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
1 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…………………......
2
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
4 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
5 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
6 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
7 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
8 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
9 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
10 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
11 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
12 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.
13 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
14 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
15 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.
16 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;
17 • 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
18 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
19 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 $42.5 million in 2006–07(Lobegeiger and Wingfield 2008).
20 2. 2 Penaeus monodon
Penaeus monodon is one of the largest penaeid species in the world, with females reaching up to 336 mm in body length (Gey et al 1983). Also called the giant tiger or black tiger, it is eurythermal and euryhaline for most of its life, and is known for its rapid growth rate (Motoh 1984).
2.2.1 Global production of Penaeus monodon
Up until 2000 Penaeus monodon was the main prawn species cultured world wide (FAO
2009), and was the highest in terms of value, of all aquaculture species (Tacon 2003). P. monodon was therefore an extremely significant aquaculture species. By 2006 P. vannamei had replaced P. monodon as the highest value aquaculture species (FAO 2009) with annual global production value nearly twice that of P.monodon.
21
Despite being the main prawn species cultured, global production of P. monodon peaked at 730 404 MT in 2003 and declined to 658 222 MT by 2006 (FAO 2009). This decrease was primarily due to disease problems that have forced growers to culture alternative species, for example P. vannamei. This trend has seen the world production of P. vannamei increase from 481 298 MT in 2002 to 2 133 381 MT in 2006; over three times the volume of P. monodon produced (FAO 2009). Despite the popularity of P. vannamei,
P. monodon remains the species of choice as it commands a higher price. For example in
2006 P.monodon was priced at US$ 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
22 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
23 • 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.
24 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.
25
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).
26 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
27 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
28 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).
29 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
30 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
31 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.
a.
b. c.
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
32 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).
33 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.
34 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.
35 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.
36
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
37 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).
Environment MIH X-organ-sinus CHH Y-organ gland complex MF Thoracic ganglion
MOIH ? GIH ECD VSH ? GIH Mandibular organ CHH GIH MF MF VSH ? VG Hepatopancreas Ovary VSH ?
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
38 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).
39 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
40 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
41 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
42 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.
43 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-
44 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.
45 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.
46
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
47 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 Male mating success depends largely on their ability to win of dominance 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 Adults live solitarily on hosts (or in other refuges), but males change attend 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.
48
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).
49 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.
50
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).
51
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
52 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
53 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
54 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
55 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.
56 • 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.
57 • 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.
58 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,
59 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
60 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.
61 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
62 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).
63 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.
64 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.
65 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.
66 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
67 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).
68 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.
69 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.
70
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
71 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.
72 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-
73 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.
74 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.
75 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).
76 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).
77 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 25.61±0.5a 26.1±0.3a 29.4±0.5b 32.6±0.8b 31.1±0.4b
(8) (12) (11) (18) (10) (8)
Captive- 21.6+0.5c 22.7±0.9c 27.4±0.3a 29.3±0.1b 29.3±0.3b 30.7±0.3b ablated (12) (7) (13) (15) (15) (14)
Captive-non 23.5+0.4 ca 24.1±1a,c 25.2±1a,c ablated (9) (7) (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)
78
500 6 a 400 6 5 5 300 4 4
200 2 3 2 3 1 2 1 b 1 1 b a 100 a a mgslipid / 100g prawn a
0 123456
1400 6 4 b 1200 5 3 1000 4 2 800 3 1 600 2 2 1 1 1 400 1 1 200 mgs protein /prawn 100 g 0 123456 GSI
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.
79
300 a 250 200
150
100 Mgs of protein of Mgs 50
0 Wild 1 to 2 2 to 3 3 to 4 Ablated 4 to 5 5 to 6 GSI
90 b 80 70 60 50 40
Mgs of lipid of Mgs 30 20 10 0 Wild 1 to 2 2 to 3 Ablated 3 to 4 4 to 5 GSI 5 to 6
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.
80 1000 1 b a 1 1,2 1 800 1 a b a a 1 600 1 1 a 2 1 1 b c a 2 1,2 400 a b a 1,2 2
mgs lipid / 100g prawn 200 b
0 123456
600 b 1 1,2 1 1 1 1 500 1 1 1 1 1 1 21 1 400
300
200
mgs protein/ prawn 100 g 100
0 123456 GSI
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.
81 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 Oocyte Oocyte diameter (µm) GSI
stage* Stage* n = 10
Ablated Wild Ablated Wild
0-I P 45.6± 1.51 52.6±0.91 1.4 ± 0.5 1 1.4 ± 0.21
(105) (96) (1.2- 2.9) (1.3-3.2)
I-III V 198.0± 1.7a2 233±1.2 b2 4.4 ± 0.9 2 5.0 ± 0.42
(111) (120) (1.9- 5.9) (3.1- 7.0)
III-IV Early CR 240.3± 2.03 261±1.92 5.9 ± 1.1 3 6.5 ± 1.0 3
(95) (87) (3.8- 7.1) (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.
82 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
83 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,
84 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 ).
85 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
86 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
87 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.
88 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)
89 both the ovary and hepatopancreas contribute nutrients (protein and lipids) to meet metabolic requirements during periods of food deprivation.
90 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
91 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.
92 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.
93 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.
94 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%.
95 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.
96 Table 5.2. Mean level of protein and lipid in the ovary and hepatopancreas tissues.
Treatment Ovary Hepatopancreas Dry matter Protein Lipid Dry matter Protein Lipid (%) (mg) (mg) (%) (mg) (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.
97 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
98 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
99 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
100 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.
101 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
102 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.
103 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
104 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
105 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.
106 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
107 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
108 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.
109 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
110 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).
111 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 Moult Ovary Development (%) weight gain (g) interval development arrested at stage (g) (days) progressed to III (%) 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.
112
a 5 3000 a a a A B 4 b 2000 3 c 2 1000 1
spawns/prawn 0 0 Natur al BIARC BIARC + eggs/gram prawn Natur al BIARC BIARC + MF MF
6000 C 80 a a a D ab a 4000 b 60 40 2000 20
0 rate (%) hatch 0 eggs/gram prawn eggs/gram Natur al BIARC BIARC + Natur al BIARC BIARC + MF MF
a 2000 a a F 100 b b E 80 a 1500 60 1000 40 20 500 survival (%) 0 0
Natur al BIARC BIARC + (No/g/spawn) zoea 1 Natur al BIARC BIARC MF +MF
a a 1.5 a G 5 a H 4 1 ab 3 b 0.5 2 1 0 0 zoea 1 (10E5/spaw n) Natur al BIARC BIARC + zoea(10E5/praw 1 n) Natur al BIARC BIARC + MF MF
113 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)
114 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
115 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
116 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
117 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
118 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).
119 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.
120 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
121 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.
122 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
123 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
124 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.
125 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.
126
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).
127
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).
128 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.
129 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
130 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.
131 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
132
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).
133 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 Egg Nauplii Protozoeal 1 per g of dry matter W A W A W A Neutral lipids 113.6a1 135.0 a1 90.8b1 71.0 c1 16.7 d1 22.0 d1 ±4.7 ±17.3 ±8.2 ±5.5 ±4.3 ±4.0
Polar lipids 73.3a2 74.0 a2 74.3 a2 54.0 b2 40.2c2 41.0 c2 ±4.4 ±7.2 ±6.1 ±5.5 ±4.3 ±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).
134 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).
135 15 * 10
5
e P 16:1n7 20:4n6
change N
Percentage 0 20:5n3 22:6n3 -5 * -10 Fatty acids
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.
136 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 Stage Neutral Polar acids Wild Ablated Wild Ablated E 16.2a1 ±0.7 9.6 b1 ±1.1 13.2a1 ±0.2 6.9b1 ±0.8
a1 b1 a1 b12 16:1n-7 N 16.0 ±1.8 9.3 ±0.7 12.5 ±1.8 6.0 ±0.2 Z 12.3a2 ±1.0 7.3b2 ±0.7 6.8 a2 ±0.9 4.0 b2 ±0.2 E 15.3a1 ±0.8 12.5a1 ±0.8 16.9a1 ±0.4 14.0a ±0.7
a1 a1 a1 18:1n-9 N 15.8 ±0.5 13.3 ±0.3 15.0 ±0.8 11.8 ±0.6 Z 15.5a1 ±0.2 13.5a1 ±0.6 11.5 a2 ±1.1 10.5 ±0.4 E 3.8 a1 ±0.1 2.3 a1 ±0.1 7.5 a1 ±0.1 3.7 b1 ±0.7
a1 a1 a12 b1 20:4n6 N 3.5 ±0.5 2.4 ±0.1 8.2 ±0.7 4.9 ±0.8 Z 4.1 a1 ±0.6 2.5 a1 ±0.1 10.0 a2 ±1.6 5.7 b1 ±0.4 a1 b E 5.3 a1 ±0.2 10.5b1 ±0.5 8.6 ±0.4 13.5 ±0.8
a1 b12 a1 b2 20:5n-3 N 4.0 ±0.5 8.1 ±0.3 9.6 ±1.0 16.1 ±0.5 a1 b2 Z 3.9 a1 ±0.7 6.1b2 ±0.8 11.3 ±1.8 16.7 ±0.4 E 6.1 a1 ±0.6 19.0b1 ±2.6 6.3a1 ±0.4 17.1b1 ±1.4
a1 b12 a12 b2 22:6n-3 N 4.8 ±0.5 16.1 ±1.4 8.0 ±0.4 19.1 ±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).
137 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
138 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.
139 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
140 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
141 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.
142 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.
143 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.
144 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.
145 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
146 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.
147 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.
148 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.
149 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
150 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.
151 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
152 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. .
153 Table 8.1: Pairings of male and female prawns placed in observation tank for videoing.
Females Males (1 pre and 1 post moult) (2 inter moult) W W Origin of prawns W D D D D W
154 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 TRAIT DESCRIPTION BEHAVIOUR MEASURE GENDER Implantation of the Observation of spermatophore transfer; Male Mating spermatophore into the male wrapping himself around the moulted females thelycum female and demonstrating rapid muscle contractions. Verified by visual examination of the female thelycum the following morning; swelling and tissue protrusion. Males showed agitation Percentage of observations showing an Pre-moult (change in location) increase in time (seconds) that the male was agitation prior to the female active (swimming or walking), during the moulting 30 minutes before the female moulted when compared to the 30 to 60 minute period prior to moult. Males showed agitation Percentage of observations showing an Post moult (change in location) increase in time (seconds) that the male was agitation after the female active (swimming or walking), during the moulted 30 minutes after the female moulted compared to the 30 minutes prior to moult. Male follows female Percentage of observations that the male Frequency of swims within 5 body-lengths of the moulted pursuit female; follows her path for a period of 3 seconds or more Males show higher Percentage of time during post moult Intensity of pursuit intensity by pursuit spent under the moulted female pursuit maintaining closeness during the first 20 minutes of the female to moulted female swimming Number of Whether one or both Number of males (one or two) that swim males that females pursued within 5 body-lengths of the moulted pursued female; follows her path for a period of 3 seconds or more Pursuit of inter- Male pursues the The male swims within 5 body-lengths of moult females intermoult female as the ‘other’ (non moulted) female; follows well as or instead of the her path for a period of 3 seconds or more moulted female Male cleaning Males pass the length Number of times a male ‘cleaned’ antennule of antennule(s) near (one or two) within the hour after female mouth area moulted Time of night when the Number of females that moulted in each 60 Female Time of moult female moults minute interval after placement in observation tank at18:00hrs
155 Table 8.3. Behavioural traits measured for each of the four experimental groups.
Origin (Female: Male) D:D D:W W:D W:W n = 7 n = 8 n = 8 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).
156
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).
157 4
3
PR female 2 W female
1 Number of moults
0 12345678910111213 Hours after 18:00
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.
158 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
159 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
160 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
161 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
162 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.
163 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
164 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
165 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
166 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
167 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.
168 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.
169 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.
170 Chapter 10
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