodstock are domesticated there are the added advantages of predictable
egg/larvae supplies and of genetic selection for improved production. Domestication, also
called ‘closing the life cycle’ has been paramount to the success of traditional terrestrial
farming such as for cattle, poultry and pigs, and is proving to be the case for many
species in aquaculture.
2.1.2 Animal species cultured in aquaculture
In 2000, there were over 210 aquatic species being cultured worldwide (Talcon 2003).
This diversity reflects the range of species available in different countries and the wide
variety of systems used. However, for a species to be commercially viable it needs to
meet a number of biological and, ultimately, economic criteria. These criteria include:
• Potential or established market;
• Capacity to be confined in culture systems;
• High growth and survival rates;
• Low production costs;
• Acceptance of artificial diets;
• Low protein requirement;
• Low incidence of disease;
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• Availability of broodstock or fry; and
• Ability to be domesticated.
Although few species meet all these criteria, it is anticipated that the development of low
cost diets and culture systems together with genetic selection to improve survival and
growth rates, should allow increasing numbers of aquatic organisms to be commercially
viable.
In recent times, aquatic crustaceans including marine and freshwater prawns, lobsters,
crabs and crayfish have become important aquaculture commodities. Specifically, in
2000 global aquaculture production of crustaceans was estimated to be 1.65 million
tonnes. While this was only 3.6% by weight of the total global aquaculture production,
crustaceans comprised 16.6% by value, estimated to be worth about US$9.37 thousand
million (Talcon 2003). In particular most crustaceans, particularly marine species, remain
high value species and are considered to be a luxury food item.
2.1.2.1 Prawn aquaculture
Prawns (or shrimps, as they are referred to in some parts of the world) are one of the most
important groups of crustaceans for aquaculture in terms of total production and value. In
particular, marine prawn culture has grown into one of the largest and most important
crustacean aquaculture crops worldwide, the significance of which is reflected in
production increases of 250% from 2000 to 2006 (FAO 2009). Furthermore, between
2002 and 2004 prawns showed the biggest (approximately 30%) increases in global
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production when compared with other aquaculture species (FAO 2006). In recent years it
has been estimated that cultured prawns represent 66% of the total weight of crustacean
aquaculture production (Talcon 2003). In 2004 Rosenberry (2004) reported that the
production of cultured prawns had reached levels equivalent to the capture fisheries, with
each industry producing an estimated 2 million tonnes per annum. Aquaculture and
capture fishery prawn production levels have continued to grow at a similar rate such that
by 2006 each was producing just over 3 million tonnes annually (FAO 2009).
The prawn aquaculture industry is primarily a land-based culture system comprised of
earthen ponds located in areas with access to brackish water. Commercial aquaculture
species are subtropical or tropical so most farms are restricted to these climatic zones.
Asia, particularly China and Thailand, are responsible for 75% of production. Latin
America, in particular Brazil, accounts for the other 25%.
Globally the black tiger prawn (Penaeus monodon) and the Pacific white shrimp (P.
[Litopenaeus] vannamei) account for over 80% of production (Talcon 2003). Annual
growth of the industry was 25% in the 1980s, slowed to between 5 and 10% during the
1990s (Globefish 2004, Rosenberry 2004, Talcon 2003) and increased again to an
average of 43% per annum between 2000 and 2006 (FAO 2009). This pattern reflects the
disease issues that plagued the P.monodon industry and the subsequent replacement
during the early 2000s of P. monodon with P. vannamei. A large part of the success of P.
vannamei as a cultured species was its readiness to breed in captivity and the resultant
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production of ‘specific pathogen free’ (SPF) broodstock which reduced the introduction
of diseases into the culture systems.
Although prawns represent only 7.7% by weight of Australia’s total aquaculture
production, it is a highly significant industry by value. Specifically, the Australian prawn
aquaculture industry (A$57 million) ranks fourth after tuna (A$255.6 million), pearl
oysters (A$175 million) and salmon (A$109 million) (ABARE 2003).
There are three species of prawn that are cultured commercially in Australia. P.
monodon is the main species accounting for most of the 3403 tonnes produced in 2002-3.
The banana prawn Fenneropenaeus merguiensis and the kuruma prawn (P. japonicus) are
also cultured commercially however, the production of F. merguiensis is limited to only
one farm and a few ponds on one or two P. monodon farms. Only 95 tonnes of kuruma
prawns were produced in 2002-3 (Lobegeiger and Wingfield 2004).
Production by Australian prawn farms has decreased by 6% from 3300 tonnes in 2005–
06 to 3085 tonnes in 2006–07. With the decrease in production and no increase in unit
price, the value of this sector has similarly decreased by 8% from $46.3 million in
2005–06 to $42.5 million in 2006–07(Lobegeiger and Wingfield 2008).
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2. 2 Penaeus monodon
Penaeus monodon is one of the largest penaeid species in the world, with females
reaching up to 336 mm in body length (Gey et al 1983). Also called the giant tiger or
black tiger, it is eurythermal and euryhaline for most of its life, and is known for its rapid
growth rate (Motoh 1984).
2.2.1 Global production of Penaeus monodon
Up until 2000 Penaeus monodon was the main prawn species cultured world wide (FAO
2009), and was the highest in terms of value, of all aquaculture species (Tacon 2003). P.
monodon was therefore an extremely significant aquaculture species. By 2006 P.
vannamei had replaced P. monodon as the highest value aquaculture species (FAO 2009)
with annual global production value nearly twice that of P.monodon.
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Despite being the main prawn species cultured, global production of P. monodon peaked
at 730 404 MT in 2003 and declined to 658 222 MT by 2006 (FAO 2009). This decrease
was primarily due to disease problems that have forced growers to culture alternative
species, for example P. vannamei. This trend has seen the world production of P.
vannamei increase from 481 298 MT in 2002 to 2 133 381 MT in 2006; over three times
the volume of P. monodon produced (FAO 2009). Despite the popularity of P. vannamei,
P. monodon remains the species of choice as it commands a higher price. For example in
2006 P.monodon was priced at US$ 4.70 per kilo compared to P.vannamei at US$ 3.60
per kilo. However growers remain reluctant to grow P.monodon because of their disease
issues.
2.2.2 Penaeus monodon farming in Australia
Australia’s prawn farming industry started in the mid 1980s with P. monodon the species
of choice after failed attempts to culture other local species. This species is endemic to
South East Asia including Australia. It therefore satisfied one essential selection criteria,
specifically, that exotic species cannot be imported live into Australia. Another big
advantage of choosing P. monodon was that it was already being cultured in other parts
of the world. Technology and feed for the culture of this species was imported directly
from Asia enabling the Australian industry to expand rapidly.
The industry continued to expand until recent years. In terms of increasing production,
several challenges now face the industry. One challenge is to overcome the issue of
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disease. By the mid 1990s the Australian industry began to suffer disease problems,
primarily viral. Despite the industry’s pro-active approach and the benefit of lessons
learnt from other countries, there has been a noted drop in production over recent years
largely due to disease problems. For example, in New South Wales the value of P.
monodon production dropped from 4.5 million dollars in 2003/2004 to 2.5 million dollars
in 2006/2007 (Wiseman 2007). Likewise, in Queensland, where most of the industry is
concentrated, production decreased from 3255 to 2861 tonnes (over 12%) from 2001/02
to 2002/3 due mainly to a virus known as GAV (Lobergeiger and Wingfield 2004).
Diseases found in Australian aquaculture prawns, including viruses, are already present in
local wild populations (Owens 1997).
A second major challenge, which is also part of the disease prevention strategy, is the
domestication of P. monodon. In an attempt to meet this objective, significant research is
now being directed towards ‘closing the life cycle’ and the subsequent implementation of
genetic selection programs for this species. It is anticipated that data from these studies
may hold the key to improved productivity and a more economically and environmentally
sustainable industry.
2.2.3 Domestication of P. monodon
Advances in production of most farmed animal species are directly related to the ability
to domesticate these animals. The advantages of domestication include:
• Removal of reliance on wild caught stock;
• The ability to improve disease control programs; and
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• The capacity for genetic improvements through selective breeding
Penaeus monodon broodstock captured from the wild vary on a temporal/seasonal basis
in terms of quality and quantity (Hansford and Marsden 1995). Reproductive
performance has also been shown to vary in size and with source of wild caught prawns
(Menasveta et al 1994). With the current industry structure, hatcheries place orders with
a limited number of specialist broodstock collectors who trawl for the prawns. In most of
Australia the beginning of the season (August) occurs at a time when the broodstock are
scarce and in poor condition. Supply then continues to vary throughout the season on an
almost daily basis in terms of both quality and quantity (Kenway pers comm.).
This unpredictability of supply makes it difficult for farm and hatchery operators to
establish reliable production schedules. For subtropical farms that have a limited growout
season and for all farms that target seasonal markets, time of stocking ponds is critical to
the economic viability of the venture.
Deviations from the ideal production schedule can result in ill prepared ponds when the
supply of Post Larvae (PLs) for stocking in ponds has been ahead of schedule and a waste
of resources in preparing ponds when supply is then delayed. At worst insufficient or
poor quality broodstock result in a severe undersupply of PLs and therefore empty ponds.
Hatchery operators suffer from wasted live feeds when eggs fail to hatch or spawners fail
to spawn along with the expense of production runs that have near zero survival of PLs.
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On the issue of disease, it has been shown that all recognised (diagnosed) diseases that
have inflicted the Australian prawn farming industry originate from the wild populations
(Owens 1997). Among the causative agents, viruses have proven to be the main threat to
the prawn faming industry. As a prawns immune system has a non-specific defence
response, prawns cannot be vaccinated (Bachere 2003). Specific pathogen free (SPF)
stocks have been bred in captivity for P (Litopenaeus) vannamei. The breeding of
pathogen free stocks has also been an objective of the P. monodon industry once
domestication has been sufficiently achieved.
2.2.4 Life cycle and reproductive biology of P. monodon
A number of aquaculture prawn species have been domesticated in Australia specifically
Fenneropenaeus merguiensis and Penaeus japonicus. To date P. monodon has proven to
be difficult to domesticate due primarily to the poor reproductive performance of
broodstock grown in captivity (Primavera 1984, Crocos et al 1997, Coman et al 2006).
To successfully domesticate any species it is necessary to have the ability to breed
successive generations in captivity. To do this it is essential to understand the life cycle of
the candidate species and the biological requirements of each developmental stage.
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Figure 2.1. Life cycle of penaeid prawns.
As for most penaeids, the ‘life cycle of P. monodon’ (Motoh 1984) consists of an
estuarine phase for the postlarvae and juvenile stages followed by a marine phase
involving an offshore migration of sub-adults. Full ovary maturation and spawning takes
place in the marine phase offshore where water quality parameters are stable for
developing eggs and early larval stages (Figure 2.1).
Spawned fertilised eggs remain suspended in the water for a few minutes then gradually
sink to the bottom. At about 28oC, hatching takes place in about 12 hours to be followed
by
• Six non-feeding, nauplii stages (1.5 days);
• Three protozoa (5 days);
• Three mysis (4-5 days); and
• Three or four megalopa substages (6-15 days).
27
Each substage requires a moult. The megalopa to early juvenile substages are usually
termed post larvae (PLs) and are given a number suffix that indicates in days the time
since metamorphosis to megalopa (eg. PL15 has been a post larvae for 15 days). It is as
PLs that migration occurs from offshore spawning grounds to inshore nursery areas
(Motoh 1984).
P. monodon is a ‘closed thelycum species’, which means it has a receptacle (thelycum)
with lateral plates that enclose the spermatophore. Mating for these species takes place at
night, within hours of a mature female moulting (Primavera 1984). It is when the female
is ‘soft’ after shedding her shell, that the male can insert the spermatophore. Moulting
interval depends on a number of factors including size, feed intake and water
temperature. At 28-30oC adult prawns will moult on average once every three weeks. The
details of the courtship are discussed in Section 4.3.
2.3 Ovary development and endocrine regulation
2.3.1 Accumulation of nutrient reserves in the oocytes of penaeid prawns
A major obstacle to the domestication of any fish or crustacean species is poor egg
quality in culture environments. As a consequence, significant research has been directed
towards identifying properties of good quality eggs and factors influencing them. A
number of factors contribute to egg quality; foremost is nutrient content for egg
28
development (cf. Abidin et al 2006). Embryos and newly hatched prawn larvae up to the
first feeding protozoa stage rely completely on nutrients in the yolk reserves accumulated
during egg development. These reserves originate from the spawner and are systematically
used during the hatching process and the early larval development until first feeding occurs.
Yolk proteins provide the basic structural components for tissues while the yolk lipids
supply energy, cell membrane components and fatty acids (Lubzens et al 1997). Proteins
and lipids are a major component of the eggs comprising 24% and 22% of prawn egg
wet-weight, respectively (Harrison 1990). In penaeid prawns protein and lipid synthesis in
the ovaries has been, and remains, a major area of research.
Studies of penaeid reproduction and egg viability have identified female specific proteins
in the haemolymph, ovary, hepatopancreas and, in some species, the adipose tissue. An
extensively studied female specific protein is vitellin, which is a high-density lipoprotein
with carotenoid pigments and is the main component of the embryonic yolk (Chang et al
1993, Avarre et al 2003). Vitellin is enzymatically cleaved into egg yolk proteins and
lipids and supplies essential nutrients to support the growth and development of the early
embryo up to first feeding larvae.
2.3.1.1 The process of vitellogenesis
As discussed above, a major protein component of the yolk in crustacean eggs is vitellin.
In Penaeus semisulcatus, vitellin constitutes 60% of the proteins that accumulate in the
mature ovary (Fainzilber et al 1989). A molecule immunologically and
29
electrophoretically indistinguishable from vitellin, called vitellogenin, has also been
detected in the haemolymph of several decapod species including P. monodon (Longyant
et al 1999). Vitellogenin is believed to be the precursor to vitellin. The rapid synthesis
and accumulation of egg yolk protein vitellin/vitellogenin by the oocytes is termed
vitellogenesis (Kung et al 2004). There is continued interest in the process of vitellin
synthesis in penaeids and its contribution to egg quality. Vitellin and vitellogenin levels
are also of interest as physiological indicators in the study of endocrine control of
vitellogenesis.
Ovarian maturation in prawns and other crustaceans is often classified on the basis of
vitellogenesis. Accordingly ovary development can be divided into three stages;
previtellogenic, early vitellogenic and late vitellogenic (Quackenbush 1986). However,
the sites and mechanisms of the egg yolk synthesis and accumulation during these stages
remain controversial. The controversy is in part due to the number of techniques/
approaches used when investigating ovary development in prawns. For example, electron
microscopy, biochemical, immunological, histochemical and more recently molecular
techniques including gene regulation and expression have all been applied in research
investigating ovary development (Chen et al 1999, Jasmani et al 2000 Kawazoe et al
2000). In addition the large number of species studied may have contributed to the
variable results reported as some aspects of ovary development appear to be species
specific (Chang et al 1993, Chen and Chen 1993, Quinitio et al 1990, Tom et al 1992,
Rankin et al 1989).
30
For P. monodon, vitellin has been isolated in the ovaries (Quinitio et al 1990, Thurn and
Hall 1999) and eggs (Chen and Chen 1993). Vitellogenin was identified in the
haemolymph (Chang et al 1993) and in the hepatopancreas (Quinitio et al 1990) and,
more recently, it has been quantified in the haemolymph (Vincent et al 2001). It is often
assumed, vitellogenin in the haemolymph is being transported to the ovary from
exogenous sources such as the hepatopancreas (Charniaux-Cotton and Payen 1988).
Vitellogenin levels in the haemolymph have therefore been used as an indicator of when
this exogenous yolk precursor is being synthesised.
Recent evidence derived from examining the expression pattern of the vitellogenin genes
during the reproduction cycle, confirmed that both the ovary and hepatopancreas play an
important role in the synthesis of the yolk precursors for P. monodon (Tseng et al 2001).
However, the total and relative contribution from each tissue has proven difficult to
determine.
Results from two studies carried out on P. monodon failed to agree on the pattern of
changes in vitellogenin concentrations with ovary development. The study by Longyant
et al (2003) showed a drop in haemolymph levels when the ovary reached maturity. This
drop was not shown in the study of Vincent et al (2001). In addition, these two studies
showed significant differences in the quantities detected at the various ovary development
stages for P monodon despite both studies using the same techniques (ELISA) to quantify
the vitellogenin. Vincent et al (2001) found vitellogenin in the haemolymph of what they
termed ‘0’ stage of ovary development, while Longyant et al (2003) showed it to be
31
undetectable in their first stage of ovary development. Some of the differences between
these two studies may be attributed to the criteria used for staging the ovary development,
which was poorly defined in both publications.
2.3.1.2 Cortical Rod formation
In addition to studies on yolk reserves, a significant body of research is now directed
towards defining and understanding the reserves that comprise the cortical rods (CR) within
mature oocytes (Fig. 2.2). The yolk reserves and CR reserves have physiologically distinct
roles and therefore impact on egg quality in different ways.
The vitellogenic stage(s) in the development of prawn ovaries include the appearance of
rod like bodies during the final stages of development (Clarke et al. 1980). After the
completion of yolk accumulation, prawn oocytes are surrounded by an ‘acellular
envelope and possess extracellular cortical rods (CR) that extend into the cortical
cytoplasm’ (Khayat et al 2001).
CRs can comprise 10% of the oocyte volume (Bradfield et al 1989), or more in prawns
where the rods are large compared to other crustaceans such as crabs (Simon Webster,
pers comm. 2005). The biochemical composition of prawn CR is not fully known,
however, precursors in the ovary of P.aztecus are 70-75 % protein and 25-30 %
carbohydrates (Lynn et al 1987). In P.vannamei it was found that CR proteins constitute
32
approximately 11% of the total ovarian proteins (Bradfield et al, 1989, Rankin and Davis
1990). CRs therefore represent a significant amount of accumulated oocyte protein.
Figure 2.2. Histological section of a Cortical oocyte (CO) showing CRs around the
periphery of the late vitellogenic oocytes (a.) (Peixoto et al 2005); SEM of eggs (b. and
c.) showing Cortical Crypts (CC) and Cortical Rods (CR) (Pongtippatee-Taweepreda et
al 2004).
CR proteins have been located in the ovary (Khayat et al 2001, Yamano et al 2003) and
Khayat et al (2001) also found the CR protein-carbohydrate complex was only present in
a.
b. c.
33
vitellogenic ovaries and that it was synthesised within the oocyte. Gene expression has
however shown that for the prawn M. japonicus, transcription of the cortical rod proteins
occurs in the previtellogenic oocytes (Yamano et al 2004). As shown by Western blotting
(as opposed to mRNA expression), this protein is concentrated in the oocyte cytoplasm
during vitellogenesis, and in the CRs during late vitellogenesis. Yamano et al (2003)
concluded that most, if not all the CR proteins are produced from early stages of oocyte
development, accumulated as yolk substances during oocyte development and finally
assembled to create the CR. Yamano et al (2004) further concluded that, transcription,
translation, and formation of the CR structure occurred at different stages of ovarian
development.
The CR proteins are used to construct a jelly layer that surrounds the fertilised eggs after
spawning. It is of critical importance during the earliest stages of embryonic development
(Yamano et al 2004) as it offers the only protection until the hatching envelop forms
(Khayat et al 2001). The jelly layer formation is believed to help maintain a suitable
microenvironment for the embryonic development and prevent polyspermy (Clarke et al
1980). Interestingly, studies on P. monodon egg activation have shown egg-sperm
interaction occurs within 1 minute of spawning (Pongtippatee-Taweepreda et al 2004).
This is very fast compared to P.aztecus were sperm-egg interaction took place between
20 and 40 minutes post spawning (Clarke et al 1980).
34
2.3.1.3 Patterns of nutrient fluctuation in hepatopancreas and ovaries
The process of vitellogenesis results in a large increase in ovary size. An immature ovary
is approximately 1% of total body weight while a mature ovary can be up to 15%. At
maturity, the dry matter of P. monodon ovaries is approximately 70% protein (Dy-
Penaflorida and Millamena 1990) and 21% lipid (Millamena and Quinitio, 1985). Apart
from the ovaries structural components, most of this protein and lipid is in the form of
egg yolk vitellin and cortical rod proteins. As discussed above both the vitellin and
cortical rods are of critical importance to egg quality.
Changes in tissue composition with ovary development have been studied for a number
of penaeid species to determine the origin of various components of vitellogenin. All
species studied have shown an increase in both ovary protein and lipids as the ovary
develops (Wolin et al 1973, Yano 1988, Rankin et al 1989, Quinito and Millamena
1992). Most of these studies are qualitative, although a few have reported on quantitative
changes that occur (Rankin et al 1989, Quackenbush, 1989, Dy-Penaflorida and
Millamena 1990, Mohamed and Diwan 1992).
It has been proposed that the accumulation of these nutrients depends on de novo
synthesis and on the continuous supply of precursors from the spawner’s diet (Souty-
Grosset 1997). In addition, Vazques Boucard et al (2002) suggests that stored reserves
play an important role but are exhausted during the early stage of ovary development in
P. indicus.
35
The hepatopancreas is a major site for protein synthesis and lipid metabolism and, as
discussed earlier has been shown to play a role in vitellogenin production in some prawn
species. Reports for a number of species have shown a decrease in hepatopancreas
reserves that coincides with a rapid increase in the same nutrients in the ovary (Teshima
and Kanazawa 1983, Rosa and Nunes, 2002). This has been reported for protein for P.
monodon (Dy-Penaflorida and Millamena 1990).
More studies are needed on P. monodon to determine when vitellogenesis is occurring in
the ovary and hepatopancreas during ovary development. This is important as it will
provide crucial information as to how the endocrine system, which may be species
specific, is operating in P. monodon. In particular, knowledge of changes occurring in the
major tissues would help identify the site and time of action by inhibitory or stimulatory
hormones. Potentially, this could be done through observation of protein and lipid
changes with a cross-referenced ovary development index (ie. ovary size and histological
evidence) to make it possible to determine at what developmental stages these changes
are occurring. This information is still missing from models detailing the endocrine
system that control reproduction in prawns.
2.3.2 Endocrine regulation of reproduction in crustaceans
There is abundant evidence that homeostasis, growth, development and reproduction in
vertebrates is coordinated by the endocrine system. Invertebrates also have endocrine
systems which vary in complexity across the diverse array of animal forms in these phyla.
36
These systems use a variety of hormones including steroids, peptides, simple amides and
terpenes. Invertebrate endocrine systems are composed primarily of neuroendocrine
components although insect and crustaceans also appear to possess true epithelial-based
endocrine glands.
In recent years considerable efforts have been made to understand the endocrine systems
of crustaceans, particularly those with commercial significance (cf. Huberman, 2000).
The diversity of species study has, again led to confusion and the emergence of very
complex models. Despite considerable efforts focussed on prawns, information on the
role of various endocrine factors shown to affect prawn metabolism, growth and
reproduction, is fragmented and remains largely hypothetical.
With advances in molecular technology the complexity of the crustacean endocrine
system model has increased. A number of crustacean hormones have been isolated and
sequenced. Some of the genes responsible for various reproductive processes have also
been identified (cf. Dircksen et al 2001). The emerging model must now incorporate
species specific hormones and the multifunctionality of some of the hormones.
Many hormones, analogous to vertebrate hormones have been studied in relation to
ovarian development in crustaceans. Circulating steroid hormones induce ovary
development in fish (Mommsen and Walsh 1988). However attempts to administer
vertebrate type hormones to stimulate reproduction in penaeids have met with varying
success.
37
As discussed below, much of the research on endocrine control of crustacean
reproduction has focussed on the inhibitory hormones; neuropeptides that negatively
control physiological processes.
2.3.2.1 The CHH family of hormones
The major neuroendocrine control centre in most crustaceans (including prawns) is the X-
organ-Sinus Gland Complex located in the optic ganglia of the eyestalk (Charmantier et al
1997). Hormones secreted by the sinus gland have profound effects on reproductive
processes (Caillouet 1972), assimilation efficiency and oxygen consumption (Rosas et al
1993), blood glucose levels (Keller et al 1985) and moult frequency (Yang et al 1996).
The effect varies with species, age and season (Adiyodi and Subramouian 1983). The
mode of action and target tissues of the sinus gland neuropeptides is still largely
unknown. However in the last 10 years much progress has been made in sequencing
individual neuropeptides and identifying some of their roles.
An accepted general model for endocrine control of reproduction begins with
environmental stimuli such as a change in temperature, photoperiod and/or diet (cf.
Adiyodi et al 1970). These stimuli influence the neurosecretory centres (X-organ-Sinus
Gland Complex) for secreted hormones (See Figure 2.3). It is known that part of the
reproductive process is under the control of a group of hormones referred to as the CHH
family of neuropeptides. They are produced and secreted by the X-organ-sinus gland
38
complex. To date they consist of the crustacean hyperglycaemic hormone (CHH), the
moult-inhibiting hormone (MIH), the gonad inhibiting hormone (GIH) and, in crabs, the
mandibular-organ inhibiting hormone (MOIH). As their names indicate these
neuropeptides exert negative control over a variety of interrelated processes (Wainright et
al 1996, Huberman 2000).
Figure 2.3 Pathways involved in the control of moulting and reproduction in crustaceans.
These four identified neuropeptides are assigned to the same family because they exhibit
a high level of amino acid homology, despite evidence that they are encoded by different
genes (Davey et al 2000). The similarity in the amino acid sequence for the members of
X-organ-sinus gland complex
Y-organ
Thoracic ganglion
OvaryHepatopancreas
Mandibular organ
GIH
MF
GIHMOIH ?
MF
MIH
ECD
MF
VG
GIHCHH
CHH
VSH ?
VSH ?
VSH ?
Environment
39
the CHH family, together with their biological activities suggests they are multifunctional
(Chang 1997). The small differences in hormone structure may affect functional activity
and/or possible receptor recognition (Davey et al 2000). The balance between stimulatory
and inhibitory hormone titres may also dictate which processes are activated.
Within the CHH group, the gonad inhibiting hormone (GIH, also known as vitellogenesis
inhibiting hormone, VIH) appears to have as its primary physiological role, the inhibition
of ovary development. Quackenbush (1989) showed that in P. vannamei eyestalk extract
suppressed protein synthesis by up to 40% in both the ovary and the hepatopancreas of
females undergoing vitellogenesis. The effect was dose dependent and restricted to the
inhibition of yolk precursor protein synthesis.
The CHH family of hormones has also been implicated in the regulation of Cortical Rod
(CR) protein synthesis (Avarre et al 2001). Yamano et al (2003) concluded that most, if
not all, the CR proteins are produced from early stages of oocyte development then
accumulated as yolk substances during oocyte development and finally assembled to
create the CR proteins. This is in agreement with Webster’s (pers. comm.) summation of
evidence to date that early vitellogenesis (previously known as primary vitellogenesis) is
associated with accumulation of CR proteins precursors from exogenous sites, and not
vitellogenin synthesis. These precursors are transported to the oocyte cytoplasm.
Synthesis of the main CR rod protein (SOP) in P. semisulcatus then occurs in situ and
was restricted to the vitellogenic stages of ovary development ovaries of (Avarre et al
2001).
40
Thus, despite SOP transcripts being found at all ovary stages, final synthesis was limited
to the later stages. Avarre et al (2001) found that Sinus Gland Extracts (SGE) and CHH
family peptides inhibited this final synthesis of the SOP. Interestingly, vitellin production
by the ovary in P. semisulcatus, decreased significantly when cortical rods appeared
(Browdy et al 1990) suggesting the oocytes changes from producing vitellin proteins to
CR proteins.
Thus CHH peptides affect the production of both vitellin and CR proteins however the
process is not fully understood. They appear to regulate through the inhibition of
vitellogenin synthesis in the hepatopancreas (and possibly other sites) and vitellin and CR
protein synthesis in the ovary. It has been proposed that GIH prevents the uptake of
exogenous vitellogenin precursors by the ovary (Charniaux-Cotton 1985). GIH or another
CHH may act in a similar way on CR protein precursors. However Avarre et al (2001)
proposes that GIH or eyestalk extracts have the potential to affect all stages of ovary
development in penaeid prawns.
2.3.2.2 The Roles of Methyl Farnesoate (MF) in prawn reproduction
Studies on crustacean reproduction have shown that a secretion from the Mandibular
Organs called Methyl Farnesoate (MF) appears to play important roles in growth and
reproduction (Laufer 1992, Jo et al 1999). It is known that the hormone methyl farnesoate
(MF), is synthesised and secreted by the Mandibular Organ (MO) and is structurally
similar to juvenile hormone III (Nagaraju et al 2004). Juvenile hormones (JH) are a
41
family of sesquiterpenoid compounds that affect crustacean metamorphosis and
reproduction. The physiological function and pathways of MF in crustaceans are not well
known. As a terpenoid hormone, however, MF appears to play at least a dual role
involved in the regulation of both moulting and reproduction. (Nagaraju et al 2004).
A number of studies on crustaceans correlated increased MF synthesis rates in the MO
with ovary development (Laufer et al. 1986, Borst et al 1987). Tsukimura and Kamemoto
(1991) and Laufer et al (1997) found that MF significantly increased the diameter of
Penaeus vannamei oocytes in vitro and MF has also been reported to increase fecundity
in P. vannamei (Laufer 1992, Laufer et al 1997). Laufer (1992) found that diets
supplemented with MF resulted in superior spawning performance and larval survival of
cultured P. vannamei.
It is not clear at what stage of ovary development MF is most active. In a review of prawn
endocrinology, Huberman (2000) interpreted the involvement of MF to be at the early
stages of vitellogenesis. It was also found that there was a marked, but transient, rise in
MF levels in the crab Cancer pagurus hemolymph at the onset of secondary
vitellogenesis (Wainright et al 1996). This is also the stage at which VIH/GIH is thought
to regulate ovary development (for review see Charniaux-Cotton 1985).
It has recently been confirmed that MF in crabs of the genus Cancer, is synthesised under
the control of the Mandibular Organ Inhibiting Hormone (MOIH). This inhibitory
hormone is a member of the CHH family (Rotllant et al 2000) and it prevents the last
42
stage enzymatic stage of MF production. Ablation decreases levels of MOIH enabling the
last stage of MF synthesis to take place in the MO (Wainright et al 1998). However, only
crabs from the genus Cancer seem to have a distinct MOIH (pers comm. Simon Webster,
2005) although there is evidence of sequence similarity between the MOIH from Cancer
and VIH/GIH from prawns. In penaeids and other crustaceans VIH/GIH could act
indirectly and involve repression of MF synthesis, that is, VIH/GIH is equivalent to
MOIH. (pers comm Simon Webster, 2004). These recent findings add to the apparent
complexity of hormonal integration of reproductive processes in crustaceans (Wainright
et al 1996) with VIH/GIH possibly targeting both ovary and MO tissues.
Regardless of the uncertainty concerning hormonal control for MF secretion, it would be
beneficial to determine whether MF has a stimulatory effect on P. monodon, as has been
shown for P. vannamei. This could directly benefit the prawn farming industry if MF
dietary supplements proved successful in increasing egg and larval production from
domesticated prawns, and also help determine if the activity of MF is species specific.
2.3.3 Endocrine manipulation strategies employed in prawn aquaculture.
In the wild, marine prawns are usually seasonal spawners with specific environmental cues
stimulating ovary development and spawning via neurosecretory centres (Khoo 1988). For
aquaculture purposes, however, captive prawns are required to spawn on demand
throughout the year. While controlling environmental conditions is an essential hatchery
protocol, it has limited success in inducing sufficient spawnings to meet commercial
43
production schedules. As a consequence industry practise relies on manipulating the
endocrine systems of prawns to improve reproductive performance (Primavera 1984).
2.3.3.1 Eyestalk ablation
Ablation is widely used in commercial hatcheries as a crude method of hormonal
manipulation to induce spawning in many crustaceans including P. monodon (Primavera
1984). The process involves the removal or constriction of (through cutting, cauterising or
tying) one eyestalk to reduce the level of GIH/ MO-IH being produced and/or secreted by
the X-organ and sinus gland complex (Longyant et al 2003). However this unilateral
ablation affects virtually all aspects of crustacean physiology that are regulated by the X-
Organ Sinus Gland Complex (Quackenbush 1986). Over time a physiological imbalance
occurs and female reproductive performance has been found to deteriorate.
Effects of eyestalk ablation
As described above, to induce P. monodon to mature and spawn in captivity on a
commercial scale requires ablation. The proportion of unablated female P. monodon to
show ovary development and/or spawn in captivity is very low (Santiago 1977, Primavera
and Borlongan 1978, Aquacop 1979, Emmerson 1983). Much of the increase in egg
production with ablation is due to an increase in spawning frequency (Browdy and
Samocha 1985, Lumare 1979, Aquacop 1979, Kelemac and Smith 1984). With ablation, P.
44
monodon can spawn 4-6 times per female per moult cycle (Beard and Wickins 1980,
Hansford and Marsden 1995, Marsden et al 1997).
Despite the increase in total egg production with ablation there are several negative
consequences of this practice. For example, there is evidence that ablation results in an
eventual decline in larval survival (Marsden et al 1997, Palacios et al 1999) and fecundity
(Beard and Wickins 1980, Emmerson 1980). Partial ovary development and spawning have
also been reported (Primavera 1984, Lumare 1979). This ‘reproductive exhaustion’
(Lumare 1979) has been attributed to the rapidity of the successive spawnings depleting
reserves for yolk production faster than they can be accumulated through dietary intake
(Aquacop 1977, Lumare 1979, Beard and Wickins 1980, Harrison 1990). It has also been
attributed to ‘time after ablation’, regardless of the number of spawns (Palacios et al 1999).
An eventual decline in spawn frequency was noted for P. vannamei (Palacios et al 2000)
implicating other physiological processes besides nutrient depletion.
Interestingly, there are reports of some aspects of reproduction improving or being
unaffected by ablation. For example, Chamberlain and Lawrence (1981) noted an increase
in fecundity for P. stylirostris after ablation. Browdy and Samocha (1985) also found no
change in fecundity or egg quality between ablated and non-ablated P. semisulcatus
spawns.
With ablation affecting glucose metabolism (CHH) and ecdysis (moulting, MIH), it is
likely to also affect mobilisation of nutrients (Harrison 1990). As the embryo and pre-
45
feeding larvae (protozoa) are lecithotrophic their nutritional quality is dependent on
maternal factors. Both quality and quantity of egg yolk will depend on maternal body
reserves, capacity for biosynthesis and dietary intake during ovary development (Harrison
1990), all of which are likely to be affected by ablation.
Studies designed to examine how ablation is affecting patterns of nutrient accumulation in
the ovaries of P. monodon could provide information for the further refinement of models
for the endocrine system in this species. Knowledge of the affect of ablation on nutrient
partitioning in the body of broodstock prawns could also prove helpful in the development
of a complete artificial broodstock diet.
2.4. Mating Behaviour of Penaeid Species
For a prawn egg to hatch and develop a number of conditions must be met. As already
discussed yolk reserves play a critical role in the development of the egg and in the
quality of the lecithotrophic larvae (that is, larvae that live off the yolk). To develop into
a larva, however, the egg must hatch and to do so requires fertilisation. Factors that
influence fertilisation include egg and sperm quality, environmental conditions under
which the fertilisation takes place, and mating success, which determines whether sperm
is available to fertilise the egg.
46
Hatch rates are often seen as a measure of fertilisation although they are different
physiological events with fertilisation being one factor that effects hatch rate. In the
industry hatch rates are also used as an indicator of whether the female has mated.
A low hatch rate of eggs is a recognised problem of naturally mated captive-bred P.
monodon broodstock. Currently artificial insemination is used to increase egg fertility and
therefore egg hatch rates. However this labour intensive process requires a high skill level
and is not the preferred option in commercial hatcheries within Australia.
Typically the natural mating process is divided into two phases; (i) securing a mate, and
(ii) transferring the sperm from the male to the female. The mechanisms involved in each
phase are different between species. Like other aspects of the reproductive process (for
example ovary development and spawning) there are indications that the mating
processes is also under the control of the endocrine system.
2.4.1 Mating strategies of crustaceans
Crustaceans represent a large and diverse taxonomic class of arthropods that include
lobsters, shrimps, and crabs, most of which are aquatic, primarily marine. They are
similar in that they have gills, ten legs, a hard exoskeleton and antennae. They are diverse
in many aspects including morphology and habitats, and as a consequence, employ
different mating strategies (Bauer 1991). Most have separate sexes which require the
coupling of male and female for the production of offspring.
47
The mating behaviour of crustacean decapods has received considerable attention during
the last few decades (Salmon 1983, Dunham 1988, Waddy and Aiken 1990). Pair
formation is an essential first step in the mating process and has been a focus of much of
the research. The mechanisms used to secure a mate depend on factors such as habitat,
resources required, physical attributes, mode of locomotion, reproductive biology and
spatial distribution (Christy 1987).
Systems
Table 2.1 shows the variation in mating systems within one family (Carcidea). This
classification system is based primarily on male behaviour. Mating systems have long
been studied with little agreement on the classificatory schemes or on the main
discriminating criteria (Correa and Thiel 2003). While male and female interactions play
an important role in mating, the competitive behaviour of males attempting to find a
receptive mate has been used as a major source of criteria for classifying reproductive
behaviour in crustaceans.
A mate can be secured by attraction or pursuit strategies and be initiated by the male or
female. Some species have elaborate courtships including displays by males such as shell
knocking in hermit crabs and claw waving in fiddler crabs
48
Table 2.1. Summary of four general mating systems in Caridea (Correa and Thiel 2003)
classified on the basis of male behaviour.
Monogamy. Adult individuals associate with a member of the opposite sex to reproduce and share one microhabitat (a refuge or host) for a long time period exceeding one reproductive cycle. Mates behave territorially towards conspecific intruders. There is usually no extra-pair mating.
Neighbourhoods of dominance
Male mating success depends largely on their ability to win aggressive encounters to overtake and defend receptive females. Pair formation is restricted to a short period (few hours) of female receptivity. During this time dominant males attend, fertilize and guard females (i.e., throughout the spawning process) after which mates separate.
Pure search Male mating success depends primarily on their ability to find (and mate with) as many receptive females as possible. To search efficiently, these males roam through the population and continually contact conspecifics until they find a receptive female. Upon locating such a female, males transfer sperm in brief and simple acts after which the pair immediately separates. There are no complex behaviours such as courtship of receptive females, nor aggressive encounters between males.
Search and attend
Adults live solitarily on hosts (or in other refuges), but males change hosts frequently in search of females close to reproductive receptivity. Upon finding such a female, males stay on the hosts and prevent takeovers by fighting. Following mating, each mate returns to a solitary life style
When are females and males ready to mate?
Development of external genitalia is a prerequisite for mating and signals sexual
maturity. It is after this stage in ontogenetic development that communication between
the male and female initiates a mating response. The age or size at which this occurs is
species specific and is influenced by environmental factors.
49
For mating to occur the mature female must be receptive and attractive to the male and
her receptivity is invariably linked to her moult cycle. For many species the female is
receptive immediately post moult when her shell is soft. For other species mating occurs
prior to egg release when the female has a hard shell. Little work has been done on males,
however, it is generally accepted that males are sexually active during their entire
intermoult hard shell phase (Correa and Thiel 2003).
Signalling
Visual cues, chemo-tactile cues and water borne chemicals, singularly or in combination,
form the bases for communication between males and females with regard to mating. In
some species such as lobsters, males visually attract females to safe shelters (Bushmann
and Atema 1997, Cowan 1991). In other species, males are attracted to the female by
water borne chemicals which in many cases have been shown to be pheromones.
Pheromones act as a non visual means of communicating between individuals of the same
species, and are usually a mixture of chemicals designed to stimulate a specific
behavioural response. The substances can be effective at minute concentrations. Sex
pheromones play a role in changing or regulating behaviours to enable each stage of the
mating process to be completed (Dunham 1988).
50
Research on crustacean sex pheromones has focused on American lobsters (McLees et al
1977, Atema and Cowan 1986) with some recent work on helmet crabs (Kamio et al
2003, 2005). For both species, mating occurs soon after the female moults. Prior to
moulting sex pheromones are released in the females urine and perceived by receptors on
male antennules (see Dunham 1978, 1988 and Salmon 1983, for reviews). This has also
been shown to occur in the blue crab, Callinecies sapidus (Gleeson, 1982). For the
American lobster research has shown that at least two pheromones are involved; one to
trigger a grasping response in the male and a second, yet to be identified, that triggers
copulation.
Control of mating behaviour
While it has been shown that visual and chemical signalling are the means of
communicating during mating, little has been reported on the system that controls the
behaviour or release of pheromones.
It has been well established that hormones play a role in the mating behaviour of fish. In
one of the few studies carried out on the effect of hormones on crustaceans, a link was
found between methyl farnesoate (MF) levels in the haemolymph and intensity of
reproductive behaviour in the spider crab (Sagi et al 1994). The authors proposed that this
could be a cause-and-effect response with the increase in MF levels being directly
responsible for the increased intensity of behaviour.
51
2.4.2 Mating strategies of closed and open thelycum species of penaeids
Most penaeids fall into the ‘pure searching’ mating system described in Table 1. It has
been hypothesised that species that are highly agile, have males that are relatively small
compared to females, lack fighting appendages and don’t possess a thick shell are suited
to this system (Bauer and Abdalla 2001). When searching male identifies a receptive
female it rapidly transfers the spermatophore while the females continue to swim and
then immediately separates from her. Such systems do not require aggressive or defensive
behaviour between competing males.
Penaeids can be divided into two groups based on the female’s thelycum. The thelycum is
an external receptacle that receives the spermatophore from the male during mating (Bliss
1982). It is located on the ventral surface and is formed by an outgrowth from the last and
next to last thoracic somites. Variously developed, two types of thelycum are discernible
in penaeid prawns; the open type with ridges and protuberances for the attachment of
spermatophores and the closed type possessing two flaps and enclosing a seminal
receptacle where spermatophores are deposited. This receptacle acts to store sperm until
the female spawns or she moults. As the thelycum is an external structure it is discarded
along with the spermatophore it holds when the female moults. The spermatophore in
open thelycum species has been shown to be more complex than in closed thelycum,
probably due to the lack of protection from the environment after attachment to the
female (Bauer and Min 1993).
52
The type of thelycum relates to when mating occurs during the females moult cycle. For
example in open thelycum species such as P. vannamei, the male deposits the
spermatophore on a hard shelled female which will spawn a few hours later (Yano et al
1997). The courtship behaviour starts in the afternoon in relation to light intensity and
some signal from the attractive female. In closed thelycum species such as P. monodon,
the males implant the spermatophore after the female moults (Primavera 1985) while the
thelycum is still soft for implantation.
2.4.3 Mating behaviour of P. monodon
Spermatophore implantation has been observed in wild-caught female P. monodon as
early as 4 months of age or 60 gram in weight. Captive-bred females as small as 40 g,
were found to have sperm in their thelycum. Both captive-bred and wild caught males at
40 g were found to have sperm (Primavera 1985).
As a closed thelycum species P. monodon mate after the female has moulted. Moulting,
and therefore mating, takes place at night. Immediately after moulting the female will
commence swimming in the water column, and if present, one or more males in the tank
will pursue her (Primavera 1985, personal observation). As described by Primavera
(1985) one male will eventually position himself parallel to and beneath the female as she
swims. As the female continues swimming the male rolls over so his ventral surface is in
direct contact with the ventral surface of the female. This step may occur a number of
53
times before the male rapidly turns perpendicular to the female and curves his body
around her. Abdominal contractions by the male follow in rapid succession for about 1-2
second(s), and thought to coincide with the insertion of the spermatophores into the
female’s thelycum. At this point all pursuit by males ceases.
2.4.4 Mating in captive-bred prawns including P. monodon
It is common practice for commercial hatcheries and research institutes both in Australia
and overseas, to use artificial insemination (AI) to improve hatch rates of eggs from
captive-bred P. monodon (M. Kenway and T. Hoang pers. comm. 2005). As hatch rates
are higher when prawns are inseminated using AI compared to natural matings, the
implication is that lack of natural matings is an issue. Further, wild-caught prawns held
under the same conditions achieve high hatch rates from natural matings suggesting
facilities are not responsible for the lower hatch rates for captive-bred broodstock.
AI has been adequate to service the small scale domestication/genetic programs that have
existed to date. However as industry moves towards full-scale domestication, AI could
develop into a rate limiting step in the expansion process. Understanding if and why
mating rates are lower in captive-bred broodstock could significantly help in the large
scale implementation of P. monodon domestication programs.
Mating vs hatch or fertilisation rates
54
A review of the literature has shown that most studies looking at the reproductive
performance of prawns include hatch rates and/or fertilisation rates (determined from
microscopic examination of the eggs hatching envelop). However there is little
information available on actual mating rates. It is therefore very difficult to determine
from the literature whether low egg hatch rates reported for captive-bred P. monodon are
due to low mating success or an egg or sperm quality issue. There have been some studies
carried out on sperm quality that would indicate this is not the issue. For example, sperm
in P. monodon showed no decline in quality over 42 days in captive wild-caught males
(Gomes and Honculada-Primavera 1993) or over 81 days with captive-bred (Fast 1993).
More importantly Fast (1993) also found no difference in sperm quality between captive-
bred and wild-caught P. monodon.
Lack of data on mating is partly due to the rarity in observing the process and, for some
species, the difficulty in visually determining if a female is fertilised/implanted.
Implantation is clearly evident in some species such as P. japonicus where the
spermatophore has ‘wings’ which extrude from the thelycum after implantation. It is also
easily observed in open thelycum species.
For P. japonicus, Hansford et al (1993) found that mating success in ponds was high
(99%) but low in tanks (30%), however, no comparison was made to wild-caught
broodstock. The low mating levels in the tanks may have been due to environmental
factors. Hatch rate of eggs from captive-bred P. japonicus held in tanks for 5 months has
been reported to be significantly lower than from wild broodstock (Preston et al 1999). In
55
this study only wild-caught P. japonicus broodstock that were fertilised were selected for
the trial and the percentage was not reported. It is not clear whether the same selection
was applied to the captive-bred broodstock making it difficult to draw any conclusions
concerning mating rates. In P. vannamei (Palacios et al 1999), an open thelycum species
wild-caught broodstock were found to have higher mating frequencies than captive-bred.
For P. semisulcatus, also a closed thelycum species, Browdy et al (1986) found a high
mating success with no difference between captive-bred and wild-caught broodstock.
Thus it is unclear whether mating is an issue in different species of captive-bred
broodstock. This is not so much due to conflicting information but rather that studies
rarely isolate mating as a reproductive performance criteria.
P. monodon
There is very little information available on mating success of captive-bred or wild-
caught P. monodon. There were earlier reports of matings occurring in ponds (Primavera
1985) and of females being unmated (Lin and Ting 1986) however there were no
accompanying details on holding conditions or percentages. Other workers have
examined the reproductive performance of captive-bred and wild-caught broodstock and
a combination of the two (Menasveta et al 1993). The data was collected from broodstock
that were naturally mated in tanks. Fertilisation rates of all eggs spawned were reported to
be high for wild-caught and captive-bred (82 and 80% respectively) but low for the cross
matings. Wild-caught females with captive-bred males had a 30% fertilisation rate while
56
captive-bred females with wild-caught males had a 39.8 %, and were not significantly
different. This result was difficult to interpret from information provided in the paper.
Summary of behavioural studies
As described above, the literature clearly indicates that hatch rates are low in captive-bred
P. monodon and some other prawn species. The key indicator that mating is a problem in
P. monodon captive-bred broodstock is the improvements obtained in egg hatch rate
using AI. It is therefore hypothesised that some stage of the mating process is suboptimal
in captive-bred prawns reducing mating success rate and contributing to the low reported
egg fertilisation and hatch rates.
To test this hypothesis studies need to be carried out to directly compare the mating
behaviour of captive-bred and wild-caught P. monodon. To make this comparison
behaviour needs to be observed in detail and a suitable classification system for different
behaviours needs to be developed. As P. monodon uses the ‘pure searching’ strategy with
male activity stimulated when the female moults, a suitable system could be based on
male behaviour.
2.5. Summary
• P. monodon is the preferred species of prawn aquaculture in much of the world.
57
• Variability in quality and quantity of wild caught broodstock and difficulty in
domesticating this species has resulted in broodstock supply being a major
bottleneck in the expansion of this industry.
• Reproductive performance criteria used to measure the quality of broodstock
includes egg quality (hatch rate and larval survival) and spawning rate.
Improvement in both these parameters will greatly assist the industry by
improving larval supply.
• Egg quality in terms of larval survival is dependent on nutrients accumulated in
the oocytes during ovary development. There is the need to know how this
process is affected by the industry practice of holding and ablating broodstock.
• Egg quality in terms of hatch rate is a major problem with domesticated (captive-
bred) broodstock. Prior to solving this problem it needs to be determined whether
low hatch rates are due to the absence of sperm due to failure to mate.
• Spawning rates of broodstock have been shown to be under the control of the
endocrine system. However the current knowledge of reproductive hormones in
prawns reveals a complex model with inherent contradictions. As the mode of
action for the different hormones may be species specific, more information is
required for P. monodon.
58
• The purpose of the following studies is to obtain data which will assist in the
development of strategies to improve the reproductive performance of P.
monodon. This will be done by investigating the contribution of the interrelated
factors of egg nutritional status, endocrinology and mating behaviour.
59
2.6. Project hypothesis and aims
The intention of this project was to investigate factors contributing to poor reproductive
performance in the cultured prawn Penaeus monodon. It was hypothesised that:
1. The quantity of nutrients accumulated in the ovaries of wild-caught prawns that
are ablated and matured in captivity differs from that of prawns matured in the
wild.
2. Ablation and captivity influences the physiology of nutrient uptake in the ovaries
and depletion in the eggs and developing lecitrophic larvae.
3. Poor mating success of captive-bred broodstock contributes to poor egg quality in
terms of low fertility and therefore low hatch rate.
4. The administration of a stimulatory hormone Methyl Farnesoate (MF) may
improve the percentage of broodstock that spawn and improve total egg
production.
In line with these hypotheses, the aims of the project are:
1. To investigate ovary development and factors affecting it by;
a. Quantifying the changes in the lipid and protein content of ovary tissue
during ovary development,
b. Classifying ovary development stages by cross referencing the gonad
somatic index (GSI) and ultrastructure changes as evidenced by histology,
and,
60
c. Comparing the biochemical composition of ovaries immediately after
capture of prawns with ovaries from prawns conditioned in tanks and
subjected to unilateral eyestalk ablation.
2. Determine if ablation can influence the composition of the early stage
(undeveloped) ovary by;
a. Causing regression of the ovaries by subjecting wild-caught female prawns
to short term starvation
b. Comparing the biochemical composition of the ovary and hepatopancreas in
ablated and non ablated prawns.
3. Determine whether ablation and captivity effects the pattern of nutrient depletion
during egg development and early larval stages by;
a. Measuring relative changes in lipid levels and fatty acid composition as eggs
and larvae develop, and
b. Comparing eggs and larvae from prawns whose ovaries matured in the
wild to those whose ovaries matured in captivity following ablation.
4. Determine if mating behaviour of captive-bred males and or females contributes
to poor egg quality by;
a. Observing time-lapse video recordings of the mating behaviour of wild-
caught prawns and detailing steps or processes involved
61
b. Comparing the observations of the wild-caught prawns with the captive-
bred broodstock.
5. Assess the effect of MF on ovary development and larval production by
conducting;
a. An in vivo study to determine the effect of dietary inclusion of MF on the
reproductive performance of P. monodon.
62
Chapter 3.
GENERAL METHODS
In this chapter, methods applied in at least two of the individual result chapters of this
thesis are described. Methods specific to individual studies are described in the relevant
result chapters.
3.1 Prawns
3.1.1 Location
There are only a few concentrations of Peneus monodon broodstock in Eastern Australia
that are accessible to commercial broodstock collectors. The prawns for the current study
were captured in waters (2 to 8 meters in depth) adjacent to Cairns in northern
Queensland by a commercial prawn fishing company (Bill Izard, Cairns Live Prawns).
3.1.2 Capture method
Prawns were captured at night using a beam trawl with an average trawl duration of 40
minutes (range 30 to 60 minutes). Upon raising the net, mature broodstock females (>
75g) and males (> 60g) were transferred to plastic tubs (100L capacity) that had ocean
water pumped through at a rate of 10L per min, and were supplied with additional
aeration. Pieces of trawl net were placed in the tubs to offer substrate for prawns to cling
63
to and to reduce disturbance from flicking prawns. The prawns remained under these
conditions until the next morning when the boat returned to Cairns port.
3.1.3 Transport method
As the spawner grounds are located approximately 1000 kms north of the Bribie Island
Aquaculture Research Centre (BIARC), it was necessary to air freight prawns. To comply
with airline regulations the prawns were packed by the spawner supplier (Bill Izard,
Cairns Live Prawns) in approved styrofoam boxes with plastic liner bags to prevent
leakage. Six prawns were then transferred to plastic bags with 10L of chilled water
(20oC) saturated with pure oxygen. The remaining two thirds of the bag was then filled
with pure oxygen and the bag sealed (tied with rubber bands) before being placed in the
box and the box lid secured with tape. To prevent the rostrum of the prawn (the sharp
protruding point on the head of the prawn) from perforating the bags, a small piece of
plastic tubing was placed over the rostrum tip prior to packing.
Boxes were transported by road to the airport and then air freighted to Brisbane (total 4-6
hours) where they were collected and driven to BIARC (1 hour).
64
3.1.4 Arrival and acclimation
Upon arrival at BIARC all boxes were opened and air stones were added to the water
while the prawns remained in the bags. Those prawns to be dissected or allowed to spawn
that night, were euthanised or transferred to spawning drums, respectively (See below).
Prawns that were to be held in captivity were acclimated while remaining in the bag.
Water from the tank that the prawns were to be released into, was then added to the bags
at a rate of 5L per 10 minutes. When temperature was within 2oC of the tank temperature
(27±1oC), prawns were released by pouring the contents of the bag into the tank.
3.1.5 Holding tanks
The maturation tanks that housed the prawns were circular fibreglass tanks (4.0 m
diameter; 0.8 m water depth). Seawater (33 ppt salinity) supplied to the tanks was filtered
to 20 µm, heated to 28oC and exchanged at a rate of 200% per day. Controlled light was
provided by suspended fluorescent fittings wrapped in green 70% 'shade cloth' (Dindas
Lew Cat No 5C7036 BL) to reduce light intensity to 5 lux as measured at the water surface
using a Licor light meter (model L1-185B) fitted with a photometric sensor (Licor model
PH4432). Day length was 14L:10D, with a ramp period of 20 minutes.
65
3.1.6 Ablation
Unilateral eyestalk ablation was carried out by cauterising one eyestalk below the eye.
This was carried out by securing the female prawn in a damp towel and pinching the
eyestalk with red hot thin pliers that had been heated over a bunsen burner (Primavera
1985)
3.1.7 Feeding
Prawns were fed one of three diets; fresh, BIARC or BIARC+MF, depending on the
experiment being conducted. The fresh diet consisted of chopped fresh-frozen green-
lipped mussel (Perna canaliculatus) and squid mantle (Loligo sp) fed alternatively. The
BIARC diets used in Chapter 6 were artificially formulated as described in the methods
section of that chapter. All diets were fed to excess, twice daily (0800, 1700).
Where the fatty acid composition of the diet was needed, daily consumption was
monitored by recording wet weight fed minus the wet weight of feed that remained in the
tank. This enabled the ratio of the squid and mussel consumed to be established.
66
3.2 Tissue, egg and larval collection, classification and biochemical analysis
3.2.1 Tissue collection
Prawns selected for extraction of ovary and hepatopancreas tissues were first euthanised by
submergence into salt water containing ice. The tissues were then removed from the prawn
by cutting along the dorsal surface just below the cuticle to ensure no perforation of tissues
occurred. The incision was then carefully opened and the tissues sections removed. After
extraction tissues were placed in small labelled containers and transferred to a -70oC freezer
until required for analysis.
3.2.2 Classification of tissues using gonad somatic index (GSI) and hepatopancreas
somatic index (HSI).
Following dissection, a Gonad Somatic Index (GSI) was calculated for each individual
prawn to determine/ assess the degree of ovary development. The GSI were calculated
using the formula;
GSI = 100 x (wet weight of the hepatopancreas or gonad / prawn wet weight).
Data were calculated to 1 decimal place or rounded up or down to the nearest whole
number.
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3.2.3 Classification of ovary developmental stage using histology
Histological changes associated with oocyte maturation in wild caught P. monodon have
been described in detail by Tan-Fermin and Pudadera (1989). In this study, following
dissection, ovaries were weighed and a small portion (2-5 mm3) removed from the anterior
abdominal region and fixed in 10% formalin and seawater. These sections were then
transferred to 70% ethanol after 24 hours, embedded in paraffin, sectioned (6 µm) and
stained with haematoxylin fuscin (Hamason, 1972). Ovary sections were then examined
microscopically and classified into three ovarian development stages (previtellogenic,
vitellogenic or cortical rod) using criteria reported by Tan-fermin and Pudadera (1989).
Measurement of oocytes (µm) was made for 6 prawn ovary sections, across the long axis of
the prominent oocytes at each GSI stage. Between 80 and 120 oocytes were counted for
each section.
3.2.4 Biochemical analysis
Proximate analysis
Moisture content of the ovary and hepatopancreas tissue was determined by oven drying a
sub-sample to constant weight at 105oC. Using freeze dried material, crude protein (N x
6.25) was derived from Kjeldahl nitrogen analysis, with copper and selenium as catalysts
(AOAC, 1990, method 988.05), was determined by Soxhlet extraction with petroleum ether
(bp 40oC to 60oC) for six hours (AOAC, 1990, method 960.39). These techniques are
68
described in detail in Marsden et al (1997). Ether extract, used here as a measure of total
lipid content, was determined by Soxhlet extraction with petroleum ether (bp 40oC to
60°C) for 6 h (Association of Official Analytical Chemists, 1990, method 960.39). For
chapter 4, to correct for variation in prawn size, the quantities of protein and lipid in each
tissue were calculated as quantities per 100g (wet weight) of prawn.
Fatty acid analysis
For fatty acid analysis, lipids were first extracted from pooled samples of each tissue by
the method of Folch et al. (1957) using the suggested modification of Christie (1982). An
aliquot of the lipid extract so obtained was separated into polar and non-polar fractions
using Sep-Pak silica cartridges (Waters Associates, MA, USA). The non-polar fraction
was eluted with 15 ml chloroform and the polar fraction with 20ml of methanol (Christie,
1982). The solvent was removed from each fraction by rotary film evaporation and the
lipids esterified to fatty acid methyl esters (FAME) using the method of Van Wijngaarden
(1967). FAME were separated by capillary gas chromatography using split injection on a
30m X 0.25 mm i.d. fused silica column coated with 0.25 µm of Durabond-23 (J and W
Scientific, Folsom, California). Column temperature was held at 160°C for 10 min and
then elevated at 3°C per min to 210°C where it was held until all FAME of interest had
been eluted. FAME were quantified by comparison with the response of an internal
standard (heneicosanoic acid methyl ester). FAME were identified by comparing their
retention times with those of authentic standards (Sigma Chemical Company, St. Louis,
Missouri).
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Chapter 4.
THE EFFECTS OF CAPTIVITY AND ABLATION ON PROTEIN, LIPID AND
DRY MATTER CONTENT OF OVARY AND HEPATOPANCREAS TISSUES IN
THE PRAWN PENAEUS MONODON.
4.0 Abstract
To investigate the effect of captivity and ablation on ovary development in P. monodon, an
experiment was conducted to quantify total protein and lipid in the ovaries and
hepatopancreas prior to and during ovary development.
Results revealed the captive conditions of this study caused a reduction in the lipid content
of previtellogenic ovaries. In addition, ablation appears to increase the hepatopancreas
contribution to lipids accumulating in the vitellogenic ovary although captive conditions
(including diet) may also play a role in this increase. Despite these significant effects on
undeveloped and developing ovaries, the current study showed that, at least for the first
post-ablation maturation cycle, captivity and ablation caused no significant change in the
levels of lipid or protein in mature ovaries. Thus, the effects of captivity on previtellogenic
ovaries and ablations’ role in regulating nutrient uptake at this developmental stage,
warrants further studies with a particular view to improving spawning frequency.
70
4.1 Introduction
As described previously (2.3.3.1), the reproductive performance of P. monodon in captivity
is characterised by (i) most females requiring ablation to induce ovary development and
spawning (Primavera 1984), (ii) regression of developing ovaries when wild caught prawns
are held in captivity (Marsden personal observation), and (iii) variable spawning frequency
and larval survival which occurs with seasons and between individuals (Hansford and
Marsden 1995). In addition, prawns whose ovaries mature in the wild have been shown to
produce better quality eggs than those matured in captivity after ablation (Beard and
Wickins 1980, Primavera and Posadas 1981, Primavera 1984, Ruangpanit et al 1984).
Female prawns are unilaterally eyestalk ablated to promote vitellogenesis in captivity.
Ablation acts by reducing levels of the vitellogenesis inhibiting hormone (VIH), one of the
sinus gland hormones. VIH prevents the onset of yolk (vitellin) production and
accumulation in the ovary. The aspects of the environment restricting ovary development
and causing regression of developing ovaries (Avarre et al 2001), have not been fully
identified, hence, the continued use of ablation by industry. However, while ablation has
enabled commercial scale hatchery production of larvae for P. monodon, its success rate is
variable (Hansford and Marsden 1995). This variation has been attributed to the
physiological condition or, more specifically, the nutritional status of prawns prior to
capture. Quackenbush (2001) suggests the function of VIH is to restrain yolk synthesis until
suitable organic reserves are in place in the hepatopancreas and/or the ovary. Accordingly
ablation may be more effective in inducing and accelerating vitellogenesis if the ovary is
already undergoing specific physiological processes related to nutrient accumulation.
71
Larval survival is also affected by the nutritional status of the spawner. It has previously
been shown that the maturation diet (fed after capture and during ovary development),
influenced both spawning frequency and larval quality in wild caught P. monodon (Marsden
et al 1997). To date, however, maturation diets cannot completely eliminate seasonal or
individual variation. This would indicate that other factors, such as the nutrient reserves in
females accrued prior to capture, may be contributing to the variation in larval survival
(Arcos et al 2003, Silbert et al 2004).
In addition to the proposed effect of nutritional status, there is evidence that ablation
negatively impacts on larval quality by accelerating the rate of ovary development.
Specifically, the hepatopancreas has been shown to make a significant contribution to
nutrients accumulated in the ovary (Dy-Penaflorida and Millamena 1990, Millamena and
Pascual, 1990, Tseng et al, 2001, Kung et al 2004). It has been suggested that ablation
results in rapid depletion of hepatopancreas reserves resulting in a shortfall of nutrients
available for transfer to the ovary (Beard and Wickins 1980, Palacios et al 1999, Vazquez-
Boucard 2004).
Protein and lipid represent 80% of dry matter in the mature ovary of P. monodon
(Millamena and Pascual, 1990, Dy-Penaflorida and Millamena, 1990). This level is high
compared with some other prawn species such as P.indicus which has a total protein and
lipid level of only 54% (Mohamed and Diwan, 1992) and confirms the significance of these
72
nutrients in P. monodon egg production (Primavera and Posadas 1981, Ruangpanit et al
1984, Harrison 1990).
To investigate whether ablation and captivity affect the protein and lipid levels of P.
monodon ovaries prior to and during development, we have conducted a study that
compares prawns dissected (i) immediately after capture from the wild (ie. natural
conditions), and (ii) after being ablated and held in captivity. Comparisons were also
made between hepatopancreas nutrient levels.
73
4.2 Methods
4.2.1 Prawns
Mature female P. monodon between 90 and 130 g in weight (108g±4g) were collected
during the first week of August by beam-trawl in Cook Bay, north Queensland and air
freighted in chilled (20oC) filtered seawater to the Bribie Island Aquaculture Research
Centre (BIARC) in southern Queensland. Upon arrival prawns were allocated randomly to
one of three groups; (i) wild caught, (ii) captive-ablated and (iii) captive-nonablated.
Wild Caught Prawns
For comparison with prawns held in captivity, 67 wild-caught female P. monodon with
ovaries at a range of developmental stages were dissected immediately upon arrival at
BIARC (approximately 20 hours post capture). Ovary and hepatopancreas tissues were
removed, weighed and stored at -70oC until required for biochemical analysis. A section of
ovary was also taken from each individual for histological examination as described below.
4.2.2 Holding Conditions for Captive Prawns
Captive prawns were held in four maturation tanks at an initial density of less than or equal
to 2 per m2. Water and light conditions were as described in Chapter 3. A diet of fresh-
74
frozen squid mantle (Loligo sp.) and mussel (Perna canaliculatus) was fed ad libitum twice
a day.
Captive Prawns: Ablated Treatment
The 80 prawns allocated to the captive-ablated treatment group were eye-tagged with
individual numbers. This group was then divided into five subgroups (each with 16
prawns) such that the average weight of prawns in each subgroup was within 5 g of all
other treatment groups. Individuals in each of the five subgroups were to be sampled during
the five described ovary development stages (0, I, II, III and IV; as described by Primavera,
1982). Prawns in the ‘0’ developmental stage were sacrificed 3 days after ablation to ensure
they did not develop beyond this designated stage.
Four prawns from each sub-group were then stocked in four experimental tanks (ie. there
were 20 ablated prawns per tank). Daily examination of ovary development was carried out
in situ by holding a submerged waterproof torch to the side of each prawn to view the
shadow of the ovary. During the four day acclimation period it was noted that the ovaries
of all individuals regressed such that they were no longer visible by external examination
(Primavera, 1982). Following acclimation, all intermoult prawns were unilaterally eyestalk
ablated while the remainder were ablated over the next two days. Upon reaching the ovary
stage (recognised by external observation) denoted by their sub-group number, prawns
were weighed, moult stage was assessed as per Promwikorn et al (2004), to ensure that all
were at intermoult stage and then individuals were euthanased by immersion in ice water.
75
The hepatopancreas and ovary tissue were then removed, weighed and prepared for
analysis. Specifically, following dissection, a Gonad Somatic Index (GSI) was calculated
for each individual to provide an assessment of ovary development (section 3.2.2.). Tissues
were also stored at -70oC pending analysis for crude protein or lipid content as described in
section 2.2.4. A section of ovary was also taken from each individual for histological
examination as described below.
Captive Prawns: Nonablated Treatment
For control purposes, 18 wild caught prawns were eye tagged and held in the same culture
conditions as those subjected to eyestalk ablation. Prawns were added to the ablated prawns
in the four experimental tanks giving 4-5 nonablated prawns per tank and a total of 24 to 25
nonablated and ablated prawns per tank. Daily examination of ovary development was
carried out as per ablated prawns. Prawns were sacrificed and samples and data collected as
per ablated treatment.
4.2.3 Statistical analysis
Lipid and protein levels were subjected to unbalanced least square, two-way ANOVA
using Genstat (2005). Treatments were GSI level (categorsied to nearest whole unit) and
origin of prawn (wild, captive ablated, captive). Significance level was set at P<0.05 and
post-hoc testing between treatment means was conducted using Tukeys test.
76
4.3 Results
Survival rate of prawns from the captive groups was 95%. Prawns were held in captivity
for a maximum of 16 days by which time sample collection was complete.
4.3.1 GSI and Biochemical Analysis
Ovaries
Ovaries with GSI values up to 9.2 were observed in wild caught prawns and up to 7.4 for
captive prawns subject to ablation. Data are only presented up to GSI 6, however, due to
low sample sizes after this stage (n<3). Table 4.1 contains ovary percentage dry matter at
each GSI stage for each treatment group and shows that ovaries of nonablated prawns did
not develop beyond GSI 3. The results also show significant increases in ovary dry matter
were observed at GSI 4 in the wild caught group and at GSI 3 in the captive ablated group.
Figures 4.1a and 4.1b show the quantity of lipid and protein in ovaries of prawns from
different treatment groups. In the wild and captive-ablated treatment groups, there were
increases in ovary lipid (Fig. 4.1a) and protein (Fig. 4.1b) levels as the GSI value
increased. Specifically, from GSI values 1 to 6, ovaries gained approximately 330mg of
lipid and 1g of protein (per 100g prawn). Importantly, ovary lipid content at GSI values 1
and 2 in wild caught prawns was significantly higher than in captive prawns (Fig. 4.1a).
77
There were, however, significant differences between the captive-ablated and wild prawns in
the rates of protein and lipid accumulation as the ovary developed. To visually illustrate the
pattern of nutrient accumulation Figure 4.2 shows the quantity of lipid and protein that
accumulated in ovary tissue between successive GSI stages (up to GSI 6) expressed as mgs of
dry matter per unit GSI. While GSI does not represent time, this scale still represents a rate of
nutrient accumulation with ovary development.
Hepatopancreas
Figs. 4.3a and 4.3b show the quantity of lipid and protein in the hepatopancreas,
respectively, in prawns from different treatment groups. One notable outcome was that at
GSI values 1-5, lipid levels in the hepatopancreas of captive-ablated prawns were
significantly higher than those from wild caught prawns (Fig 4.3a.), however, this could be
attributed to starvation during the 20 hours (duration of capture and transport) prior to
tissue collection of the wild treatment prawns. Consequently, these results are not
considered to be a treatment effect. The major findings presented in Figure 4.3 was that for
the wild and ablated treatment groups, the level of lipid in the hepatopancreas of prawns
with a GSI value of 2 was significantly lower than those from prawns with a GSI value of
1. A second decrease in hepatopancreatic lipids was also significant between GSI values 5
and 6 for ablated prawns. A significant reduction in hepatopancreas protein level was also
observed at GSI 2 in the captive-ablated prawns (Fig.4.3b).
78
4.3.2 Histology
Table 4.2 shows GSI values at which the three ovarian histology stages occur in ablated
and wild groups of prawns. The histological status of oocytes at each GSI stage was
determined using the criteria of Tan-Fermin and Pudaderas’ (1989) for developmental
stages of previtellogenic, vitellogenic and early cortical rod. Mean oocyte diameter was
significantly different between ablated and wild groups in the vitellogenic stage of
development.
Table 4.1. Average percentage values of dry matter in prawn ovaries with different GSI values.
Treatment GSI
1 2 3 4 5 6
Wild caught 26.3+0.4a
(8)
25.61±0.5a
(12)
26.1±0.3a
(11)
29.4±0.5b
(18)
32.6±0.8b
(10)
31.1±0.4b
(8)
Captive-
ablated
21.6+0.5c
(12)
22.7±0.9c
(7)
27.4±0.3a
(13)
29.3±0.1b
(15)
29.3±0.3b
(15)
30.7±0.3b
(14)
Captive-non
ablated
23.5+0.4 ca
(9)
24.1±1a,c
(7)
25.2±1a,c
(2)
Dry matter is expressed as a percentage of the wet tissue weight. Sample size is indicated by the
number enclosed within parentheses (n). Dry matter percentages with the same superscript within
both rows and columns are not significantly different (P<0.05)
79
0
100
200
300
400
500
1 2 3 4 5 6
mgs
lipi
d / 1
00g
praw
n
a a a abb
a
12
2
1
31
1
3
4
5
45
2
6
6
0
200
400
600
800
1000
1200
1400
1 2 3 4 5 6
GSI
mgs
pro
tein
/ 10
0 g
praw
n b
1 111
1
2
2 23
5
4
1
6 4
3
Figure 4.1
Lipid (a) and protein (b) content (mg ± se per 100g wet prawn) at successive GSI stages in
the ovary of wild caught (black), ablated (white) and nonablated (grey) prawns. Bars at the
same GSI stage with different letter superscripts are significantly different from each other
(p<0.05). Across GSI stages, within treatment (wild caught, ablated, or non-ablated) bars
with different number superscripts are significantly different.
80
1 to 2 2 to 3 3 to 4 4 to 5 5 to 6
AblatedWild
01020
30
40
50
60
70
80
90
Mgs
of l
ipid
GSI
Figure 4.2.
Average quantities (mgs per wet weight ovary) of protein (a), and lipid (b) in captive-
ablated and wild P. monodon accumulated between successive GSI stages.
1 to 2 2 to 3 3 to 4 4 to 5 5 to 6Ablated
Wild0
50
100
150
200
250
300M
gs o
f pro
tein
GSI
a
b
81
0
200
400
600
800
1000
1 2 3 4 5 6
mgs
lipi
d / 1
00g
praw
n
a
c
b
aa
b
aa
b aa
bb
a
1
1,2
11
2
2
1
1,2
1,2
1
1
1
1
2
1
0
100
200
300
400
500
600
1 2 3 4 5 6GSI
mgs
pro
tein
/ 10
0 g
praw
n
b1
12 1
11
1
1 1
1
1
1,211
1
Figure 4.3
Lipid (a) and protein (b) content (mg ± se per 100g wet prawn) at successive GSI stages in
the hepatopancreas of wild caught (black), ablated (white) and nonablated (grey) prawns.
Bars at the same GSI stage with different letter superscripts are significantly different from
each other (p<0.05). Across GSI stages, within treatment (wild caught, ablated, or non-
ablated) bars with different number superscripts are significantly different.
82
Table 4.2. GSI values and externally identified development stages (0-IV) for the three
histological stages, P (previtellogenic), V (vitellogenic) and ECR (early cortical rod) of
ovaries from wild and captive-ablated P. monodon.
External
stage*
Oocyte
Stage*
Oocyte diameter (µm) GSI
n = 10
Ablated Wild Ablated Wild
0-I P 45.6± 1.51
(105)
52.6±0.91
(96)
1.4 ± 0.5 1
(1.2- 2.9)
1.4 ± 0.21
(1.3-3.2)
I-III V 198.0± 1.7a2
(111)
233±1.2 b2
(120)
4.4 ± 0.9 2
(1.9- 5.9)
5.0 ± 0.42
(3.1- 7.0)
III-IV Early CR 240.3± 2.03
(95)
261±1.92
(87)
5.9 ± 1.1 3
(3.8- 7.1)
6.5 ± 1.0 3
(5.4- 9.2)
GSI mean ± se and (range) and oocyte diameter mean± se and (cells counted) are presented
for each developmental stage.
* Stages as determined by the criteria of Tan-Fermin and Pudadera (1989).
Mean values in the same row with different letter superscripts are significantly different
(P<0.05) between ablated and wild for the variable measured. Mean values in the same
column with different number superscripts are significantly different (P<0.05) between
stages.
83
4.4 Discussion
A significant finding of the current study was that undeveloped ovaries appear to be
affected by short term changes in environmental conditions (captivity). Specifically, the
captive conditions of this study appeared to cause a reduction in the lipid content of
previtellogenic ovaries, presumably by causing depletion of lipids (ovaries regressed from
their pre-capture condition) or by reducing lipid accumulation. Lipid levels in mature
ovaries, however, were not affected by ablation and captivity. The factors and mechanisms
responsible for the observed changes in the previtellogenic ovary remain to be determined.
It is possible the early accumulation of lipids is triggered by specific seasonal environmental
cues, including dietary factors (Crocos et al 1997), via a stimulatory hormone (for review see
Khoo 1988 and Huberman 2000, Mendoza et al 1997). For P. monodon these cues are
evidently lacking in the captive environment as not only were lipid levels low but ovaries
failed to enter the vitellogenic stages of development.
In accordance with Quackenbush’s (2001) suggestion that adequate reserves are necessary
before spontaneous ovary development will occur, the low pre-vitellogenic lipid levels may
prevent the onset of vitellogenesis (yolk accumulation) in P. monodon. Examination of
oocytes at GSI 1 and 2 (previtellogenic ovaries) showed most were in the perinucleolus
stage of development (Yano 1988) with no significant differences in the average diameter
for the captive and wild treatments. The increased size of follicle cells surrounding oocytes,
as was observed for some of the larger oocytes in the ovaries of wild prawns at GSI 2 (data
84
not shown), however indicates that the ovaries were entering the oil globule stage that
occurs prior to yolk accumulation (Yano 1988).
While further studies are required to verify the significance of early ovary lipid levels, there
is an increasing body of evidence to support the hypothesis that the spawners undergoing
spontaneous ovary development are ‘primed’ and have already undergone specific
physiological changes (Adiyodi and Adiyodi 1970, Thurn and Hall 1999, Vincent et al
2001, Arcos et al 2003, Tsutsui et al 2005) which possibly involve oil globule formation
and lipid accumulation (Yano 1988). Similarly, the previously reported variation in ovary
response to ablation (Hansford and Marsden 1995) may be related to the completion of this
step which in turn is influenced by genetic, age-related and/or environmental factors
(Crocos et al, 1997, Palacios and Racotta 2003, Arcos et al, 2004, 2005).
In addition to the previtellogenic lipid content, this study revealed differences in the pattern
of nutrient accumulation between ovaries matured in the wild and ovaries matured in
captivity following ablation. Timing of nutrient accumulation may also be critical to final
egg yolk quality as the ovary composition has been found to vary with stage of
development. For example, in studies on P. semisulcatus the types of protein accumulating
(Avarre et al 2001) as well as lipid classes and the percentage of lipid synthesised in the
ovary that was bound to vitellin, were found to change during ovary development (Shenker
et al 1993, Ravid et al 1999). Similarly research to date indicates the synthesis of the two
major yolk components, vitellin and CR proteins are separate and stage dependent
processes in penaeid prawns (Rankin and Davis 1990, Quinitio and Millamena 1992,
85
Kawazoe et al 2000, Avarre et al 2001, Khayat et al 2001, Quackenbush 2001, Yamano
et al 2003, 2004). In addition, Vazques-Boucard et al (2002) suggests that the
hepatopancreas and ovary in Fenneropenaeus indicus have separate but complimentary
roles in vitellogenin synthesis. This also appears to be the case for P. monodon where ovary
synthesis of vitellogenin is high during previtellogenic and early vitellogenic stages then
decreases (Thurn and Hall 1990) as vitellogenin levels increase in the haemolymph
indicating an increase in hepatopancreas vitellogenin synthesis (Vincent et al 2001,
Longyant et al 2003) or its retention in the haemolymph.
Hence it is feasible that the change in nutrient accumulation patterns caused here by
captivity and or ablation, could affect egg quality by compromising the completion of one
or more of the processes involved in vitellogenesis. Interestingly, while captivity and/or
ablation did affect the GSI stage at which the peaks in protein and lipid accumulation
occurred in the ovary, it did not affect the stage at which lipids were mobilised from the
hepatopancreas. Thus there may be some independence in terms of how these factors affect
the ovary and hepatopancreas tissues. Based on findings that ablation increased levels of
vitellogenin mRNA levels in prawn ovaries but not in the hepatopancreas tissue, Tsutsui et
al (2005) suggest that ovary development in response to ablation will differ to when it
occurs naturally and this may contributing to poor egg quality. The changes demonstrated
in the current study may in part account for the inferior egg production reported for captive
ablated P. monodon prawns when compared to prawns spawned immediately after capture
from the wild (Coman et al 2006 ).
86
The role of the hepatopancreas in supplying nutrients to the developing ovary has been
confirmed for a number of prawn species including P. monodon (Dy-Penaflorida and
Millamena, 1990; Millamena and Pascual, 1990, Tseng et al, 2001). In support of these
findings, the current study showed a decrease in hepatopancreas lipid content between GSI 1
and 2 (242 mg) with an associated increase in the lipid content of spontaneously developing
ovaries between GSI 2 and 3 (33 mg). Hepatopancreas protein levels also showed a decrease at
this stage despite the rapid turnover of hepatopancreas protein making its production and
transfer difficult to quantify (Hewitt 1992). The amount of lipid mobilised in P. monodon was
in excess of immediate ovary uptake. This is in contrast to the findings for P.indicus where
lipid mobilised from the hepatopancreas was insufficient to account for increases in the ovary
lipid content (Galois, 1984, Vazques-Boucard et al, 2002). The excess lipid from the P.
monodon hepatopancreas may be; 1) contributing to the general energy requirements of the
prawn at that specific ovary development stage or, 2) combined with vitellogenin proteins and
stored in the haemolymph until ovary uptake after GSI 3. This latter proposal is supported by
Thurn and Hall’s (1999) finding that vitellogenin levels in the haemolymph were high during
pre and early vitellogenesis in P. monodon.
Ablation and captivity appeared to increase the amount of lipid mobilised from the
hepatopancreas in prawns undergoing ovary development when compared to those
developed in the wild. Despite the low levels of lipids in the hepatopancreas at all GSI
stages in the wild treatment group compared to the captive (which is credited to starvation
during the 18 hr transportation period), the differences in lipid content between each GSI
stage are taken to represent stage-specific mobilisation. Based on this premise, the amount
of lipid mobilised between GSI 1 and 2 for the captive-ablated prawns (439 mg) was nearly
87
twice the amount mobilised in wild prawns (242 mg). This increase is thought to be
necessary to meet the lipid requirements of the rapidly developing ovaries in ablated
prawns (although, as with spontaneous developing ovaries, it was in excess of associated
ovary increase of 80mg). With this high mobilisation rate of hepatopancreas lipids it is
possible that ablation, and/or captive conditions, could cause a shortfall in lipids available
from the hepatopancreas for ovary development during later maturation cycles as has
previously been proposed (Beard and Wickins 1980, Palacios et al 1999, Vazquez-Boucard
2004).
A second significant finding in relation to hepatopancreas changes was a drop in the lipid
levels at GSI 6 in ablated-captive prawns. Despite this also appearing as a trend in prawns
developing in the wild, this second decrease has not previously been reported and its
significance is unknown. It occurs when oocytes are at the early cortical rod (CR) stage of
ovary development, and as CRs have no structural requirements for lipids (Khayat 2001) it
is not clear why there is a higher demand for lipids at this GSI stage. It is, however, a stage
that has generated recent interest in terms of hormonal control, as captive breed prawns can
suspend ovary development at this stage (Yamano et al 2004, Qui et al 2005).
Conclusions
The captive conditions of this study and the process of ablation did not impact on the
quantity of protein and lipid in ovaries that mature during the first post ablation cycle of
ovary development. They do, however, alter the lipid content of previtellogenic ovaries and
88
mobilisation of hepatopancreas reserves. Together these factors determine whether ovary
development proceeds in P. monodon.
The early stages of ovary development require further investigation as the condition of the
immature ovary may help determine whether development proceeds. The influence of the
eyestalk inhibitory hormones in early nutrient accumulation is of particular interest. In
addition, further study of lipid quality and quantity in mature ovaries is warranted for this
species. In particular, the impact of this nutrient on reproductive performance (ie. egg and
larval quality) is not fully understood.
89
Chapter 5.
THE EFFECTS OF ABLATION AND STARVATION OF THE PRAWN PENAEUS
MONODON ON PROTEIN AND LIPID CONTENT IN OVARY AND
HEPATOPANCREAS TISSUES.
5.0 Abstract
To further investigate factors effecting previtellogenic ovaries of Penaeus monodon, an
experiment was conducted whereby wild caught prawns were held for ten days in captivity
and allocated to one of four treatments groups; (i) fed, (ii) fed and ablated, (iii) starved, and
(iv) starved and ablated. Prawns were held in a confined space which had previously been
shown to prevent ovaries from advancing beyond the previtellogenic stage of development.
Results showed that when prawns were held in captivity, their ovaries regressed from the
pre-capture development stage of early vitellogenesis. Starvation increased the extent of
this regression and also caused a decrease in the size of the hepatopancreas. Most
importantly, ablation reduced the depletion of nutrients from the ovary and hepatopancreas
that was caused by starvation. Specifically, final levels of protein and lipid in the ovary
and protein levels in the hepatopancreas of prawns in the starved and ablated treatment
group were not significantly different to the fed treatment group. These findings suggest
that (i) eyestalk neuropeptides are involved in regulating tissue reserves prior to
vitellogenesis and that this is an endocrine control point for ovary development and, (ii)
90
both the ovary and hepatopancreas contribute nutrients (protein and lipids) to meet
metabolic requirements during periods of food deprivation.
91
5.1 Introduction
In chapter 4 we confirmed that, when held according to current industry practises, Penaeus
monodon broodstock rarely undergo spontaneous ovary development and that ablation is
required to artificially trigger this process. Importantly, it was also determined that captivity
resulted in a reduction in the lipid content of previtellogenic ovaries. After ablation,
vitellogenesis proceeded (ovaries matured) and the rate of synthesis and/or accumulation
was such that there was no detectable effect of captivity and/or ablation on the lipid levels
by the time the ovary had reached maturity (Ch 4). As discussed previously in Ch 4, the
level of lipid at this stage may influence spawning rate. It has been proposed that the
nutritional status of the broodstock is indicative of an individual being ‘primed’ for
breeding. For example, priming may be a necessary step before ablation can trigger
vitellogenesis (Quackenbush 2001) and may also involve oil globule formation and lipid
accumulation (Yano 1988). The variable effect of ablation on P. monodon reproductive
performance (Hansford and Marsden 1995, Marsden et al 1997) may therefore be related to
the extent of the priming.
Thus the previtellogenic ovary and factors that influence its development were considered
worthy of further investigation. Of particular interest was the impact of ablation on the
ovary at this stage as most of the research to date focuses on the vitellogenesis inhibiting
hormone (VIH) and its regulation of the later vitellogenic stage of development (for review
see Huberman 2000). Information on the endocrine regulation of early nutrient
92
accumulation is considered necessary to increase control over prawn reproduction and
thereby the commercial viability of this aquaculture species.
Accordingly, the aim of the current study was to determine 1) the impact of short term
starvation on the size and composition of previtellogenic ovary and hepatopancreas
tissues, and 2) if ablation alters the effect of short term starvation on tissue size and
composition.
93
5.2 Methods
5.2.1 Prawns
P. monodon females (90 to 100 g) were captured during September and air freighted to
BIARC. Upon arrival prawns were weighed and their moult-stage assessed (Promwikorn et
al 2004). Forty prawns in post moult and early intermoult (B-C) stage were selected for
the experiment.
5.2.2 Holding conditions and experimental design
To ensure prawns were previtellogenic and to arrest ovary development at this stage, each
prawn was placed in a confined space that had previously been shown to cause developing
ovaries to regress and to prevent immature ovaries from developing (data not shown).
Under these conditions vitellogenesis was prevented even if prawns were ablated.
Eight prawns were sacrificed on arrival at BIARC to act as controls and their ovary and
hepatopancreas tissues removed for biochemical analysis. The remaining 32 prawns were
allocated to one of four treatment groups: (1) fed, (2) fed and ablated, (3) starved and (4)
starved and ablated giving eight prawns per treatment such that mean weight of prawns in
each group was within 5 g.
94
Prawns housed individually in a confined space; black Polyethylene tanks (0.91m x 0.5m x
0.6m). Seawater was maintained at 28oC and 36 ppt salinity, continuously exchanged at a
rate of 100% per day. Fed groups received a diet of fresh-frozen squid mantle (Loligo sp.)
and mussel (Perna canaliculatus). Feeding was ad libitum twice a day and food intake was
monitored to confirm that feeding had occurred.
On day 10, prawns were sacrificed and their ovary and hepatopancreas tissues were
removed for biochemical analysis (3.2.4). The GSI & HSI was determined and the tissued
were stored for later measurement of dry matter, lipid and protein as described previously
(3.3.2).
5.2.3 Statistical Analysis
Experimental treatment effects were assessed using ANOVA and Tukey post-hoc tests with
significance level set at P<0.05.
95
5.3 Results
Survival of treatment prawns was 100%. Table 5.1 shows that captivity significantly
decreases (p<0.05) GSI values in all treatment groups when compared with the wild
caught controls that were in the early vitellogenic stage of development. Interestingly,
prawns that were starved, but also ablated, had GSI values which were not significantly
different from fed-captive animals.
Dry matter, protein and lipid levels in the ovaries of captive-held prawns were generally
significantly lower than those in the ovaries of prawns sacrificed immediately on arrival
at BIARC (Control group) (Table 5.2). Furthermore, protein and lipid values in ovaries
from starved nonablated prawns were significantly lower than those from captive-fed
prawns. Interestingly, however, ovarian protein and lipid levels in starved prawns subject
to ablation were not significantly different from fed, nonablated captive individuals.
The HSI values shown for the control prawns (Table 5.1 and 5.2) were strongly
influenced by the period of starvation (approximately 20 hrs) during transportation. It is
included to indicate the starting condition of prawns. HSI (Table 5.1), dry matter, protein
and lipid content (Table 5.2) in the hepatopancreas of captive fed prawns were
significantly reduced by ten days of starvation. Specifically, when captive prawns were
starved for the 10 days dry matter, protein and lipid content was significantly reduced
(p<0.05). In particular, lipid content decreased by approximately 90%.
96
Table 5.1 Influence of Starvation and ablation on GSI and HSI values.
Treatment GSI HSI
Control 3.7± 0.5 c 2.4 ± 0.1 b,c
F 2.2 ± 0.3 b 2.6 ± 0.2 c
FA 2.4 ± 0.5 b 2.3 ± 0.1 b
S 1.2 ± 0.1 a 1.6 ± 0.1 a
SA 2.0 ± 0.3 b 2.1 ± 0.3 b
Values are mean ±se (n=8). Control = initial (wild caught) condition, F = fed; FA = fed and
ablated; S = starved; SA = starved and ablated. Identical superscripts denote treatment
means that are not significantly different (P<0.05) within columns.
97
Table 5.2. Mean level of protein and lipid in the ovary and hepatopancreas tissues.
Treatment Ovary Hepatopancreas
Dry matter
(%)
Protein
(mg)
Lipid
(mg)
Dry matter
(%)
Protein
(mg)
Lipid
(mg)
Control 27.8±1.1 c 632±32 c 200±26 d 31.5±1.3 b 198±23 a 173±42 b
F 24.1±1.4 b 346±23 b 74±16 bc 37.8±1.0 c 353±24 b 345±47 c
FA 26.6±1.2 b,c 400±32 b 99±14 c 37.5±1.1 c 320±23 a,b 300±55 b,c
S 19.2±0.8 a 134±19 a 15±6 a 20.0±0.9 a 189±15 a 30±8 a
SA 20.3±0.9 a 283±25 a,b 38±12 a,b 21.6±0.9 a 297±18 ab 43±7 a
Values are (mean mg per 100 g prawn ± se) (n=8). F = fed; FA = fed and ablated; S =
starved; SA = starved and ablated. Identical superscripts denote treatments that are not
significantly different (P<0.05) `within columns.
98
5.4 Discussion
A significant finding of the current study was that ablation increased or retained nutrient
levels in P. monodon ovaries despite the ovaries remaining arrested in the previtellogenic
stage of development. This was evident in both fed and starved prawns. In the fed prawns
the slight increase in the dry matter and lipid content of ovaries was consistent with the
findings of Palacios et al (1999) who showed ablation caused an increase in the number
of lipid droplets in the immature ovaries of P. vannamei. In starved prawns, ablation
dramatically reduced the decline that starvation caused in ovary nutrient levels.
Specifically, protein and lipid levels in previtellogenic ovaries of starved prawns were not
significantly different to those of the fed prawns.
The increase in nutrient levels in the vitellogenic ovary as a result of ablation indicates
that the removal of the eyestalk reduces a factor inhibiting development at this stage. This
factor may be the vitellogenesis inhibiting hormone (VIH), one of the eyestalk inhibiting
hormones, which is known to regulate synthesis and accumulation of egg yolk (vitellin)
and its precursors (vitellogenin) in both the ovary and hepatopancreas, respectively
(Tsutsui et al 2005, Okumura et al 2004, Brady in prep., Thurn and Hall 1999, Coman et
al, 2006). Alternatively, the elevated nutrient levels may be due to one or more of the
other hormones in the sinus gland (SG) which have been shown, individually or in
combination, to negatively regulate a number of physiological processes including lipid
metabolism. SG extracts have recently been shown to influence synthesis of non-vitellin
proteins in previtellogenic prawn ovaries (Avarre et al 2001, Tsutsui et al 2005). Most of
99
these proteins have associated lipids (Shenker et al 1993). Regardless of whether these
nutrients are components of vitellin, their early accumulation may represent an important
control point for hormones affected by ablation. Progression beyond this point depends
on levels of SG inhibitory hormones and other factors such as holding conditions as was
demonstrated by the arrested development of ovaries in the current study.
The current study also showed that ablation affected the nutrient levels in the
hepatopancreas of previtellogenic prawns. Notably, in starved prawns it increased
protein, and to some extent lipid, content in the hepatopancreas. The hepatopancreas is a
multifunctional tissue involved in a diverse range of metabolic activities including,
protein, lipid and carbohydrate metabolism and lipid storage (Yepiz-Plascencia et al
2000, Sánchez-Pa et al 2007). An increase in metabolism as a result of ablation (Chen
and Chia 1995) may have increased the synthesis and storage or, alternatively, reduced
the depletion rate of proteins and lipids. It is possible that the nutrients are components of
egg yolk and are being retained for later mobilisation to the ovary. For example, ablation
may result in the transcription and translation of vitellogenin genes in the hepatopancreas
with nutrients being preferentially supplied to the tissue for this purpose. However,
whether at this very early stage of development the hepatopancreas reserves are destined
for the ovary (Tiu et al 2006, Tseng et al 2001, Thurn and Hall 1999, Ch 4) remains to be
determined.
In contrast to ablations’ affect on the hepatopancreas of starved prawns, in fed prawns it
caused a decrease in HSI which was also reflected in both the protein and lipid content of
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the tissue. It is possible that ablation triggered mobilisation of vitellogenin components
destined for the ovary despite the GSI (average 2.2) showing vitellogenesis had not
commenced. This mobilisation prior to uptake of nutrients by the ovary (which marks the
onset of vitellogenesis) was previously noted in Ch 4. In addition, the effect of ablation
on the hepatopancreas of both fed and starved prawns supports recent evidence that the
SG hormones can act independently on the hepatopancreas and ovary tissues. For
example, based on gene expression studies, Okumura et al (2004) suggests prawn
vitellogenin synthesis is regulated separately in the ovary and the hepatopancreas.
The study also highlights the ability of captive environments to prevent ablation from
initiating vitellogenesis. In contrast to results presented in Ch 4, where conditions were
based on industry best practise, no ablated prawns in the current study advanced to the
vitellogenic stage of development. The environment of the captive held prawns was
evidently stressful and/or lacking in a required stimulatory factor. While the ‘suitability’
of the environment has been defined for a number of water quality parameters (Primavera
1984) and dietary components (for review see Wouters et al 2001), there remain
unidentified elements that evidently prevent spontaneous development. Quackenbush
(2001) emphasised the importance of diet by suggesting the function of VIH is to restrain
yolk synthesis until suitable organic reserves are in place in the hepatopancreas and/or the
ovary.
It has previously been proposed that the physiological outcome of ablation is a function of
both inhibitory and stimulatory hormones (Fingerman 1987). A number of hormones have
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been shown to have a stimulatory affect on ovary development (for example Charniaux-
Cotton 1985, Quackenbush 1986, Huberman 2000). The critical environmental or dietary
factors lacking in the captive environment, may be active through the endocrine system
and involve a stimulatory hormone produced in situ in response to environmental change,
and/or provided by the broodstock diet (for review see Harrison 1990 and Wouters 2000).
Future studies need to further investigate whether the captive environment is arresting
ovary development though the presence of stressful conditions or through the lack of
essential stimuli. Proposed stimulating hormones are a logical next step in research aimed
at increasing our knowledge of hormonal regulation of ovary development.
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Chapter 6
METHYL FARNESOATE AS A POTENTIAL HORMONE FOR STIMULATING
OVARY DEVELOPMENT AND INCREASING EGG HATCH RATE IN THE
BLACK TIGER PRAWN, PENAEUS MONODON
6.0 Abstract
There is mounting evidence that the terpenoid hormone methyl farnesoate (MF) plays
important roles in regulating reproductive processes in crustaceans. In particular, MF has
been shown to increase early stage ovary development and mating success. It was
therefore considered a good candidate for improving reproductive performance as it was
these criteria that were reduced by holding or rearing of Penaeus monodon. To this end,
and to gain further information on its roles and possible modes of action, MF was orally
administered to ablated Penaeus monodon at a concentration of 5.5 um per gram of diet,
and a range of reproductive performance criteria measured. Results confirmed that MF
can influence the reproductive process of this species. Specifically, under the conditions
of this study, MF inhibited late stage ovary development and reduced fecundity in ablated
prawns. The impact of the artificial diet (without additional MF), relative to a squid-
mussel diet, was also assessed in this study and although it increased the quality of larvae
produced, it also increased inhibition of late stage ovary development. Thus while the
current study has increased our knowledge of MF by isolating an ovary developmental
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stage at which MF regulates reproduction in P. monodon, factors that determine the
extent of its effect and whether it has a stimulatory or inhibitory effect, remain unknown.
Until these factors are identified, the application of MF as a means of predictably
manipulating egg production in captive prawns remains problematic.
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6.1 Introduction
Penaeus monodon is one of the most difficult penaeid species to breed in captivity,
indicating that it is sensitive to environmental conditions. As previously discussed,
unilateral eyestalk ablation is used routinely by hatchery operators to accelerate ovary
development (Primavera 1984). Nevertheless this crude method for reducing levels of
inhibitory neuropeptides (specifically vitellogenesis inhibiting hormone (VIH); for
reviews see Keller 1992 and Huberman 2000) is not always effective in inducing prawn
ovarian development and spawning (Aquacop 1977, 1979, Beard and Wickens 1980,
Arnstein and Beard 1975, Hansford and Marsden 1995, Marsden et al 2007). It has been
proposed that in addition to the requirement for VIH levels to be reduced, stimulatory
hormones are required to promote ovary development (Charniaux-Cotton 1985,
Quackenbush 1986, Huberman 2000) and that they operate in response to environmental
cues that for P. monodon are lacking in captive environments (Tsutsui et al 2005). Earlier
studies (Chapter 4 and 5) suggested that cues supplied through the natural environment
may be critical to ensuring the adequate nutritients are present in the ovary (and possibly
the hepatopancreas) before yolk accumulation (vitellogenesis) can commence. Based on
earlier research, it has been proposed that a stimulatory hormone is involved in this early
stage of ovary development.
Many stimulatory hormones have been proposed as regulators of crustacean reproduction
(Huberman 2000) including methyl farnesoate (MF), a terpenoid hormone synthesised in
the mandibular organ (MO). MF has been implicated in a wide range of hormonally
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regulated processes in crustaceans (Kuballa et al 2007, Nagaraju et al 2004, Lovett et al
2001, Soroka et al 1993, Liu et al 1997, Sagi et al 1994, Freeman and Costlow 1980) and
there is a significant body of evidence to show MF stimulates or enhances various aspects
of crustacean reproduction (Nagaraju et al 2004, for review see Laufer and Biggers
1992). For example, in vitro increases in levels of MF have been correlated with
increased prawn oocyte diameter (Tsukimura and Kamemoto 1991, Laufer et al 1997)
and with increased Vg (vitellogenin) gene expression in the hepatopancreas and ovary of
the prawn Metapenaeus ensis (Tiu et al 2006) and the red crab Charybdis feriatus (Mak
et al 2005). In addition, male gonad size and mating rates have been shown to increase in
various crustacean species following administration of MF in vivo (Homola et al 1991,
Sagi et al 1994, Laufer et al 1993, Nagaraju et al 2004).
Of particular significance are the results of previous in vivo studies that demonstrated that
inclusion of MF in broodstock diets induced a dose dependent increase in fecundity
(Laufer 1992 and Laufer et al 1997), spawning frequency and larval survival (Laufer
1992) in ablated L. vannamei and increased fecundity, hatch rate and fertility in ablated
P. monodon (Hall et al 1999). These findings suggest that the combination of eyestalk
ablation with orally administered MF may have the potential to improve the quantity and
quality of larvae produced in commercial P. monodon hatcheries.
In terms of practical application, little is known of the mechanism by which MF regulates
specific aspects of reproduction such as ovary development, fecundity and hatch rate.
Also unclear is, the stage of ovary development at which MF has the most significant
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regulatory effect (Wainright et al 1998, Nagaraju et al 2004, 2006). For instance, while
the MO’s secretion of MF in prawns is highest during the vitellogenic stage of ovary
development (Laufer et al 1986, 1987), immature oocytes have been shown to increase in
size in response to MF administration (Tsukimura and Kamemoto 1991). Likewise, in
crab haemolymph, MF levels were shown to be highest during pre and early
vitellogenesis (Nagaraju et al 2004, Ruddell et al 2003).
A lack of conformity in the results achieved to date, both within and between prawn
species (Laufer 1992, Hall et al 1999) indicates a need for further studies to evaluate the
potential of orally administrated MF as a practical means for improving egg and larvae
production in P. monodon broodstock. A high level of predictability will be essential for
any commercial application of MF in the culture of this species.
The current study aimed to test whether inclusion of MF in broodstock (male and female)
diet, in conjunction with eyestalk ablation, provides a method for increasing larval
production from P. monodon broodstock.
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6.2 Methods
This study compared the effect of three diets on the reproductive performance of ablated
prawns. A formulated diet (BIARC) was used as a vector for oral administration of MF.
Two control diets were included; the BIARC diet without MF and a fresh diet of fresh
frozen seafood (as described below). The natural diet was included to isolate the effect of
the artificial diet.
6.2.1. Prawns and holding conditions
After arrival at the Bribie Island Aquaculture Centre (BIARC) individual prawns were
weighed, eye-tagged and carapace tagged (for monitoring moult intervals). Prawns were
then allocated to weight classes (60±5 g, 70±5 g etc) and representatives from each
weight class were then allocated randomly to each diet treatment group.
After 14 days, ovary development was assessed visually by shining a torch from the
ventral side of the prawn and observing the shadow caste by the ovary from the dorsal
side. This was to ensure ovaries showing signs of development at time of capture had
regressed over this period. As all prawns were found to be at 0 stage of development
according to Primavera’s (1985) classification, inter-moult prawns were immediately
ablated, while remaining prawns were ablated within the next few days. Ovary
development, egg and larvae monitoring were carried out according to methods described
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in Marsden et al (1997) for 42 days post-ablation. Two days after moulting, prawns had
their carapace tags replaced in accordance with their eye-tag number.
There were 28 female and 14 male prawns per diet treatment (Natural, BIARC,
BIARC+MF), divided equally between 2 replicate tanks (4m diameter, 0.8m water depth)
giving a tank density of 1.75 prawns per m2. Water was maintained at 28°C, filtered to
25µm and exchanged at 200% per day. Light was provided by suspended fluorescent
fittings wrapped in green 70% ‘shade cloth’ (Dindas Lew Cat No. 5c7036 BL) to reduce
light intensity to 0.5µEm-2 sec-1. Day length was set at 14 hours with a 20 minute ramp
period.
6.2.2 Diets
Three diets were evaluated: a fresh diet, a formulated maturation diet (named BIARC)
and the BIARC+MF diet. The fresh diet consisted of chopped, fresh-frozen green-lipped
mussel (Perna canaliculus) and squid mantle (Loligo sp) fed alternatively (for estimates
of biochemical analysis see Marsden et al 1992). The formulated diet (BIARC) was
processed into moist, ‘spaghetti like’ (4mm diameter) strands. The proximate analysis of
this diet has been described previously (Marsden et al, 1997). It is important to note that
in previous studies P. monodon broodstock fed this artificial diet demonstrated equivalent
or superior reproductive performance to those fed a fresh diet (Marsden et al, 1997). For
the BIARC+MF diet, MF (2E6E) dissolved in acetone was added to the lipid component
of the BIARC ingredients during diet preparation to attain a final concentration of 5.5µg
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MF per gram of wet weight diet. The equivalent volume of acetone was also included in
the BIARC diet without the MF. This MF concentration was chosen to maintain
consistency with the study of Hall et al (1999), and Laufer (1992). Prawns were fed to
excess at 0900hr and 1700hr daily.
6.2.3. Statistical analysis
Differences in spawning performances criteria were analysed using one-way ANOVA
with replication (tanks). Differences between treatment means were analysed using a
LSD pair wise comparison of means. The level of significance for results was set at
P<0.05.
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6.3 Results
As shown in Table 6.1, there was no significant difference (p>0.05) in average weight
gain or moult interval of female prawns in response to any of the experimental diets.
Survival rates for all diets were very high, ranging from 96% to 100%. By contrast, both
diet and the addition of MF to the artificial diet affected late stage ovary development.
Specifically, the number of prawns arrested at stage III of ovary development was 5 times
higher when the BIARC diet replaced the fresh diet. The inclusion of MF in the artificial
BIARC diet induced an additional two fold increase in the number of prawns with ovary
development arrested at stage III.
The reproductive performance criteria measured for prawns fed the three diets is shown
in Figure 6.1. The addition of MF to the artificial diet significantly reduced the number of
spawns per prawn from an average of 3.0±0.4 (BIARC) to 1.8 ± 0.3 (BIARC+MF) (Fig
6.1A). The addition of MF to the BIARC diet also significantly reduced average
fecundity of the first three spawns from 4,100 to 3,200 eggs per gram of prawn (Fig
6.1C). Dietary MF, however, had no significant effect on average egg hatch rate or larval
survival (averaged over the first three spawns), fecundity of the first spawn and the
number of protozoea 1 (Z1) per gram of prawn or per spawn (Figs 6.1D, 6.1E, 6.1B,
6.1F, 6.1G).
Figure 6.1H shows the total Z1 output per prawn fed the BIARC diet (3.0±0.4million)
was not significantly different to that obtained for the Natural diet (4±0.5 million). Z1
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output from prawns fed the BIARC+MF diet (1.9±0.4 million), however, was
significantly lower than from prawns fed the Natural diet although not significantly
different from prawns fed the BIARC diet.
Analysis of data also indicated significant differences in reproductive performance of
prawns fed fresh or artificial diets. For example, the number of spawns per prawn
obtained using the Natural diet was significantly higher than obtained using the BIARC
diet (Fig 6.1A). By contrast, the survival rate of larvae to Z1 obtained using the BIARC
diet was significantly higher than that obtained using the Natural diet (Fig 6.1E).
112
Table 6.1. The mean (±SE) survival, start weight, percentage weight gain (average
weight gain (g)/starting weight x 100), moult interval, the percentage of prawns that
developed to stage III and the percentage that did not spawn for female P. monodon
(n=28 per treatment) 42 days after ablation in each of the three diet treatments.
Diet Survival
(%)
Start
weight
(g)
% weight
gain (g)
Moult
interval
(days)
Ovary
development
progressed to
stage III (%)
Development
arrested at stage
III (%)
Natural 96.4 74.4±4.1 25.6±2.3 18.5±3.9 100 3.6±0.01c
BIARC 100 74.9 ± 4.7 20.3±3.5 18.3±3.1 96.4 17.8±0.9b
BIARC+MF 100 76.2 ± 4.8 26.1±2.7 18.0±2.9 100 39.3±1.2a
Values with different superscripts within columns indicate significant (P<0.05)
differences between diet treatments.
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G
0
0.5
1
1.5
Natural BIARC BIARC +MF
zoea
1 (1
0E5/
spaw
n)
a a a
D
02040
6080
Natural BIARC BIARC +MF
hatc
h ra
te (%
) aa
aC
0
2000
4000
6000
Natural BIARC BIARC +MF
eggs
/gra
m p
raw
n
ab ab
B
0
1000
2000
3000
Natural BIARC BIARC +MF
eggs
/gra
m p
raw
n a a a
020406080
100
Natural BIARC BIARC +MF
surv
ival
(%) a
b b E F
0
500
1000
1500
2000
Natural BIARC BIARC+MF
zoea
1 (N
o/g/
spaw
n) a a a
H
012345
Natural BIARC BIARC +MF
zoea
1 (1
0E5/
praw
n) a
abb
A
012345
Natural BIARC BIARC +MF
spaw
ns/p
raw
n ab
c
114
Figure 6.1 (Previous page) Spawning performance criteria for Penaeus monodon broodstock fed
either a fresh (squid and mussel), an artificial (BIARC) or an artificial diet supplemented with
methly farnesoate (BIARC+MF). (A) number of spawns per prawn; (B) egg output per gram of
prawn, first spawning; (C) egg output per gram prawn, first three spawns; (D) mean hatch rate of
eggs for first three spawns; (E) mean survival to protozoeal 1 for the first three spawns; (F) mean
protozoeal 1 output per gram prawn for the first three spawns; (G) mean protozoeal 1 output for
the first three spawns; (H) mean total protozoeal 1 output per prawn. Mean and standard error
(n=28) with the same superscripts are not significantly different (p>0.05)
115
6.4 Discussion
This study has shown that MF can inhibit aspects of penaeid prawn reproduction in vivo.
Specifically oral administration of MF reduced the number of spawns per prawn and
relative fecundity (averaged over the first three spawns) in ablated P. monodon. Closer
observation showed that the reduction in the number of spawnings resulted from MF
inhibiting ovary development during late vitellogenesis or during the final stages, termed
‘prematuration’ and ‘maturation’ by Yano (1988, 1995). Despite considerable evidence
that MF is a stimulatory hormone (for reviews see Borst et al 1987, Huberman 2000,
Laufer and Biggers 2001 and Tsukimura 2001), other studies support the current findings
which demonstrate that MF can also function to inhibit some aspects of crustacean
reproduction (Tiu et al 2006, Tsukimura et al 2006, Mak et al 2005).
Previous studies have shown that orally administered MF can stimulate aspects of prawn
reproduction. For example, at similar dietary inclusion levels to those administered in the
current study, MF was shown to increase fecundity, egg fertility and hatch rate in ablated
P. monodon (Hall et al 1999) and spawning, fertility and hatch rates in ablated
Litopenaeus vannamei (Laufer 1992). With increasing evidence of the complexity of
crustacean endocrine systems (for a review see Okumura 2004), it is likely that an array
of factors contribute to the variable outcomes between the studies. For example, species
specific differences in MF function or mode of action may contribute to conflicting
results of the current study on P. monodon and the study on L.vannamei (Laufer 1992).
Alternatively, MF concentration in the haemolymph may explain the apparent differences
116
in the responses of crustaceans to this hormone. For example, Mak et al (2005) showed
that during specific stages of crab ovary and egg development, low levels of MF
stimulated hepatopancreas Vg gene expression while high levels inhibited expression. In
the case of orally administrated MF, it is possible that differences in diet consistency,
formulation, ingestion and acclimation period prior to ablation (for example, 3 weeks for
Hall et al (1999) and 2 weeks for the current study) could have affected the
concentrations of MF in the haemolymph. Further, the half-life of MF in the haemolymph
is less than one hour (Tsukimura 2001) suggesting concentration may have fluctuated in
accordance with feeding frequency. Thus experimental methods, via their impact on MF
haemolymph concentration, may be critical to the specific physiological response
generated by exposure to MF.
Alternatively, or in addition to haemolymph concentration of MF, prawn size or stage of
sexual maturity, and the pre-capture condition of the prawns (Primavera 1984, Marsden
et al 2007) may have contributed to the observed differences in results. These factors may
influence whether the active role of MF is to regulate reproduction, moulting (Abdu et al
1998, Chang 1997, Tamone and Chang 1993) or juvenile development (Borst and Laufer
1990, Rotllant et al 2000, Tsukimura 2001). Inhibition of late ovary development or
spawning by MF via stimulation of ecdysis, however, is unlikely to have occurred in the
current study as prawns fed the three treatment diets showed no difference in their
percentage weight gain or moult interval over the eight week experimental period.
Moreover, while the female prawns in the current study were significantly smaller than in
the Hall et al (1999) study (average 75 and 120 g (Hall pers. comm.) respectively), the
117
high rate of advanced ovary development in all prawns in the current study confirms that
the cohort was sexually mature (Primavera 1985). Thus the inhibition of ovary
development and fecundity is unlikely to be due to a juvenilising effect of MF as was
recently found in immature freshwater shrimp where MF caused a decrease in ovary
weight and oocyte diameter (Tsukimura et al 2006). We therefore suggest that MF’s
inhibition of late stage ovary development (and/or spawning) and fecundity is not due to
the hormone acting as a juvenilising or a moulting capacity.
While other studies have shown MF regulates development in early stage ovaries
(Tsukimura and Kamemoto 1991, Nagaraju et al 2004, 2006), the current study showed
that MF can directly, or indirectly, regulate late stage ovary development. MF’s mode of
action at this developmental stage may be via control of the gene(s) responsible for Vg
synthesis in the ovary and hepatopancreas (Tiu et al 2006, Mak et al 2005). As both these
tissues synthesise Vg in P. monodon (Tseng et al 2001, Thurn and Hall 1999), inhibition
of synthesis in either tissue could conceivably arrest ovary development. If this mode of
action is operating however, it is interesting that MF’s effect was not evident until
vitellogenesis was nearing completion, or possibly complete. Alternatively, MFs mode of
action may be via control of Vg uptake rather than, or in addition to, synthesis.
Specifically, MF has been shown to activate protein kinase C (PKC), an isoenzyme
involved in Vg uptake by oocytes and follicle cells whose isotypes vary during ovary
development in the freshwater crayfish Cherax quadricarinatus (Soroka et al 2000). This
regulatory pathway would operate at the later stage of ovary development in P. monodon
when Vg components are being actively accumulated (Thurn and Hall 1999, Tseng et al
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2001). In view of the late stage at which MF appears to be operating in the current study,
it is also possible that the hormone is acting in conjunction with a recently identified egg-
laying hormone (ELH) (Liu et al 2006). As with MF, levels of ELH were shown to
decrease greatly just prior to spawning in P. monodon.
Regardless of the mode of action, results of the current study provide additional evidence
that a critical control point in egg production occurs during late stage ovary development.
Previous studies have noted the occurrence of arrested ovary development and either
premature or partial spawning at stage III of ovary development (Tan-Fermin 1989)
particularly in domesticated prawns (Yamano 2004, Makinouchi and Hirata 1995).
Events taking place during this stage warrant further investigation. Cytological studies
that relate phases of meiosis (Anderson et al 1984, Cledon 1986, Yano 1988, 1995) to
arrested development could help isolate the processes being regulated. MF concentration
in the haemolymph have previously been shown to decrease prior to egg release (Laufer
and Biggers 2001) and it may be that, in addition to affecting Vg synthesis and uptake,
MF affects germinal vesicle breakdown (GVBD) or ovulation (Laufer and Biggers 2001).
Alternatively it has been proposed that arrested development is related to incomplete
cortical rod (CR) formation (Yano 1988). Regulation of these processes could also affect
fecundity, which the MF treatment in the current study reduced after the first spawn.
Without ultrastructural examination of ovaries, however, it remains to be determined
which of these processes are inhibited by the dietary inclusion of MF in P. monodon.
An additional finding of this study was that the artificial broodstock diet (BIARC)
induced similar growth performance to the fresh diet as assessed by weight gain, survival
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and moult interval of female prawns. Nevertheless, significant differences in ovarian
development and reproductive performance occurred with diet. As previously reported
(Marsden et al 1997), the BIARC diet resulted in a higher mean survival rate for larvae to
protozoeal 1 developmental stage (averaged over the first three spawns) than the squid-
mussel diet. However, in contrast to previous comparisons, the spawning frequency of
prawns fed the BIARC diet was lower than for the squid-mussel. This occurred primarily
because, for this study, the squid-mussel diet resulted in a higher than average number of
spawns per prawn (Marsden et al 1997, Hansford and Marsden 1995). This difference
between studies may be due to the quality of squid and mussel fed although variation in
results has also been linked to seasonal changes and the resultant pre-capture condition
(including nutritional status) of the prawns (Hansford and Marsden 1995).
While the reproductive output of the P. monodon is affected by pre-capture condition
(Marsden et al 2007), the post-capture maturation diet has long been shown to have a
major influence on a number of performance criteria for prawns (for review see Harrison
1990). However, it is possible the difference in ovary development for the two control
diets (squid-mussel and artificial) may be due to a dietary factor that is not in itself a
nutrient but rather a component within the diet (such as a hormone or hormone precursor)
that may influence ovary development/spawning at critical stages (such as cortical rod
formation or egg release). This has previously been proposed; for example, a low-
molecular weight peptide extracted from live short-necked clam was effective in inducing
ovary maturation in prawns (Kanazawa 1990). Similarly, a squid extract was effective at
inducing secondary vitellogenesis in P. vannamei (Mendoza and Revol 1997).
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Accordingly, for the prawns used in the current study, it is possible that a stimulatory
factor is present at a higher level in the squid-mussel diet than in the artificial diet.
Evidently, MF (at the concentration and method of inclusion used in this study) is not this
missing stimulatory factor.
Therefore the results of this study, in conjunction with previous studies on P. monodon
(Hall et al 1999) and other species (Laufer 1992, Tsukimura and Kamemoto 1991, Mak
et al 2005, Tiu et al 2006), have confirmed that MF can play a role in regulating prawn
reproduction. Further, they indicate that for P. monodon, MF can be active during late
stage ovary development. In contrast to the results of other studies (Hall et al 1999),
however, MF was shown to inhibit certain aspects of reproduction indicating that its role
may be complex and variable. To achieve a predictable outcome requires a greater
understanding of MF’s target tissues and of its interaction with other hormones in
regulating specific physiological processes (Gunawardene et al 2002 , Kuballa et al 2007,
Rodriguez et al 2001, 2002, Mak et al 2005, Tiu et al 2006 or for a review see Huberman
2000). Until the interplay between hormones, tissues and the environment is better
understood, the practical application of single hormones for the regulation of
reproduction in crustaceans is likely to remain problematic.
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Chapter 7.
THE IMPACT OF CAPTIVITY AND ABLATION ON LIPID AND FATTY ACID
PROFILES OF PENAEUS MONODON EGGS AND EARLY LARVAL STAGES
7.0 Abstract
This study assessed the combined effect of captivity and ablation on lipid quality and
percentage composition in eggs and developing lecitotrophic larvae (nauplii 2 and
protozoeal 1), from first post ablation spawnings of wild caught P. monodon. Results
showed that captivity and/or ablation significantly affected fatty acid profiles in eggs and
larvae. Specifically, when compared to eggs from prawns with ovaries that matured in the
wild, the eggs obtained from ablated prawns held in captivity for 5 to 10 days showed
higher levels of the HUFAs 20:5n3 and 22:6n3. By contrast, levels of most MUFAs and
the n6 fatty acids were decreased by captivity and/or ablation.
The study also examined the changes in lipid percentage composition that occurred with
development. Specifically, it was shown that as prawns from both treatment groups
progressed from egg to protozoeal 1 stages of development there was a similar overall
decline in lipid levels. A key finding of this study, however, was that in ablated animals
this decline was evident during egg development and hatching and during nauplii
development and metamorphosis to protozoeal 1. By contrast, with the wild treatment
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group a significant decline in lipid content occurred only during nauplii development and
metamorphosis to protozoeal 1.
Interestingly, in both treatment groups a relatively uniform depletion of fatty acid was
observed as eggs developed to protozoeal 1. The MUFA, 16:1n7 was an exception being
selectively depleted during the progression from nauplii to protozoeal 1. There were also
significant differences in the relative levels of specific fatty acids when larvae from
different treatment groups were compared. In particular, a major impact of captivity
and/or ablation was to promote selective depletion of 20:5n3 and 22:6n3 in the neutral
lipid fraction at each stage of development studied.
Based on these findings, we suggest that changes in the levels of total lipids and/or
specific fatty acids, captivity and/or ablation may significantly impact on the quality of
eggs and larvae obtained in aquaculture environments.
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7.1 Introduction
The quality of eggs and larvae from ablated prawns whose ovaries mature in captivity is
often inferior to prawns whose ovaries mature in the wild (Emmerson 1980, Yano and
Wyban 1993, Bray and Lawrence 1992). The egg yolk is of critical importance to larval
quality as it provides the nutrients necessary for embryogenesis, egg hatching and, in
Penaeus monodon, the development of six lecitotrophic larval stages (nauplii) and
metamorphosis to the first feeding stage of protozoa 1 (protozoeal 1).
Protein and lipid are the major components of prawn egg yolk. The lipids are the main
energy source during embryogenesis and also fulfil essential roles in cell membrane
structure, nutrient transport and hormone formation (Chu et al 1994). In Ch. 4 we
determined that, total lipid levels in mature ovaries are resistant to changes associated
with captivity and ablation. Nevertheless, in other crustacean species, these lipids have
been shown to vary significantly in quality in response to captivity and/or ablation and,
accordingly, are believed to be key nutritional factors influencing egg hatch rate and
larval survival (Laven and Sorgeloos 1991, Xu et al 1994, Wickin et al 1995, Palacios et
al 1999, Huang et al 2008). In terms of quality, the class of lipid (eg. neutral
triglycerides, polar phospholipids) and associated fatty acids, are of particular
significance as they determine the physiological role(s) of the lipid (Harrison 1990,
Cavalli et al 1999, Racotta et al 2002). For example, polar lipids typically have functional
roles in cell membranes while the neutral lipids provide a major source of energy. A
number of studies have also linked levels of individual fatty acids with specific aspects of
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larval quality including egg hatch rate and fertilization (Millamena 1989, Cahu et al 1994,
Xu et al 1994, Marsden et al 1997, Perez-Velazquez et al 2003, Huang et al 2008).
Probably the best known influence on egg lipid quality is broodstock diet (for review see
Harrison 1990). In particular, it has been shown that the fatty acid profile of the eggs
frequently reflects that of the diet. This is largely due to the limited ability of prawns to
synthesize highly unsaturated fatty acids (HUFAs) in both the n3 and n6 families
(Kanazawa et al 1979, Teshima et al 1992) and the consequent need for these essential
fatty acids (EFAs) to be supplied by the diet.
Ablation of broodstock has also been shown to affect lipid quality (Beard and Wickins
1980, Primavera and Posadas 1981, Ruangpanit et al 1984, Yano and Wyban 1993). For
example, Palacios et al (1999) found ablation of P. vannamei prawns caused a significant
change in levels of some egg lipid classes. Teshima et al (1988) also reported that ablation
of P. japonicus caused an increase in the proportion of 22:6n3 (docosahexaenoic acid,
DHA) and a decrease in 20:4n6 (arachidonic acid, AA) and 20:5n3 (eicosapentaenoic acid,
EPA) in prawn ovaries.
Determination of the optimum fatty acid profiles of eggs and larvae is considered to be a
necessary step for the improvement of larval quality (Harrison 1990) and, on a larger
scale, the economic viability of domesticated P. monodon. Eggs from ovaries that
develop spontaneously in the wild are considered representative of the ideal fatty acid
profile. This is based on larval survival studies (Lytle et al 1990, Millamena and Pascal
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1990) and the assumption that ovary development is triggered by optimal environmental
conditions, including nutrition. Depletion patterns (relative decreases in fatty acid levels)
as larvae develop provide another indication of the significance of individual fatty acids
(Cahu et al 1988).
The aim of the current study is to compare total lipid levels, lipid classes (polar and
neutral) and fatty acid profiles in eggs and larvae from (i) prawns whose ovaries matured
in the wild and, (ii) prawns whose ovaries matured in captivity following ablation. In
addition the patterns of lipids and their fatty acid depletion as development progresses
from eggs to first feeding larval stage will be examined.
126
7.2 Materials and methods
7.2.1 Prawns
Prawns were sourced from Cook Bay (See Chapter 3 Methods) and consisted of ten gravid
(ready to spawn) females, ten females with immature ovaries and ten males.
Gravid female prawns that were ready to spawn were placed individually in spawning
drums (see below). Non gravid females (showing no ovary shadow when observed
externally through the dorsal surface) and males were held in maturation tanks as described
in Chapter 3. After one week acclimation, females were unilaterally eyestalk ablated.
Ovarian development was monitored every afternoon using a submerged light to reveal the
shadow of the ovary on the dorsal exoskeleton.
While in captivity prawns were fed a diet consisting of squid and mussel (1.3:1). The fatty
acid profile of this diet is detailed in Table 7.1.
7.2.2 Egg and larval collection and processing
If the female’s ovary had developed fully the individual was placed in a spawning drum.
Spawning drums of 150L (1m diameter) were filled with filtered (1 µm) seawater heated
to 28°C and lightly aerated.
127
Spawning drums were checked for spawning every 2 hours after midnight (0:00) using a
red light torch. This was to get an estimated spawning time to be able to calculate when
successive developmental stages must be collected. Pilot studies demonstrated that fatty
acid profiles of eggs did not change significantly (p<0.05) within the first two hour
period after spawning (data not shown). If a spawning occurred the spawner was
immediately removed. The water was then agitated using a plastic paddle (to ensure eggs
were evenly distributed in suspension) and 4 x 80ml samples were taken for counting and
estimation of total egg number (fecundity). Approximately 2 g of eggs were siphoned
from the spawning drum for biochemical analysis. Eggs were rinsed with distilled water to
remove remnants of salt that could affect dry weight and ash analysis. Eggs were drained
and transferred to labelled jars for freezing at -70oC until biochemical analysis.
To enable later estimate of hatch rate, a second set of 4 samples was taken (as described
above) for counting to determine the number of eggs remaining after the sample of analysis
was taken. The eggs were also microscopically examined to establish the hatch time and,
when possible, if eggs were fertilized (Hall et al 2000 AIMS web site). Inspection
frequency was increased to every hour prior to the expected hatch time, until all viable
eggs had hatched. Larvae (nauplii and protozoeal 1) were sub sampled for counting and
collected for analysis (as per eggs).
128
7.2.3 Biochemical analysis
Biochemical analysis was carried out to determine total lipid, polar and fatty acids in the
polar and neutral lipid fractions of the eggs as described below.
In the current study the changes in lipid content with development were not measured on a
per egg/larvae basis. This was primarily due to the difficulty associated with counting the
large number of eggs and larvae required to ensure accurate biochemical analysis for each of
the 48 samples collected. Pilot studies to estimate the number of individuals per unit wet
weight showed large variation due to the entrapment of water in the samples and this
approach was therefore considered inaccurate (data not shown). Consequently, samples
were collected of sufficient wet weight for chemical analysis and results were expressed on
a dry matter basis.
Proximate analysis
Total lipid content was determined by Soxhlet extraction with petroleum ether (bp 40-
60°C) for 6 hr (Association of Official Analytical Chemists, 1990, method 960.39).
129
Polar and neutral fatty acid analysis
For fatty acids, lipids were extracted by the method of Folch et al., (1957) using the
modification of Christie (1982). An aliquot of the lipid extract was separated into polar
and non-polar fractions using Sep-Pak silica cartridges (Waters Associates, MA, USA).
The non-polar fraction was eluted with 15 mL chloroform and the polar fraction with 20
mL of methanol (Christie, 1982). The solvent was removed from each fraction by rotary
evaporation and the lipids esterified to fatty acid methyl esters (FAME) by the method of
Van Wijngaarden (1967). FAME were separated by capillary gas chromatography using
split injection on a 30 m x 0.25 mm i.d. fused silica column coated with 0.25 m of DB-23
(J & W Scientific, Folsom, California). Column temperature was held at 160°C for 10
minutes and then increased at 3°C min-1 to 210°C where it was held until all FAME of
interest had been eluted. FAME were quantified by comparison with the response of an
internal standard (heneicosanoic acid methyl ester). FAME were identified by comparing
their retention times with those of authentic standards (Sigma Chemical Company, St.
Louis, Missouri).
7.2.4 Statistical analysis
Data on individual fatty acids were first summarised and scanned for outliers. Statistical
analysis involved ANOVA and Tukeys test to assess stage and treatment effects of first
captive spawnings. Results were regarded as significant at the 5% level.
130
7.3 Results
As shown in Fig. 7.1, the relative amount of lipid detected in samples declined
dramatically as prawns progressed from eggs to protozoeal 1. The captivity/ablation
treatment, however, did not appear to influence the proportion of lipid in the dry matter at
egg and protozoeal 1 stages. By contrast in nauplii from captive/ablated females the total
lipid level was significantly less than that in those samples obtained from females whose
ovaries matured in the wild. Subsequently, it was demonstrated that this reduction in lipid
content in nauplii from ablated females was evident in both the polar and neutral lipid
fractions (Table 7.2).
A general trend observed in the current study was that for both treatments (wild and
ablated) fatty acid levels in the neutral lipid fractions of eggs and nauplii were
significantly higher (p<0.05) than those detected in the polar fraction. By contrast, fatty
acid levels in the polar fraction of protozoeal 1 lipids were significantly higher (p<0.05)
than those detected in the neutral fraction.
As shown in Table 7.3, the relative levels of eight of the twenty seven fatty acids
measured in the egg samples were significantly changed by the captivity/ablation
treatment (data for other fatty acids not shown). In particular, it was demonstrated that
within the neutral and polar lipid fractions of eggs obtained from ablated females there
was significantly less (p<0.05) 16:1n7, 20:1n11, 20:4n6, 22:4n6 and 22:5n6 than in
samples obtained from females whose ovaries had matured in the wild (Table 7.3 and
131
Fig. 7.2). By contrast, levels of 20:1n9, 20:5n3 and 22:6n3 were significantly higher in
the lipid fractions of eggs obtained from captive/ablated females than in those obtained
from the wild controls.
As shown in Table 7.4, the impact of captivity and ablation on lipid quality extended
beyond the egg stage of development. For example, significantly less (p<0.05) 16:1n-7
was detected in the neutral lipid fraction of eggs, nauplii and protozoeal 1 obtained from
ablated females than in samples from females whose ovaries had matured in the wild.
Likewise the captivity/ablation treatment appeared to significantly increase (p<0.05) the
proportion of 20:5n3 and 22:6n3 detected in the neutral lipid fraction of eggs, nauplii 2
and protozoeal 1.
Table 7.4 also shows how the relative level of specific fatty acids changed as eggs and
larvae developed. Of particular note is the decrease in 16:1n7 which occurred in both
treatment groups and in both lipid fractions (neutral and polar), between developmental
nauplii (N2) and protozoeal ( Z1). The main effect of captivity and/or ablation on the
depletion pattern of fatty acids with development was to increase the rate of depletion of
20:5n3 and 22:6n3 relative to other fatty acids. Despite the higher depletion rate,
however, the levels remained higher in the Z1 from the captive-ablated group than in the
wild caught group.
132
Table 7.1 Fatty acid (FA) profiles (% dry matter of total fatty acids) of the squid-mussel
diet
FA Neutral Polar
14 1.9 2.8
16 13.6 22.5
16: In7 2.1 1.5
18 3.4 3.9
18: In9 5.2 1.7
18: In7 1.8 1.5
18: 2n6 0.8 0.6
18: 3n3 1.1 0.6
18: 4n3 2.0 0.8
20: 1n11 0.1 0.3
20: 1n9 2.7 3.1
20: 1n7 0.4 0.4
20: 2n6 0.3 0.2
20: 4n6 0.3 1.2
20: 5n3 11.7 13.6
22: 1n9 0 0.2
22: 1n6 0.1 0.4
22: 1n3 0.2 0.1
22: 5n3 0.3 0.8
22: 6n3 28.0 35.5
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Figure 7.1 Total lipid levels (% dry matter ±SE; n=8) in eggs (E), nauplii 2 (N) and
protozoeal 1 (Z) from prawns whose ovaries matured in the wild (W) or matured in
captivity following ablation (A). Data points with the same letter superscripts are not
significantly different from one another (p<0.05).
134
Table 7.2 Total fatty acid (mg) per gram of dry matter, in neutral and polar fractions of
lipids ± SD for wild (W) and ablated (A) treatment groups.
Mg total fatty acid
per g of dry matter
Egg Nauplii Protozoeal 1
W A W A W A
Neutral lipids 113.6a1
±4.7
135.0 a1
±17.3
90.8b1
±8.2
71.0 c1
±5.5
16.7 d1
±4.3
22.0 d1
±4.0
Polar lipids
73.3a2
±4.4
74.0 a2
±7.2
74.3 a2
±6.1
54.0 b2
±5.5
40.2c2
±4.3
41.0 c2
±1.6
Average values in rows within each developmental stage (Egg, Nauplii, Protozoeal 1)
with different letter superscripts are significantly different. Values within in each column
(W or A) with different number superscripts are significantly different (P<0.05).
135
Table 7.3 Average (±SD) level (% total fatty acids) of selected fatty acids in the neutral
and polar lipid fractions of egg lipids, for Wild (W) and Ablated (A) treatment groups.
Fatty acids Neutral Fraction Polar Fraction
W A W A
16:1n-7 16.2 a ±0.7 9.6 b ±1.1 13.2a ±0.2 6.9b ±0.8
20:1n-11 1.5 a ±0.2 0.6 b ±0.1 1.7a ±0.2 0.8b ±0.1
20:1n-9 0.5 a ±0.1 1.3 b ±0.2 0.7a ±0.0 2.2b ±0.4
20:4n-6 3.8 a ±0.1 2.3 b ±0.5 7.5a ±0.1 3.7b ±0.7
20:5n-3 5.3 a ±0.2 10.5 b ±0.5 8.6a ±0.4 13.5b ±0.8
22:4n-6 1.9 a ±0.1 0.6 b ±0.1 2.1a ±0.0 0.6b ±0.2
22:5n-6 1.0 a ±0.1 0.5 b ±0.1 1.0a ±0.0 0.4b ±0.1
22:6n-3 6.1 a ±0.6 19.0 b ±2.6 6.3a ±0.4 17.1b ±1.4
Different superscripts within rows indicate significant differences (P<0.05).
136
Figure 7.2 Mean (±SD) changes (P<0.05) in the percentage composition of selected
neutral and polar fatty acids in eggs caused by captivity and ablation. An asterisk
indicates a significant difference (P<0.05) between the neutral (N) and polar (P) values.
-10
-5
0
5
10
15
16:1n7 20:4n6
20:5n3 22:6n3
Fatty acids
P N
Perc
enta
ge
e ch
ange
*
*
137
Table 7.4 Average (±SD) levels (% of total fatty acid DM) for selected neutral and polar
fatty acids in eggs (E), nauplii 2 (N) and protozoeal 1 (Z) for captive held ablated prawns
and wild caught prawns.
Fatty
acids
Stage Neutral Polar
Wild Ablated Wild Ablated
16:1n-7
E 16.2a1 ±0.7 9.6 b1 ±1.1 13.2a1 ±0.2 6.9b1 ±0.8
N 16.0a1 ±1.8 9.3b1 ±0.7 12.5 a1 ±1.8 6.0 b12 ±0.2
Z 12.3a2 ±1.0 7.3b2 ±0.7 6.8 a2 ±0.9 4.0 b2 ±0.2
18:1n-9
E 15.3a1 ±0.8 12.5a1 ±0.8 16.9a1 ±0.4 14.0a ±0.7
N 15.8a1 ±0.5 13.3a1 ±0.3 15.0 a1 ±0.8 11.8 ±0.6
Z 15.5a1 ±0.2 13.5a1 ±0.6 11.5 a2 ±1.1 10.5 ±0.4
20:4n6
E 3.8 a1 ±0.1 2.3 a1 ±0.1 7.5 a1 ±0.1 3.7 b1 ±0.7
N 3.5 a1 ±0.5 2.4 a1 ±0.1 8.2 a12 ±0.7 4.9 b1 ±0.8
Z 4.1 a1 ±0.6 2.5 a1 ±0.1 10.0 a2 ±1.6 5.7 b1 ±0.4
20:5n-3
E 5.3 a1 ±0.2 10.5b1 ±0.5 8.6a1 ±0.4 13.5b ±0.8
N 4.0a1 ±0.5 8.1b12 ±0.3 9.6 a1 ±1.0 16.1 b2 ±0.5
Z 3.9 a1 ±0.7 6.1b2 ±0.8 11.3 a1 ±1.8 16.7 b2 ±0.4
22:6n-3
E 6.1 a1 ±0.6 19.0b1 ±2.6 6.3a1 ±0.4 17.1b1 ±1.4
N 4.8 a1 ±0.5 16.1b12 ±1.4 8.0 a12 ±0.4 19.1 b2 ±0.3
Z 4.8a1 ±0.8 13.1b2 ±0.3 11.3 a2 ±1.7 20.5 b2 ±0.2
Values in rows within either the neutral or polar lipid class, that have the same letter
superscript are not significantly (P<0.05) different. E, N and Z values for each fatty acid
that have the same numerical superscript are not significantly (P<0.05).
138
7.4 Discussion
Egg lipids
In this study, egg lipid content was approximately 30% of dry matter; similar to levels
previously reported for P. monodon (Crocos et al 1997). Comparisons with other species
indicate that lipid levels may be species specific (Palacios et al 1999, Teshima et al 1989).
In the current study, captivity and ablation had no significant effect on the percentage of
lipid in the dry matter of eggs from the first post ablation spawn. Lipid quality, in terms of
total neutral or polar fractions in the egg, also appeared largely unchanged by captivity or
ablation. This supports earlier findings that indicated total lipid levels are resistant to change
and to some extent, could be regarded as a conservative component of egg composition
(Cahu et al 1994, Marsden et al 1997, Marsden et al 2007).
A major determinant of the quality, and therefore the functional role, of a lipid is its fatty
acid content. Only eight of the twenty seven fatty acids measured in eggs, changed as a
result of captivity and/or ablation. These fatty acids included highly unsaturated fatty acids
(HUFAs) and mono-unsaturated fatty acids (MUFAs). One HUFA whose levels were
lowered by captivity and/or ablation was the essential fatty acid (EFA) arachidonic acid
(AA; 20:4n6). The relative level of this fatty acid, along with the other n6 fatty acids, was
lowered by ablation. AA is thought to be a precursor for hormone like prostaglandins
shown to be essential for reproduction in a number of animals and has previously been
positively correlated with fecundity and egg production in P. monodon (Huang et al
139
2008). By contrast, in this study the level of another EFA, eicosapentaenoic acid (EPA,
20:5n3) was doubled by captivity and/or ablation. Levels of this fatty acid have been
positively correlated with fecundity (Xu et al 1994), larval survival (Crocos et al 1997)
and hatch rate. Likewise it was shown in this study that levels of docosahexanoic acid
(DHA; 22:6n3) were trebled by captivity and/or ablation suggesting there may be a
deficiency in n6 fatty acids but not n3.
The decrease in AA and the associated increase in EPA and DHA levels contributed to an
increase in relative levels of n3:n6 observed in this study. Although there is little
consensus on the relevance of this ratio, 3:1 in nauplii of P. vannamei has been
recommended (Wouters et al 2001). In the eggs from prawns whose ovaries developed in
the wild the ratio was 2:1 while in eggs from prawns that developed in captivity after
ablation it was 10:1. Thus the captive ablated group may have n3 to excess; however, the
significance of the high levels remains to be determined.
Although not considered EFAs, monounsaturated fatty acids (MUFAs) are a major
energy reserve for embryogenesis (Figueiredo et al 2008). The level of palmitic acid
(16:1n7), comprising approximately 13% and 16 % of the fatty acids in the egg neutral
and polar fractions respectively was significantly decreased by captivity and/or ablation.
Interestingly, a recent study showed that its level was higher in eggs from domesticated
(pond reared) P. monodon than from the wild caught broodstock with the implication
being that lower levels were preferable.
140
Thus while the current study showed that captivity and ablation together caused
deviations from the “natural” fatty acid profile (as demonstrated by eggs from the wild
treatment group) the separate contribution of captivity and ablation to these changes
remains unknown. Due to the difficulty in inducing ovary development in non-ablated P.
monodon, it was not possible to get sufficient spawnings from a non-ablated captivity group.
Hence the effect of ablation could not be isolated from that of captivity to determine if their
effects are summative or opposing. It may be possible that there were short term (ten
days, by which time all spawns were collected) differences in the diet of the two study
groups that contributed to the change in lipid quality. The diet fed to the captive ablated
broodstock demonstrated low levels of AA and palmitoleic acid (16:1 n7), and high
levels of EPA and DHA, relative to other fatty acids and was reflected in the eggs of this
group. The diets consumed during ovary development in the wild group could not be
accurately determined although it has been shown to consist largely of molluscs and to
exhibit seasonal variation (Crocos et al 1997).
Ablation of prawn broodstock has also been shown to change fatty acid profiles of egg and
larvae. Lipid metabolism is known to be under endocrine control (O’Connor and Gilbert
1968), possibly through enzyme activity (Gonzalez-Baro and Pollero 2000), and to respond
to ablation (Santos et al 1997). Ablation was previously shown to increase DHA and
decrease AA in prawn eggs (Teshima et al 1988) which also occurred here as a result of
captivity and/or ablation. Surprisingly, Teshima et al (1988) also found that ablation
decreased the relative level of EPA yet in the current study its level was doubled as a result
141
of captivity and/or ablation. The basis for these apparent contradictory results remains to be
determined.
Egg and lecitotrophic larval development
A second aim of this study was to examine the depletion pattern of lipids as the embryos and
lecitotrophic larvae of P. monodon developed, and how this was affected by captivity and
ablation. To this end a comparison was made of the percentage of lipid and fatty acids in the
dry matter at three developmental stages; that is eggs (E), early nauplii (N2) and protozoeal
1 (Z1). In comparing these stages we were able to determine the lipid depletion (relative to
other dry mater components) during embryonic development and hatching (E-N2) and
during nauplii moults and metamorphosis to first feeding protozoa (N2-Z1).
As eggs from prawns whose ovaries matured in the wild, developed and hatched into
nauplii, there was no significant change in the percentage of lipids in the dry matter. During
development of embryos and nauplii there is an estimated 30-40% loss of dry matter
(Hollan, 1978, Chu and Ovsianico 1994, Herna´ndez-Herrera 2001, Pandian 1970, Pillai
and Clarke 1987). Thus, no change in the percentage lipid indicates the contribution of
lipids to any loss of dry matter during development to nauplii is equal to the other dry matter
components combined. Protein and carbohydrates have also been shown to contribute to
energy requirements and exuvia (Horst 1989). In the current study, captivity and/or ablation
resulted in a 10% lipid decrease with development from eggs to nauplii and this decline was
apparent in both the neutral and polar classes. The conditions therefore appear to have either
142
increased the relative contribution of lipids to energy demands or loss with the shell or
exuvia during hatching or moulting respectively. These results are in contrast to findings for
P. esculentus where ablation caused an increase in nauplii lipid levels (Rothlisberg et al
1991); possibly reflecting a species related difference in energy metabolism.
In the wild group it was not until the progression from nauplii to protozoeal 1 that a
preferential use or loss of lipids occurred. In particular, between early nauplii and protozoeal
1 there was a 17% reduction in the percentage lipid with the decline most evident in the
neutral class. A loss of lipids also occurred in the captive-ablated group (9%) but was less
dramatic possibly due to the earlier decline between eggs to early nauplii. However to fully
interpret the difference in the pattern of lipid utilization between the two groups (captive-
ablated and wild) requires further studies to provide accurate measurements of
developmental time, wet weight of eggs and accompanying changes in other dry matter
components of the eggs and nauplii.
Interestingly this study also showed a relatively consistent depletion of fatty acids with
development from eggs to protozoeal 1, although there was some variation in the polar
lipid fraction. A noted exception was the MUFA, 16:1n7 which was selectively depleted
during development from N 2 to Z1. The main effect captivity and/or ablation had on this
trend of fatty acid depletion during development, was to cause selective depletion of
20:5n3 and 22:6n3. Despite this the levels of both fatty acids remained above the levels in
the Z1 of the wild group suggesting they remained in excess.
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Summary
The current study shows that ablating and holding broodstock in captivity (as per conditions
of this study) for between five and ten days, caused significant changes in egg fatty acid
profiles and in the lipid depletion patterns as eggs developed into first feeding larvae
(protozoeal 1) for the first post ablation spawn. Specifically, the relative level of MUFAs
and n6 fatty acids in the eggs were reduced while n3 HUFAs were increased. Captivity
and/or ablation also resulted in increased use of lipids when eggs developed and hatched
into nauplii but decreased it use as nauplii developed into protozoeal 1, such that the total
use was unchanged. However, the role of MUFAs (which were selectively depleted) and the
significance of lipids during egg development and hatching, both warrant further
investigation. In addition, there is a need to isolate and characterize the specific
contributions of ablation, culture environment and broodstock diet on the fatty acid profiles
of P. monodon eggs and larvae.
144
Chapter 8
REPRODUCTIVE BEHAVIOURAL DIFFERENCES BETWEEN WILD
CAUGHT AND POND REARED PENAEUS MONODON PRAWN
BROODSTOCK.
8.0 Abstract
Time lapse video observations were carried out to compare the mating behaviour of
different combinations of domesticated (pond reared) and wild caught prawn broodstock
of the important aquaculture prawn species, Penaeus monodon.
Copulation was observed for the wild x wild mating pairs, but not within the pond reared
group. Precopulation behaviour, primarily the male pursuit of moulted females, was
lower for groups involving pond reared males or females.
We consider whether the domestication process, comprising both genetic and husbandry
effects, have reduced the ability of the female to attract a male and the male’s ability to
detect and respond to female cues.
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8.1 Introduction
There are a number of programmes dedicated to closing the life cycle of the important
aquaculture prawn species, Penaeus monodon in culture. Diseases, believed to be
introduced with wild caught broodstock have been responsible for cases of dramatic
decline in recent P. monodon production. (Globefish 2004). Domestication of this species
has the potential to relieve industry dependence on wild caught broodstock and provide
specific pathogen free (SPF) broodstock, capable of producing genetically improved
offspring.
To date P. monodon hatchery operators have demonstrated a preference for wild caught
prawns due to the relatively poor reproductive performance of domesticated broodstock
(Coman et al 2006). Low egg hatch rates and significantly reduced larval production are
recognised problems of domesticated P. monodon. As described previously (2.3.1) a
major factor contributing to egg development is nutrient content. In particular, there is
complete reliance on nutrients in the yolk reserves until the first feeding protozoa stages.
In chapters 4 and 5 we demonstrated that captive environments impact on the nutrient
profiles on P. monodon ovaries. Furthermore, in chapter 7 we provided evidence that
changes in the levels of total lipids and/or specific fatty acids associated with captivity
and/or ablation may significantly impact on the quality of eggs and larvae obtained in
aquaculture environments.
146
As described previously, (2.3.4) there are factors other than nutrient status that
profoundly influence the reproductive process in Penaeid prawns. For example, as with
virtually all multicellular animal species, the creation of new individuals is accomplished
by the process of fertilisation which involves the fusion of male and female gametes.
Typically, the fertilisation process in multicellular animals is accompanied by distinct
mating behaviours. Evidence has been presented that low fertility in P. monodon is not
simply an outcome of using a tank mating environment as matings of wild caught P.
monodon in tanks can result in high egg hatch rates (Marsden et al 1997, Hansford et al
1995, Coman et al 2006). Likewise, low fertility of domesticated P. monodon is not fully
explained by underlying egg and sperm quality, as the use of artificial insemination (AI)
can increase fertility (Nimrat et al 2005). Based on such AI results, it is tempting to
speculate that low egg hatch rates associated with domesticated P. monodon may be a
function of reduced mating success.
Mating behaviour has been observed and described for many penaeid species (Aquacop
1977, Brisson 1986, Browdy 1989, Yano et al 1988) including P. monodon (Primavera
1979). Nevertheless, there is a general paucity of data about the mating behaviours
associated with this species in captivity. Observing the events and stages at which
interruption of the mating process may occur has the potential to provide insights into the
causative agents of low fertility.
In order to redress the limited data on domesticated P. monodon mating behaviour, we
observed and described the behaviour of pond reared and wild caught P. monodon males
147
and females under laboratory conditions using time lapse video recordings. We then
considered whether domestication affects the behaviour of male and female broodstock,
and whether mating behaviour is associated with low egg hatch rates of domesticated P.
monodon.
148
8.2 Methods
8.2.1 Experimental prawns
Prawns originated from two sources: (i) pond reared domesticated third generation
prawns, (D) and (ii) wild caught prawns (W) captured after reaching sexual maturity from
coastal Queensland waters. The D stock consisted of 14 month old male and female
prawns that were harvested from a 200m2 plastic lined pond at the Bribie Island research
Centre (BIARC) located in southern Queensland, Australia. Prawns had been reared at an
average density of 4 m2 and were fed twice daily on a diet consisting of a high protein
pellet (Higashimaru-Marsupenaeus japonicus diet) with a twice weekly supplement of
fresh-frozen mullet and squid. For the W treatment group, twenty five females and twenty
males were captured from fishing grounds off Cairns in north Queensland and air
freighted to BIARC in southern Queensland (See Chapter 3). It should be noted that the
original stock for the D lines was from the same spawning grounds as the W stock.
The average size for the domesticated male and female prawns was 78.4±1.2 g and
94.6±2.0 g, respectively, and for the wild caught it was 84.2±1.8 g and 105.6±0.9 g,
respectively.
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8.2.2 Holding facilities
Prior to being transferred to holding tanks, prawns were physically examined for
abnormalities (including external genitalia and antennule damage), eye tagged for
individual identification, weighed and moult staged according to Promwikorn et al
(2004). After rejection of any damaged prawns the remainder were transferred to a tank
and held for a seven day acclimation period at a density of 2 m-2. Water temperature in all
holding tanks was maintained 28oC and exchanged at a rate 150% daily. Prawns were fed
twice daily on a diet of fresh frozen squid or mussel.
8.2.3 Observation tanks
The three observation tanks (diameter of 1.5m, 1.2m depth) were housed in a temperature
and light controlled room; each with a time lapse video surveillance camera (Sony)
mounted above. Every afternoon the tanks were filled with filtered (20µm), preheated
water (28oC) to a depth of 1 metre. There was no water exchange and the air temperature
in the room was heated to 28oC to maintain the water temperature. One air stone released
a fine stream of bubbles that maintained O2 levels at 8.0 mg/L without visually disturbing
the water surface. Lighting was supplied by red bulbs positioned adjacent the cameras
above each tank. The observation tanks were cleaned and refilled daily.
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8.2.4 Observations
At 18:00 hrs all acclimatised females in Tank A (pre-moult stage) were again moult
staged. When a female was predicted to moult that night she was transferred with two
inter-moult males (see Table 8.1) and one inter-moult female (not expected to spawn) to
an observation tank for overnight video surveillance. Care was taken to ensure that both
intermoult and premoult females were from the same original experimental group (ie. W
or D). Videoing commenced at approximately 19:00 hours. The following morning males
and inter-moult females were returned to their respective tanks. If a female had moulted
by the following morning, the video cassette was coded to enable viewing by two
independent assessors (with no prior knowledge of prawn origin) so that behavioural
criteria could be assessed.
8.2.4.1 Behaviour classification
Descriptions of behaviour traits, and methods used to measure male (2 intermoult) and
female (1 pre-moult and 1 intermoult) prawns in observation tanks are shown in Table
8.2.
8.2.5 Statistical analysis
Generalized linear models (McCullagh and Nelder 1989) were used to analyse the data in
GenStat (2000), with a two-way model of 'female source', 'male source', and their
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interaction. Continuous variables assumed a Normal distribution, with the log-
transformation used if necessary. Binary variables assumed a Binomial distribution with a
logit link. Differences between the means were determined using Tukey post–hoc test
with significance levels set at p<0.5. Multinomial logistic regression was also used to
identify predictive behaviour(s) and assign each with an accuracy rating according to the
percentage of cases (potential mating events) correctly assigned to one of the four
treatment groups.
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8.3 Results
Matings: Matings occurred only where the female originated from the wild group (W
female x D male or W female x W male). (Table 8.3) but was significantly lower
(p<0.05) when W females were combined with D males.
Pre moult agitation: Pre-moult agitation of males was observed more frequently in
parings with D females, irrespective of male origin. Post moult agitation of males,
however, was the same for all mating combinations.
Pursuit: Domesticated males showed lower levels of pursuit of females than W males.
For example, the level of pursuit exhibited by D males towards D females was 70% less
than the level of pursuit exhibited by W males towards D females (Table 8.3).
Percentage of the time the male spent under the female: The origin of the prawn (D or W)
was associated with the percentage of the time the male spent under the female. For
example, if a W male was paired with a W female (W:W) most pursuit time (55%) was
spent under the female (Fig.8.1 ). By contrast, in a pairing of D male with a D female
(D:D) the male spent only 2% to 5% of his pursuit time under the female.
Observations of moulting frequency indicated that moulting occurred significantly later
(P<0.01) for D females than for W females. The average time of moult was 23:30 ±00:34
for W females and 02:06±00:47 for D females (Figure 8.2). Also, the range of times over
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which individual females moulted was greater for D females than for W females.
Specifically, D females moulted between 20:15 and 07:35, while W females moulted
between 20:20 and 02:40. .
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Table 8.1: Pairings of male and female prawns placed in observation tank for videoing.
Females
(1 pre and 1 post moult)
Males
(2 inter moult)
Origin of prawns
W W
W D
D D
D W
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Table 8.2 Description of behaviour traits, and methods used to measure male (2
intermoult) and female (1 pre-moult and 1 intermoult) prawns in observation tanks.
PRAWN GENDER
TRAIT DESCRIPTION
BEHAVIOUR MEASURE
Male
Mating
Implantation of the spermatophore into the females thelycum
Observation of spermatophore transfer; male wrapping himself around the moulted female and demonstrating rapid muscle contractions. Verified by visual examination of the female thelycum the following morning; swelling and tissue protrusion.
Pre-moult agitation
Males showed agitation (change in location) prior to the female moulting
Percentage of observations showing an increase in time (seconds) that the male was active (swimming or walking), during the 30 minutes before the female moulted when compared to the 30 to 60 minute period prior to moult.
Post moult agitation
Males showed agitation (change in location) after the female moulted
Percentage of observations showing an increase in time (seconds) that the male was active (swimming or walking), during the 30 minutes after the female moulted compared to the 30 minutes prior to moult.
Frequency of pursuit
Male follows female Percentage of observations that the male swims within 5 body-lengths of the moulted female; follows her path for a period of 3 seconds or more
Intensity of pursuit
Males show higher pursuit intensity by maintaining closeness to moulted female
Percentage of time during post moult pursuit spent under the moulted female during the first 20 minutes of the female swimming
Number of males that pursued
Whether one or both females pursued
Number of males (one or two) that swim within 5 body-lengths of the moulted female; follows her path for a period of 3 seconds or more
Pursuit of inter-moult females
Male pursues the intermoult female as well as or instead of the moulted female
The male swims within 5 body-lengths of the ‘other’ (non moulted) female; follows her path for a period of 3 seconds or more
Male cleaning Males pass the length of antennule(s) near mouth area
Number of times a male ‘cleaned’ antennule (one or two) within the hour after female moulted
Female
Time of moult
Time of night when the female moults
Number of females that moulted in each 60 minute interval after placement in observation tank at18:00hrs
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Table 8.3. Behavioural traits measured for each of the four experimental groups.
Origin (Female: Male)
D:D
n = 7
D:W
n = 8
W:D
n = 8
W:W
n = 9
Matings (%) 0a 0a 20a 60b
Pre moult agitation (%) 86a ±0.13 75b ±0.15 43d ± 0.18 67c ± 0.16
Post moult agitation (%) 100 100 100 100
Pursuit (%) 29a ± 0.17 100b ± 0.01 71 c ± 0.17 89 bc ±0.10
Number of males pursuing 1.5 a ± 0.02 1.6 a ± 0.01 1.9 b ± 0.01 1.6 a ± 0.01
Pursuit of inter-moult female (%) 0 12.5 0 0
Male cleaning 4.7 a b ± 1.9 8.1b ± 2.3 3.3 a ± 0.1 2.6 a ± 0.6
Means along rows with same superscript are not significantly different (P<0.01).
157
Figure 8.1. The mean (± se) percentage of the time males spent under the female during a
40 minute post moult interval. Means with same superscript are not significantly different
(P<0.01).
158
0
1
2
3
4
1 2 3 4 5 6 7 8 9 10 11 12 13
Hours after 18:00
Num
ber o
f mou
lts
PR femaleW female
Figure 8.2 The number of moults for W (n=15) and D (n=15) females in each 60 minute
time interval after transfer at 18:00 hrs.
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8.4 Discussion
The likelihood of successful mating occurring under the experimental conditions of this
study was significantly lower for domesticated broodstock than for wild caught
broodstock. Specifically, no mating occurred when domesticated prawns were paired
while 60% of the wild pairings mated successfully. Thus for the prawns observed in the
current study, lack of natural mating success would have contributed significantly to low
egg hatch rate frequently reported for domesticated Penaeus monodon stocks (Kenway
pers com., Coman et al 2006)
Behavioural comparisons among wild and domesticated stock showed that for the
domesticated stock the male rarely pursued the moulted female and the intensity of
pursuit (as measured by the percentage of pursuit time the male spent under the female)
was significantly less than for the wild stock. In observations where the domesticated
males did pursue females, they did not advance to the stage of copulation (rotation,
embrace and spermatophore transfer). Thus one observable difference in mating
behaviour between wild and domesticated males was a decrease in the stimulation of
males in response to female moulting.
Cross-matings (between D and W) also showed a reduction in pursuit intensity when
compared with wild crosses (W:W). That is, a reduction occurred when either a W female
was paired with a D male or D female was paired with a W male. This result suggests that
D females may be less attractive (able to stimulate a male response) and D males are less
160
receptive (able to detect and/or respond) to cues from the D females than their W
counterparts. Based on this evidence, we suggest that both sexes contributed to the poor
mating rate of domesticated P. monodon prawns observed here.
Outcomes of the current study are in general agreement with a previous study that
showed males contribute significantly to reduced mating rates in domesticated P.
monodon broodstock. For example, Makinouchi and Hirata (1995) reported that
spermatophore implantation of wild caught females decreased from 66.7% to 32.0%
when wild caught males were replaced with pond reared (domesticated) males. In the
current study results suggest that females also contribute to the decline in mating rate by
domesticated stock. There is also the possibility that the female may be making a slightly
greater contribution to the decline than the male. Notably, when the wild caught female
was replaced by a domesticated female no matings occurred, whereas when the wild
caught male was replaced by a domesticated male 20% mated. Moreover, mating
intensity tended to be slightly lower in pairings with domesticated females than with
domesticated males.
Thus it appears that domestication of P. monodon can result in changes in behaviour that
reduces successful mating responses. While it is not known exactly why this occurs it is
likely that factors that have been shown to influence other aspects of reproduction,
including fertilisation and egg hatch rate may play a role. Factors include genetic
background, age, diet, stocking density and a range of environmental parameters (Crocos et
al 1997, Marsden et al 1997, Palacios and Racotta 2003, Arcos et al 2004, 2005). No
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physical abnormalities were visible in the external genitalia of domesticated prawns. One
factor tested in the current study was prawn size as it is an indictor of sexual maturity and
mating only occurs between sexually mature prawns (Primavera 1984). The size (g wet
weight) of males and females were examined as a covariate and it was found that for the
size range tested, size did not influence behaviour of male or female prawns. This result
suggests all experimental prawns were sexually mature, an observation that was verified
for the domesticated prawns in a separate spawning trial (data not shown). Specifically,
manually extracted spermatophores were examined and classified as mature (Pratoomchat
et al 1993) and, following ablation, over 60% of the domesticated females spawned with
an average hatch rate of 40%. Thus physical/sexual immaturity is unlikely to be the cause
of reduced mating ability.
The mechanism by which the mating process is controlled in penaeid prawns remains
unknown, however, studies on other species of marine crustaceans have shown release of
sex pheromones can act as physiological cues to direct specific mating behaviours
(Breithaupt and Eger 2002). It is hypothesised that in prawns one or more sex
pheromones, released by the female, may regulate mating behaviour in the male
(Primavera 1984, Wyban and Sweeney 1991). Further, based on the absence of observed
physical contact between the sexes during the mating process in P. monodon, chemicals
are likely to be soluble pheromones that act at distance. The release of such pheromones
during the moulting process by a sexually mature P. monodon female may be necessary
to direct the male to receptive female and then to stimulate copulatory behaviour (Zhang
and Lin 2006). ‘Antennule cleaning’ could be a means by which males concentrate
162
chemical signals from the water (Lin et al 2000). Domesticated females may release
lower volumes of pheromone as evidenced by the increased antennule cleaning by males
paired with domesticated female when compared with males paired with wild females. If
pheromones are regulating mating behaviour then the poor response of domesticated
males may result from a reduced ability to detect water born chemicals. Thus while the
presence and mode of action of the female pheromones in P. monodon is yet to be
confirmed, this study lends some support to the hypothesis that water born chemicals play
a role in regulating mating behaviour in P. monodon and that domestication can interfere
with some aspects of this physiological process.
A change in the physiology of the domesticated prawns used in this study was further
indicated by the pre-moult agitation of the males, presumably due to the female’s early
release of male stimulating cues. While the moulting process in the domesticated females
(exit from shell and subsequent flicking motions) did not appear to differ from that of
wild females, pre-moult stimulation of males by domesticated females may also relate to
a change in the systems that control pheromone release during the moulting process. It
has been well established that moulting is under the control of the endocrine system and
involves a number of different hormones (Huberman 2000).
Additional evidence that domestication can affect prawn physiology is provided by a
significant time difference during which moulting occurs as noted in female prawns in the
current study. Like most crustaceans, P. monodon moults at night in response to diurnal
cues via a number of regulating hormones. Interestingly, on average, domesticated
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females moulted significantly later in the night (02:30) than did wild caught females
(23:30). The delay may, in part, be explained by geographical origin and the associated
differences in day length (Chung et al 1994) however it does not explain the extended
period over which moults occurred in domesticated females (11hrs 20mins) compared
with wild females (4 hrs 20 mins). It is possible that endocrinal mediation of the moulting
process has been altered by the domestication process that responses to environmental
cues, including light intensity, delayed moulting.
Thus results of this study have shown that both male and female domesticated prawns can
exhibit reduced mating behaviour. We hypothesise that such reductions in mating
behaviour would contribute to poor hatch rates in cultured P. monodon. Cues required to
stimulate a male to vigorously pursue and mate with a female are evidently poorer in
domesticated females (possible release of pheromones) and are not being detected by
domesticated males (possible inadequate receptors such as antennules, the periopod
dactyls, and the mouthparts which are primary chemoreceptor organs (Kamio et al 2005,
Lin et al 2000)). To improve mating success in prawns reared incaptivity and reduce the
industries need to artificially inseminate domesticated prawns, the environmental and
endocrinological factors that control or influence the processes involved in successful
natural mating require further investigation.
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Chapter 9
GENERAL DISCUSSION AND CONCLUSIONS
As detailed in the introduction, the sustainability of the P. monodon aquaculture industry
is hampered by a reliance on wild-caught broodstock whose supply is limited both in
quantity and availability, and also has the potential to introduce diseases to the culture
environment. In an effort to address this problem, the work conducted in this thesis
sought to identify factors which contribute to poor reproductive performance of captive
stock. Of particular interest were the mechanisms of, and factors influencing, ovary
development and egg quality including the industry practice of holding prawns captive
and ablating them to induce spawning.
Overall, the findings presented in this thesis demonstrated that the captive environment
(and associated husbandry practises) had a profound influence on physiological and
behavioural processes that are fundamental to P. monodon reproduction.
Initial studies confirmed the significance of protein and lipid in P. monodon egg
production. As described previously (2.3.1) protein and lipids comprise approximately
80% of P. monodon egg dry matter. Interestingly, it was shown in Chapter 4 that the
levels of protein and lipid in mature ovaries were not affected by captivity (as per the
conditions of the study) and ablation. As a consequence, we suggest that any negative
effect on the quality of eggs from the first post-ablation cycle which results from industry
165
practises (ie. captivity or ablation) cannot be attributed to levels of these major nutrients.
Nevertheless, upon closer examination it was clearly demonstrated that captivity and/or
ablation had a major impact on several aspects of ovary development. Most notably,
(i) Captivity caused ovary regression
(ii) Captivity caused a reduction of lipid levels in previtellogenic ovaries
(iii) Ablation initiated secondary vitellogenesis, and
(iv) Ablation and/or captivity caused a change in the pattern of nutrient
accumulation in the developing ovary.
The low lipid level in previtellogenic ovaries (iii) was considered a particularly
interesting finding because it reflects the arrested development of early stage ovaries in
captive females. To further investigate the potential roles that SG hormones were playing
at this early stage(s), an additional study was conducted to isolate the effects of ablation
from captivity in previtellogenic ovaries (Chapter 5). A significant finding was that
ablation reduced the depletion of nutrients from the ovary and hepatopancreas that was
associated with starvation. This outcome strongly suggests that SG hormones are involved
in the earlier stage(s) of ovary development in P. monodon. Findings of the study also
indicated that the SG regulation at this early stage may be independent of secondary
vitellogenesis, which did not proceed under the environmental conditions of this
particular study.
An additional finding of Chapter 4 and 5 was that ablation increased the protein and lipid in
the hepatopancreas during early ovary development, providing evidence that SG
166
hormones are also involved in regulating hepatopancreas reserves during this stage.
Furthermore, the observed mobilization of hepatopancreas reserves (notably of lipids) at
the onset of secondary vitellogenesis supports existing evidence that, the hepatopancreas
is involved in the synthesis of vitellogenin (egg yolk precursor) in P. monodon (Vincent
et al 2001, Longyant et al 2003).
The indication that the SG hormones are involved in regulating development in
previtellogenic ovaries is a major finding of the current investigations since relatively little
is known about the hormonal control of ovary development at this stage (Thurn and Hall
1999). The results also confirmed that ablation can stimulate secondary vitellogenesis,
which has been attributed to the vitellogenesis inhibiting hormone (VIH), one of the SG
hormones (for reviews see Quackenbush, 1986, Charnaux-Cotton, 1986).
Whether early accumulated reserves are components of the egg yolk vitellin (Thurn and
Hall 1999) or are accumulated to perform other functions during embryogenesis (Avarre
et al 2001, Tsutsui et al 2005) remains to be determined. Thus it is not clear whether
these changes represent ‘primary vitellogenesis’ which by definition is the endogenous
synthesis of vitellin by the ovary. Regardless of their function, their accumulation
evidently requires a decrease in SG hormones, which can be achieved through ablation.
Importantly, the findings of the current studies also showed that the ability of ablation to
instigate early or late stage development is influenced by culture environment. More
precisely, environmental conditions can have a stage specific affect. For example, the
167
captive environment used in Chapter 5 prevented ablated prawns from undergoing
secondary vitellogenesis but enabled them to progress to earlier stage(s). Based on these
findings, we hypothesise that for spontaneous development to occur, as it does seasonally
in the wild, specific environmental factors are required that provide essential signals or
resources at each developmental stage.
A possible reason the captive environment can arrest ovary development is that it lacks
some essential stimuli which, in addition to the decreased level of SG inhibitory
hormones, are required for ovary development to proceed. Based on previous published
studies, it was proposed that the terpenoid hormone methyl farnesoate (MF) was the
missing stimuli (for review see Huberman 2000). Accordingly MF was orally
administered to ablated prawns and results confirmed that MF has a role in regulating
reproduction in P. monodon. However contrary to previous results (Laufer 1992, Hall et
al 1999), MF as administered in this study, inhibited the final stage of ovary development
and reduced fecundity. Broodstock diet was also shown to affect development at this
stage. Thus while MF failed to stimulate development of early stage prawn ovaries, the
study identified a third stage at which ovary development can be arrested in P. monodon
and implicated both hormones (notably MF) and diet as regulatory factors.
As described previously, the other major finding of interest in Chapter 4 was that, for the
first post ablation cycle, captivity and ablation had no effect on total lipid and protein
levels in mature ovaries. It remains to be determined, however, if these treatments
affected lipid quality and/or pattern of utilization as eggs and larvae develop. Therefore
168
an additional study was conducted (Chapter 7) which measured relative levels of specific
fatty acids. Results showed that captivity and /or ablation increased levels of the HUFAs
20:5n3 and 22:6n3 and reduced levels of most MUFAs and n6 fatty acids in eggs. A
second key finding of this study, was that the eggs from the ablated-captive prawns
showed a major decline in lipids (%DM) during development and hatching. By contrast,
eggs from the wild treatment group only showed a significant decline in lipids during the
later nauplii development stage. Based on these findings, we deduced that lipid quality
and metabolism in eggs and lecitotrophic larvae are significantly altered by captivity
and/or ablation.
Having determined that standard culture industry practises (ie. captivity and ablation)
have major impacts on the physiology of P. monodon ovaries, eggs and larvae, the final
study saught to determine what impact domestication had on other important reproductive
processes in this species. Specifically, the final study (Chapter 9) aimed to isolate mating
success as a factor contributing to the low hatch rate (HR) of eggs from domesticated P.
monodon broodstock. Video observations of the mating behaviour of different
combinations of pond-reared domesticated (D) and wild caught (W) prawn broodstock
revealed copulation occurred for the W x W mating pairs, but not within the D group. In
addition, precopulation behaviour, primarily the male pursuit of moulted females, was
lower for groups involving D males or females. Thus one observable difference in mating
behaviour between W and D males was a decrease in the male’s response to the female.
169
Cross-matings of W and D male and female prawns further showed that a reduction in
pursuit occurred when either a W female was paired with a D male or D female was
paired with a W male. This result suggests that D females may be less attractive (able to
stimulate a male response) and D males are less receptive (able to detect and/or respond)
to cues from the D females than their W counterparts. Based on this evidence, we propose
that both genders contribute to the poor mating rate frequently reported for domesticated
P. monodon prawns.
We propose that a lack of natural mating success would contribute significantly to low
hatch rate of eggs from the domesticated P. monodon stock observed in the current study.
As there were no external structural abnormalities visible in the domesticated prawns, we
hypothesise that the factors responsible for the changed behaviour are physiologically
driven. In particular, that the physiological processes underlying mating behaviour have
been compromised by the captive rearing of the prawns. This hypothesis is further
supported by the significant delay that occurred in the time the domesticated females
moulted in the current study. It will be interesting in future studies to determine if the
noted changes in physiology and mating behaviour of prawns held or bred in captivity
have the same underlying cause, for example lack of appropriate environmental stimuli
during prawn or ovary development.
170
Conclusion
Thus, the series of studies comprising this thesis have improved our understanding of
reproduction in P. monodon. Most notably the findings provided new or further evidence
that
• the levels of protein and lipid in mature ovaries of wild caught broodstock is not
altered by the industry-based conditions of captivity or the process of ablation,
• patterns of ovary nutrient accumulation, particularly during early ovary
development, are altered by captive conditions,
• the SG hormones together with the environment regulate both previtellogenic and
secondary vitellogenic stages of ovary development,
• the final stages of ovary development (which represents a third stage at which
development can be controlled in P. monodon) is influenced by MF, diet and yet
to be defined aspects of domestication
• domestication of P. monodon can cause a significant reduction in mating success
due to apparent changes in the physiology and hence, mating behaviour of both
male and female prawns.
Until the interplay between hormones, tissues and the environment is better understood,
the practical application of single hormones (such as MF) for the regulation of
reproduction in crustaceans is likely to remain problematic.
171
Chapter 10
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