FRESHWATER PRAWN Macrobrachium Rosenbergii (De Man, 1879) (Crustacea: Decapoda) AQUACULTURE in FIJI: IMPROVING CULTURE STOCK QUALITY

FRESHWATER PRAWN Macrobrachium rosenbergii (De Man, 1879) (Crustacea: Decapoda) AQUACULTURE IN FIJI: IMPROVING CULTURE STOCK QUALITY

Shalini Singh

A thesis submitted in fulfilment of the requirements for the degree of Master of Science in Marine Science

School of Marine Studies Faculty of Science, Technology and Environment The University of the South Pacific

July, 2011

Acknowledgements

I would like to thank my sponsors, the Australian Center for International Agricultural Research (ACIAR) and The University of the South Pacific (USP), for providing the opportunity to study for my master’s degree. My sincere thanks and gratitude to my supervisors, Dr. William Camargo (USP), Professor Peter Mather, Dr. Satya Nandlal, and Dr. David Hurwood (Queensland University of Technology - QUT) whose support, encouragement and guidance throughout this study is immensely appreciated. I am also very grateful to the following people for critically editing my thesis: Prof. Peter Mather, Dr. Satya Nandlal, Dr. William Camargo and Dr. Carmen Gonzales. Thank you all for your enormous support and guidance, which led to the successful completion of the project. I thank you all for allocating time from your very busy schedules to read my thesis.

I would also like to thank the Fiji Fisheries Freshwater Aquaculture Section, particularly Naduruloulou Research Station, the staff for their immense effort and assistance, particularly in the hatchery and grow-out phases which could not have been successfully carried out without your support. I acknowledge the support, and assistance provided by ACIAR Project Officer Mr. Jone Vasuca and Kameli Lea for their immense effort during this project. Thank you for all the hard work while conducting fieldwork at Naduruloulou. I would also like to thank Dr. Simon Hodge of the Biology Department at USP for his suggestions, advice and assistance in carrying out the statistical analysis and data presentation in this thesis.

I deeply appreciate the support and assistance provided by my families and friends. I am deeply thankful to my parents and my brothers for their assistance in providing transportation during my fieldwork to Naduruloulou Research Station especially in the weekends. I would also like to acknowledge the support provided by Temalisi Koroi, Monal Lal, Parnesh Kishore, Emmanuel Ram, Vinod Kumar, Avinash Singh and many others who have helped me in successfully carrying out my studies.

Abstract

A comparative growth trial of the giant freshwater prawn (GFP), Macrobrachium rosenbergii (De Man, 1879) was conducted to evaluate the most suitable strain from three (Vietnam, Indonesia and Malaysia) high performing stocks to be introduced in the Republic of Fiji as a means to improve local culture stock quality. Advanced postlarvae (PL) of each strain (130 from Indonesia, 250 from Malaysia and 300 from Vietnam) were reared to maturity, under quarantine conditions, in separate tile-lined cement tanks at the Fiji Fisheries Naduruloulou Research Station. Berried females from each exotic strain and from the local strain (Fijian-control) were selected randomly and PL was generated in a single hatchery cycle for strain evaluation trials. PL (PL 8 to 10, average weight range of 0.01-0.02 g) were stocked at a density of 5 PL/m2 into 12 earthen ponds (4 strains x 3 replicates), and grown for 147 days (d) while being fed with a locally produced (29 percent protein) sinking pellet at a set ratio to mean body weight. No significant difference (P = 0.083) was observed for mean final body weight among strains (Fiji 25.89 ± 0.57, Vietnam 26.86 ± 2.59, Indonesia 24.66 ± 1.68 and Malaysia 23.38 ± 1.18 g). However, a significant difference was observed among the different morphotype stages (P = 0.001). Survival ranged from 69 to 84 percent, but no significant difference (P = 0.05) was evident among strains (Fiji 69 ± 14, Vietnam 84 ± 7, Indonesia 79 ± 2 and Malaysia 75 ± 5 percent). Specific growth rate (SGR) ranged from 4.84 - 5.28 percent (Fiji 4.87, Vietnam 4.89, Indonesia 4.84 and Malaysia 5.28 percent), food conversion ratio (FCR) ranged from 1.86 - 2.75 (Fiji 2.42 ± 0.64, Vietnam 2.75 ± 0.41, Indonesia 2.44 ± 0.42 and Malaysia 1.86 ± 0.67 percent), sex ratio ranged from 1:1.0 - 1:1.7 and marketable prawn (>20 g) ranged from 56 - 70 percent (Malaysia 56 ± 34, Fiji 59 ± 40, Indonesia 62 ± 10 and Vietnam 70 ± 27 percent). In this study physical water quality parameters (temperature, pH and dissolved oxygen) showed no significant difference among ponds or strains. Results showed that there was no significant difference in mean growth performance among the strains (Vietnam, Indonesia, Malaysia and Fiji) when environmental factors were suitable for growth. While not significant, the Vietnam strain was the top ranked strain based on final survival, mean body weight gain, evenness of different morphotype and sex and percentage marketable

ii prawn. The Indonesian strain was ranked second just below the Vietnamese strain. While the Malaysian strain was ranked third, when compared to the Fijian strain however, selection of strain should also consider the hatchery phase because this strain performed the poorest in the hatchery. This study provides baseline data for establishing an informed choice on strains options to take into future and to identify a superior strain for the local culture industry. Additional experiments on larval development and growth performance among the four strains with appropriate replication need to be conducted at other sites to allow genotype by environment (G x E) effects in Fiji to be assessed in a comprehensive manner.

Keywords: Freshwater prawn, Macrobrachium rosenbergii, strain evaluation, Fiji, Vietnam, Malaysia, Indonesia

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Abbreviations and acronyms

ACIAR - Australian Centre for International Agricultural Research d – Day

D.O. – Dissolved oxygen

FAO – Food and Agricultural Organization

FCR – Food conversion ratio

GFP – Giant Freshwater Prawn

G and E (G x E) – Genotype by environment h - Hour

JICA – Japan International Cooperation Agency kg – Kilogram

LRT – Larval rearing tank

MT – Metric tons

MFF – Ministry of fisheries and Forest mg/L – Milligrams/Liter

Ne – Effective population size

NRS – Naduruloulou Research Station

PICs – Pacific Island Countries

PL – Post larvae ppt – Part per thousand

QUT – Queensland University of Technology

SAS – Sigatoka Agricultural Station

SGR – Specific growth rate

SPC – Secretariat of Pacific Community

SPIFDA – South Pacific Islands Fisheries Development Agency

TF – Tebara Farms Ltd t – Tons

UNDP – United Nations Development Programme

USP – University of the South Pacific

Table of Contents

Acknowledgments……………………………………………………………………….i Abstract…………………………………………………………………………………..ii Abbreviations and acronyms……………………………………………………………..iv Table of Content…………………………………………………………………………vi List of Tables ……………………………………………………………………………ix List of Figures……………………………………………………………………………xi Chapter 1 Introduction ...... 1 1.1 World aquaculture production ...... 1 1.2 Regional status of aquaculture in the Pacific ...... 2 1.3 Brief history of aquaculture in Fiji ...... 5 1.4 Global status of M. rosenbergii culture ...... 6 1.4.1 M. rosenbergii introduction in the Pacific ...... 7 1.4.2 M. rosenbergii culture in Fiji ...... 9 1.5 Problems confronting GFP industry in Fiji ...... 10 1.6 Development of high performing GFP culture lines ...... 12 1.7 Why culture GFP? ...... 13 1.8 Objectives of the study ...... 13 1.9 Thesis overview...... 14

Chapter 2 Literature Review…………………………………………………………..15 2.1 Nomenclature and morphology……………………………………………………...15 2.2 Distribution of M. rosenbergii……………………………………………………….16 2.3 Biology……………………………………………………………………………….17 2.3.1 Food habitat……………………………………………………………………..17 2.3.2 Life cycle and larval development……………………………………………...18 2.3.3 Hygiene and diseases………………………………………………………….. 19 2.3.4 Water quality management……………………………………………………...20

2.3.5 Growth and Molting ...... 21 2.4 Larviculture of M. rosenbergii ...... 21 2.4.1 Green water culture ...... 22 2.4.2 Clear water culture ...... 23 2.5 Grow-out, harvesting and marketing ...... 23

Chapter 3 Comparative grow-out trials of four GFP strains in earthen pond ...... 26 3.1 Introduction ...... 26 3.2 Materials and Method...... 27 3.2.1Study site ...... 27 3.2.2 Larval production ...... 28 3.2.3 Grow out ...... 31 3.2.4 Sampling design ...... 33 - Pond sampling ...... 33 3.2.5 Measurement of Prawn Growth ...... 33 3.2.6 Evaluation of growth parameters ...... 34 3.2.7 Statistical Analysis ...... 35

Chapter 4 Results…………………………………………………………………….... 36 4.1 Water Quality ...... 36 4.2 Prawn Growth ...... 38

Chapter 5 Discussion and Conclusion ...... 45 5.1 Water parameters...... 45 5.2 Comparison of growth performance ...... 45 5.3 Survival ...... 48 5.4 Genotype-environment interaction ...... 51 5.5 Feed ...... 52 5.6 Body traits ...... 54 5.7 Size variation ...... 55 5.8 Sex ratio...... 57

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5.9 General discussion...... 58 6.0 Conclusion ...... 62 7.0 Recommendation ...... 64 Literature cited ...... 65 Appendix 1 ...... 79 Appendix 2 ...... 81 Appendix 3 ...... 83

viii

List of Tables

Table 1. Annual value of aquaculture production per country………………… ...... 2

Table 2. Number of farm units and persons involved in aquaculture……… ...... 5

Table 3. Feeding ratio based on body weight of PL………………………………...... 18

Table 4. Water parameters recommended for prawn grow-out…………………… ...... 20

Table 5. Initial weights, length, of berried M. rosenbergii female’s representative of the four strains in pond on 7 January 2011……………………………………… ...... 30

Table 6. Initial M. rosenbergii PLs weights, stocking density, pond area and strains from four countries stocked in triplicates in 12 earthen ponds……………… ...... 31

Table 7. Formulated feed composition of pellet diet fed to freshwater prawns M. rosenbergii over the five months ...... 32

Table 8. Water quality parameters recorded in the 12 experimental M. rosenbergii giant freshwater prawn culture ponds over 5 months (mean ± se) ………………… ...... 36

Table 9. Least square means of body traits for each strain of the giant freshwater prawn M. rosenbergii cultured over five months…………………………… ...... 38

Table 10. Summary of stocking density, final body weight, specific growth rate, survival rate and food conversion ratio and percent marketable prawn (>20g) of the giant freshwater prawn M. rosenbergii cultured over five months in earthen ponds...... 41

Table 11. Summary of outcomes of tests for body traits on the giant freshwater prawn M. rosenbergii strains ...... 42

Table 12. Growth performance of male and female for strains of the giant freshwater prawn M. rosenbergii cultured over five months…………………………...... 44

List of Figures

Figure 1. Crustacean production in Pacific region ...... 4

Figure 2. Main producer countries of M. rosenbergii ...... 7

Figure 3. Geographical boundary separation between Asia and Australia ...... 17

Figure 4. Life cycle of M. rosenbergii ...... 19

Figure 5. Map of study site...... 28

Figure 6. Biometric measurement of M. rosenbergii ...... 34

Figure 7. Temperature of four strains of M. rosenbergii cultured over five months ...... 37

Figure 8. D. O. of four strains of M. rosenbergii cultured over five months ...... 37

Figure 9. pH of four strains of M. rosenbergii cultured over five months ...... 38

Figure 10. Weight gain in four strains of freshwater prawns M. rosenbergii, cultured over five months in earthen ponds ...... 39

Figure 11. Body weight of four strains of freshwater prawns M. rosenbergii, cultured over five months in earthen ponds ...... 40

Figure 12. Body weight of four strains of freshwater prawns M. rosenbergii, classified into male and female morphotype...... 40

Figure 13. Survival of four strains of freshwater prawns M. rosenbergii, cultured over five months in earthen ponds ...... 41

Figure 14. Percentage morphotype proportions of four strains of freshwater prawns M. rosenbergii, cultured over five months ...... 43

Figure 15. Percentage berried female and mean weight of berried and spent females of the giant freshwater prawn M. rosenbergii strains after 63 and 143 days of culture ...... 43

Figure 16. Least square means of body traits for male and female of the giant freshwater prawns M. rosenbergii, cultured over five months ...... 44

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Chapter 1. Introduction

1.1 World Aquaculture Production

The Food and Agriculture Organization (FAO) defines aquaculture as the farming of aquatic organisms and aquatic plants with some form of intervention in the rearing process to enhance production, such as regular stocking, feeding, protection from predators and individual or corporate ownership of the stock being cultivated (FAO, 2003).

Aquaculture is currently the world’s fastest growing animal food-producing sector and is outpacing human population growth, with per capita supply from aquaculture increasing from 0.7 kg in 1970 to 7.8 kg in 2006, an average annual growth rate of 6.9 percent (FAO, 2008). Aquaculture is playing an increasing role in meeting demand for human consumption of fish and fishery products. In the past few years, major increases in the quantity of fish consumed have originated from aquaculture. The average contribution of aquaculture to per capita fish available for human consumption rose from 14 percent in 1986, to 47 percent in 2006, and is expected to reach 50 percent in the next few years. Aquaculture has also had a major role in terms of meeting food security needs in several developing countries, particularly in Asia, with significant production of some low-value freshwater species, which are mainly destined for domestic consumption (FAO, 2008). Further, the contribution of aquaculture to global supplies of fish, crustaceans, molluscs and other aquatic animals has continued to grow, increasing from 3.9 percent of total production by weight in 1970 to 36.0 percent in 2006 (FAO, 2008). In 2006, 47 percent of the world’s fish food supply came from aquaculture which is likely to overtake capture fisheries as a source of food fish. From a production of less than 1 million tons per year in the early 1950s, aquaculture production in 2006 was reported to be 51.7 million tons with a value of USD78.8 billion, representing an annual growth rate of nearly 7 percent (FAO, 2008). World production will need to increase however, by 30- 40 million tons from its current production level by 2030 to meet growing global 1

Chapter 1. Introduction demand for fish. World aquaculture is heavily dominated by the Asia-Pacific region, a region that accounts for 89 percent of production in terms of quantity and 77 percent in value (FAO, 2008). The Asia–Pacific region accounts for 98 percent of carp, 95 percent of oyster production, and 88 percent of shrimps and prawns. Furthermore, the contribution of farmed products to international trade has grown considerably, with export growth rates for species such as catfish and tilapia exceeding 50 percent per year (FAO, 2008).

1.2 Regional Status of Aquaculture in the Pacific

In the Pacific, aquaculture is a new phenomenon dating back less than 50 years, with the introduction of Mozambique Tilapia from Africa for mosquito control and aquaculture (Ponia, 2010). There is no traditional history of culturing fish and shellfish thus aquaculture skills are limited and there is little infrastructure developments in the region. Compared with fishing, aquaculture is currently of little commercial significance (Table 1) in the Pacific with one important exception–black-lip pearl farming which is confined to eastern Polynesia (Adams et al., 2000). Development is needed before aquaculture can be considered economically sustainable elsewhere in the Pacific.

Table 1. Annual value of aquaculture production per country (USD thousands; source: Ponia, 2010). Country 1998 2007 American Samoa 10 Cook Islands 6,315 2,473 FSM Fiji Islands 217 2,244 French Polynesia 155,290 173,598 Guam 757 1,391 Kiribati 421 17 Marshall Islands 4 128 Nauru 15 New Caledonia 16,230 28,835 Northern Mariana Islands 205 Palau 24 PNG 1,477 1,725 Samoa 33 Solomon Islands 214 74 Tonga 141 180 Vanuatu 495 Total 181,065 211,646

Chapter 1. Introduction

Shrimp (Penaeus spp.) farming has been a focus of commercial development in several islands over the past 30 years, but with varying degrees of success; Tilapia (Oreochromis niloticus) aquaculture has contributed to the subsistence economy in some areas, and seaweed (Kappaphycus spp.) is considered a future commercial export (Adams et al., 2000). Culture of a number of other marine and freshwater species is still at an experimental or essentially ‘backyard’ stage. Pacific Island Countries (PICs) now recognize that aquaculture provides one of the few long-term, sustainable ways of deriving benefits from inshore fisheries resources (Adams et al., 2000). Profitable aquaculture of penaeid shrimps and blacklip pearl oysters has been established in some areas by commercial interests, enterprises producing for export markets that are firmly established in New Caledonia, Fiji and Solomon Islands and applying technology developed originally in Japan, Taiwan and France. Pearl’s are the region’s most valuable commodity. In 2007, the industry was valued at USD176 million (Ponia, 2010). Crustaceans made up the second most valuable commodity in 2007, valued at USD31 million - mainly from marine shrimps but with some contribution from freshwater species (Fig. 1).

Figure 1. Crustacean production excluding New Caledonia shrimps in Pacific regions (Source: Ponia, 2010).

Chapter 1. Introduction

The crustacean species farmed are blue shrimp (Litopenaeus stylirostris), giant tiger shrimp (Penaeus monodon), white shrimp (Litopenaeus vannamei), giant freshwater prawn (Macrobrachium rosenbergii), monkey river prawn (Macrobrachium lar) and red crawfish (Cherax quadricarinatus) (Ponia, 2010). Recent industry growth in Fiji has been due to freshwater prawn M. rosenbergii whereas French Polynesia is concentrating solely on marine shrimp (Ponia, 2010). New shrimp farms in Vanuatu, Northern Mariana Islands and Guam have also contributed to peak production in 2007 of 204 t worth USD2.7 million.

There are major opportunities for import substitution in the region, for example in marine shrimp. French Polynesia imports around 500 t but produces only 50 t and Fiji’s consumption is around 900 t of which it imports around 600 t. High cost of feed and poor supply of juveniles are the major impediments to meeting demand compared with the low price of imported shrimp (Ponia, 2010). In 2007, 1,230 t of edible products were sold locally, mainly consisting of marine shrimp and other commodities. In 2007, production unit analysis was carried out in countries that were major producers of pearl, shrimp and tilapia. It was estimated there were 3,200 farm units providing livelihood benefits to 9,290 persons involved in aquaculture (Table 2). Table 2. Number of farm units and persons involved in aquaculture (Source: Ponia, 2010). Farm units Persons Cook Islands 80 450 Fiji Islands 50 280 French Polynesia 530 5,000 New Caledonia 40 560 PNG 2,500 3,000 Total 3,200 9,290

1.3 Brief History of Aquaculture in Fiji

Aquaculture in Fiji dates back to 1940 when the possibility of freshwater fish culture was first presented. Hall (1949) suggested there was a general lack of animal protein in Fiji thus fish farming was a need. In 1949, first stock of Tilapia (Oreochromis

Chapter 1. Introduction mossambicus) was brought to Fiji and stocked at Sigatoka Agriculture Station (SAS) (Villaluz, 1972; Uwate et al., 1984). In 1954, a total of 54 fingerlings were imported from Malaysia by Dr. W. J. Payne and stocked at SAS (Holmes, 1954; van Pel, 1955; Villaluz, 1972; Rabanal et al., 1981; Uwate et al., 1984; Gillett, 1989; Gulick, 1989). This species was initially introduced to provide animal protein for pig stocks however, possibility of using Tilapia for human consumption was also examined (Holmes, 1954; Payne et al., 1954). In 1962, Fiji government introduced the Inland Fisheries Programme that included fish culture. In 1970, the United Nations Development Programme (UNDP) sponsored a project to assist the PICs in fisheries development called the South Pacific Islands Fisheries Development Agency (SPIFDA) with the objective to examine potential of aquaculture in the region. In the same year, the Fiji government had in its budget a five year aquaculture development programme with the aim of producing molluscs for local and tourist market as well as to provide livelihoods for local people (Uwate et al., 1984). In 1974, the Fiji government initiated a follow up activity to the SPIFDA program to develop fish and oyster culture in the country (Uwate et al., 1984; Eldredge, 1994). One objective of the Fisheries Division was to carry into the commercial phase the culture of aquatic plants and animals, in addition to weed control. In 1981, freshwater, brackish water and marine sites for aquaculture development were identified (Uwate et al., 1984). Aquaculture in Fiji was still far from being established; there was no private industry nor was there any stable source of seed, attributed to inadequate training and limited local resources. The UNDP/FAO projects assisted in initiating brackish water aquaculture and marine shellfish culture. By 1981, three freshwater prawn farms and seven fish farms were in operation and the Fisheries Division was providing technical advice on fish and prawn farming (Uwate et al., 1984). Major aquaculture facilities were constructed in July 1968 at Lami fisheries with four ponds which were stocked with aquatic macrophytes and grass carp. In 1948, an experimental pond was established at Naduruloulou which later in 1975 was expanded due to space limitations at Lami. The primary function of Naduruloulou Research Station (NRS) was to culture and spawn grass carp, this station was perceived to be the ‘Fiji Freshwater Aquaculture Program Center’ and in 1983 a freshwater prawn hatchery was established with the help from Japan International Cooperation Agency (JICA) 5

Chapter 1. Introduction

(Uwate et al., 1984). In 1973, fish culture ponds were constructed at Raviravi, Ba, as part of a Fiji Agricultural Department mangrove reclamation scheme. Culture trials were initiated on rabbit fish, mullet, milkfish and tilapia for demonstration of their commercial feasibility (Uwate et al., 1984). However, after five and half years of research, the government of Fiji and France Aquaculture established a joint shrimp farming project which later was terminated.

1.4 Status of M. rosenbergii Culture

Crustaceans makeup the second most valuable commodity in aquaculture with two groups cultured on a large commercial scale, namely, freshwater prawns and marine shrimp. While freshwater crayfish, lobsters and mud crabs (Scylla serrata) are also cultured on a smaller scale (Avault, 1996). Marine shrimp dominate crustacean aquaculture with production reaching 66 percent valued at USD6.8 billion in 2000 (FAO, 2003). The main cultivated species are Penaeus monodon, P. chinensis and L. vannamei, with these three species accounting for over 86 percent of total world shrimp production. The first recorded global data for farmed GFP (M. rosenbergii) production was 5,246 t in 1984 and in 1989 production had increased by three folds to 17,608 t valued at USD75 million (FAO, 2003). Global production of M. rosenbergii in 2007 was over 221,000 t, 2.7 times greater than a decade earlier (FAO, 2009). The three major species, which are commercially cultured for prawn production, are: M. rosenbergii valued at USD452 million, M. nipponense valued at USD698 million and M. malcolmsonii valued at USD118 million (FAO, 2009; New et al., 2010). By 2007, global farm-gate value of all species of freshwater prawn farming had reached almost 460,000 t in quantity with a total value of USD1.86 billion per year (FAO, 2009). The major areas of production in the different region of the world are divided into (1) Asia and the Pacific, (2) North and Latin America, and (3) Europe and Africa. The five major producing countries are China (56.3 percent), Thailand (12.5 percent), India (12.3 percent), Taiwan (4.5 percent), and Vietnam (3.6 percent) (New et al., 2010). Some small PICs also produce M. rosenbergii though not for export but for local markets, these include Fiji Islands, French Polynesia and New Zealand. It appears from data 6

Chapter 1. Introduction available that south, southeast and east Asia are the center of production of prawns. GFP farming is thus a major contributor to global aquaculture, both in terms of quantity and value (Wowor and Ng, 2007).

Figure 2. Main producer countries of M. rosenbergii (Source: FAO, 2006)

1.4.1 M. rosenbergii Introduction in the Pacific

The origin of freshwater prawn M. rosenbergii culture was initiated first by rearing wild-caught juveniles or by wild trapping (New and Valenti, 2000). However, the first experience of rearing M. rosenbergii from hatchery produced PL dates to the early 1960s. Ling (1977) and Ling and Costello (1979) noted that many experiments were conducted by Fisheries biologists all over the world directed at rearing prawn larvae but had been unsuccessful. In 1961 a major breakthrough was achieved at the Marine Fisheries Research Institute at Glugor, Penang (Malaysia), when the FAO expert Shao- Wen Ling discovered that freshwater prawn (M. rosenbergii) larvae required brackish conditions for their development and maturation (FAO, 2003). While Ling’s discoveries were fundamental, it was the work of another pioneer, Takuji Fujimura that made the commercial development of freshwater prawn culture possible. Fujimura’s research in Hawaii commenced in 1965 with the introduction of broodstock from Malaysia (Fujimura & Okamoto, 1972; Ling and Costello, 1979). Within three years, the work of Fujimura and his team at the Anuenue Fisheries Research Center in Honolulu developed mass rearing techniques for commercial scale hatchery production of prawn postlarvae 7

Chapter 1. Introduction

(PL) (Fujimura and Okamoto, 1972). Availability of PL for stocking initiated grow-out experiments and eventually led to commercial farms in Hawaii and elsewhere during the 1970s. Based on the success of the Hawaiian hatchery and grow-out research and commercial experience, broodstock were introduced both from Southeast Asia and from Hawaii, into many countries where M. rosenbergii was not indigenous. Introduction were made to other Pacific nations for example, in 1973 to Tahiti (Aquacop, 1977; Eldredge, 1994), in 1975 to Fiji (Uwate et al., 1984; Eldredge, 1994), in 1979 to W. Samoa (Popper, 1982; Eldredge, 1994), in 1983 to Solomon Islands (Nichols, 1985; Eldredge, 1994), in 1992 to Cook Islands (Eldredge, 1994) and in 1974 to Palau (Eldredge, 1994). Farming was carried out in Tahiti (Aquacop, 1983), Solomon Islands (Nichols, 1985), Samoa, Cook Islands and a few other countries at various times since the late 1970s. Farming has since been abandoned mainly due to closure of hatcheries because grow-out farms were too small to run hatcheries at full capacity or profitability (Aquacop, 1983) which lead to unavailability of PL’s. Currently in the Pacific, it is farmed in Fiji, Hawaii and New Zealand (FAO, 2003; Nandlal and Pickering, 2005). However, there is increasing interest in the region to re-start M. rosenbergii culture.

1.4.2 M. rosenbergii Culture in Fiji

Aquaculture of freshwater prawns in Fiji has come a long way since its first introduction in 1970s. Although M. lar is the indigenous species captured in Fiji, M. rosenbergii is the species first experimentally cultured. GFP was introduced in the country in 1973 from Hawaii by government officer and two other Fijians after their training in Hawaii (SPC, 2002; J. Vasuca, pers. comm., 2011). Initial introduction was done by the government to develop farming technology for small-scale rural farmers. Other introductions were made from Hawaii in late 1970 and early 1980 (Uwate et al., 1984). From 1982 to 1987, JICA volunteers assisted aquaculture research and development projects based at Lami in 1981 and in 1983 shifted to NRS with one of the objectives being to try and successfully spawn GFP (SPC, 2002). Larval rearing techniques used were adapted from Anuenue in Hawaii and Aquacop in Tahiti. Studies were successfully 8

Chapter 1. Introduction carried out on larval rearing, nutrition and grow-out techniques to produce market size prawns routinely. This success later resulted in the commodity being used for freshwater aquaculture in the country. Later introductions were made from Taiwan in 1993 and Tahiti in 1996 (S. Nandlal, pers. comm., 2011). Even though the seed production technique was successfully developed in the mid 1980s, farming extension did not occur before 1998 (FAO, 2003). Currently the Fijian stocks in use are the introduced stock from Tahiti which is maintained at NRS. The hatchery has a capacity to produce 1 million PLs annually and these are distributed free to Fijian farmers (semi commercial) and sold to commercial farmers (M. Dawai, pers. comm., 2010). Currently the country has two hatcheries; one government own and one privately owned by Tebara Farms Ltd (TF). M. rosenbergii are mostly cultured in earthen ponds and sometimes in combination with Tilapia and carps. Fiji currently practices monoculture of GFPs which are harvested 4-5 months after stocking with PLs. Several types of formulated diets including Tilapia pellet, powdered mash consisting of fish meal or meat meal, wheat bran or mill mix and copra meal can be used for feeding purposes. M. rosenbergii culture plays an important role in food production, diversifying economy and increasing employment opportunities in Fiji. Over the last 14 years, GFP farming has attracted considerable attention from around the country and the region. Initially, GFP farming was concentrated in the central part of the main island with 12 operational prawn farms where farmers practice a semi commercial level of farming (K. Vodo, pers. comm., 2011). Recently increasing demand of prawns locally has attracted many farmers to venture into GFP farming. Expansion of GFP farming however, depends on the availability of PLs, the supply of which is the main bottleneck. TF in Navua, specializes in GFP farming, it produced 19 t in 2007 valued at FJD0.25 million (A. Singh, pers. comm., 2010). According to the farm manager, there is potential to expand to meet local demand, however, reluctance shown by local investors is mainly due to two reasons; the low availability of PL and a lack of a locally produced feed. Production figures reflect the history of species following introduction and experimental phase indicating that the sector is gradually moving from experimental stages to production phase. Furthermore, statistics reported in FAO (2000); New and Valenti, 2000) production of farmed freshwater prawn for Fiji from 1989 to 1992 ranged from 1 – 6 metric tons, from 1993 to 1996, 85 – 93 metric 9

Chapter 1. Introduction tons and in 1997 to 1998 40 metric tons was produced. However, freshwater prawn farming has never been boosted in Fiji until 1998. According to Fiji Fisheries Division the supplier of this wrong information is unknown (J. Vasuca pers. comm., 2011). Freshwater prawn has not been produced at NRS from 1991 to 1997 until introduction of M. rosenbergii from Tahiti was made in 1998. Annual production dramatically increased from USD0.2 million in 1998 to USD2.2 million in 2007 (Ponia, 2010).

1.5 Problems Confronting GFP Industry in Fiji

Problems hindering expansion of GFP industry in Fiji are mainly due to the inconsistency of larval production, small size of prawns, low production outputs and no locally produced low cost feed for prawn. The development of prawn farming into a large scale industry for export market are plagued by such problems listed above together with the main concern raised by farmers on the quality of PLs supplied to them. It is assumed that the current culture stock may have been inbred due to the small number initially introduced. While domestication of freshwater prawn has been established since 1980s, the number of foundation populations and information on mating scheme are unknown. Over the years the same population has been used to produce PL. Despite a long aquaculture history, the genetic structure of these local stocks has not been determined. The issue of inbreeding and genetic deterioration of local stocks was raised by both prawn farmers and government officers as the probable cause of declining farm production. To support this contention, Fiji Fisheries staff reported that many females appear to be reaching maturation at smaller size than in the past, a likely consequence of inbreeding and poor management of culture stocks. There are some factors that might cause low levels of prawn production, including slow growth rate, size variation at harvest and deterioration of pond environments. Genetic deterioration in the local hatchery stocks due to inbreeding may also be responsible for low productivity of prawns. Thus, as the industry is about to expand in the Pacific region it is imperative that the quality of existing culture stocks be assessed and decisions made about whether new stocks are required to maintain industry development before stocks are translocated to new culture environments. To date very few breeding programs have 10

Chapter 1. Introduction been undertaken on M. rosenbergii to improve culture traits but this situation is changing quickly as many groups around the globe have considered developing faster growing and disease resistant stock for culture. In a study conducted by Australian Center for International Agricultural Research (ACIAR) project (FIS/2002/083) (de Bryun et al., 2004) genetic diversity in GFP wild stocks were examined across the species’ natural range. Data generated in this study provided the starting point for sourcing genetically diverse stocks in Asia to be evaluated against the Fijian domesticated stock to identify the best stock for the culture industry in Fiji. As GFP is not native to Fiji or the region (except in Papua New Guinea and Australia) new culture lines to augment genetic diversity in Fijian culture stocks, must be sourced from elsewhere and evaluated under quarantine conditions in Fiji is the best option for providing the best culture stock to farmers in the future. Therefore, the Fisheries Division has introduced non-native prawn PL for broodstock from Vietnam, Indonesia and Malaysia to replace or upgrade existing stocks due to inconsistencies in prawn production in Fiji. Other pressing issues affecting the industry include marketable size of prawns which must be addressed to ensure this new industry remains viable.

1.6 Development of High Performing GFP Culture Lines

Introduction of freshwater prawns for farming purposes to countries where it was exotic has occurred, usually from farm ponds or research centre. Thus it seems highly likely that prawn culture in countries where M. rosenbergii is not indigenous has been based on very small initial broodstock number. Therefore, this makes the industry in those countries vulnerable to genetic degradation a phenomenon which has been observed in commercial farms in Taiwan (New and Valenti, 2000). China’s huge culture industry has recognized this danger and regularly introduces new broodstock from abroad. Genetic degradation has also been observed in countries where this species is indigenous but where broodstocks are obtained only from grow-out ponds rather than from the wild. While there is room for increasing aquaculture production through better farm management, the increases in production needed to meet the demand will have to come from the use of genetically improved breeds/strains as has been the case in crops and 11

Chapter 1. Introduction livestock (Gupta and Acosta, 2001). Genetic research and the application of breeding programs have been responsible for increased production efficiency and improved productivity in crops and livestock. In recent years there has been growing interest in developing high performing culture lines for the industry via stock improvement programs and directed selection (Dobkin and Bailey, 1979; Doyle et al., 1983; Chareontawee et al., 2007 and Thanh et al., 2009). These selective breeding programs have typically been done in favorable environments where growth is high. In these environments, prawns receive high protein supplementary feeds formulated commercially. A few studies aiming to evaluate the performance of M. rosenbergii strains have been conducted in Philippines, Vietnam, Indonesia, China, Thailand and other countries (Sulit et al., 2005). Other strain improvement studies have also been conducted to improve growth rate and increase productivity on farms in Indonesia, Vietnam and other Asian countries. Strain selection however, has never been conducted in the Pacific region and this is the first time this type of study has been undertaken for GFP. The Fiji Fisheries Division recognized the need to actively manage genetic quality of farmed aquatic species to maintain their productivity since involvement in an ACIAR project on Tilapia stock improvement carried out from 1993 to 2001 (ACIAR Project Document, 2005). It was therefore decided to source highly divergent genetic stocks of GFP and carry out strain selection in the Fijian environment. This study is the first of its kind in the region because baseline information on diversity of hatchery stocks is not available. The purpose of this study is to compare the productivity of strains from other geographical areas with the current stocks available in the country as a reference.

1.7 Why Culture GFP?

The infrequent supply of seeds and costly production of shrimp by coastal communities has raised interest in freshwater prawn farming to utilize otherwise unusable coastal land. Farming of freshwater prawn is not as highly technical or capital intensive as shrimp farming therefore, is more accessible industry for small-scale operators. Interested locals are fortunate to start up prawn farming with assistance from government in expenditure and other technical areas. Furthermore, farming systems 12

Chapter 1. Introduction employed are similar to those utilized for Tilapia but with more profitable returns (Nandlal and Pickering, 2005). A publication by FAO in 2006 stated that aquaculture sectors like this provides employment and generates income for any nation. Lecouffe (2005) wrote that prawn farming presents a substantial amount of benefits for the poor fish farmers by raising income from their available water resources and by achieving a more positive impact on poverty alleviation. Furthermore, a low external input method brings a high income in a few months without hampering other household daily activities. Other benefits include attenuation in the pressure on the native stocks that are exhausted by years of harvesting.

1.8 Objectives of the Study

The overall aim of the project was to evaluate the genetic diversity of introduced M. rosenbergii lines and improve, if required, genetically these lines to secure the further development of the GFP industry in Fiji. Additionally, high yielding strains with good adaptation to environmental challenges and a high survival rate will be developed. This is the first attempt to genetically improve this prawn species. Improving the quality of produce and increasing farm yields can give Fijian prawn farmers a real market advantage.

Specific Objectives:

1. Culturing of introduced strains for second generation (G2) PL production for comparative strain evaluation trial in ponds. 2. Carry out strain evaluation experiment using a low technology semi-intensive culture system. 3. Compare performance based on highest growth and survival rates of genetically different strains of introduced freshwater prawn. 4. Identify the best performing strain for the Fijian conditions based on comparative grow-out trials.

Chapter 1. Introduction

1.9 Thesis Overview

This thesis is organized into five chapters in order to accomplish research aims. The first chapter covers an introduction to the aquaculture production in the world, then, in the Pacific region, a detailed history of the development of aquaculture and the introduction of M. rosenbergii in Fiji. The second chapter has a detailed literature review on the nomenclature, distribution, biology, larviculture and grow-out of M. rosenbergii. Chapter three describes the methodology that was followed in the study that was undertaken to determine the most suitable strain from three (Vietnam, Indonesia, Malaysia) high performing stocks introduced in Fiji to improve local culture stock quality. Chapter four presents the results obtained in the strain evaluation study through the presentation of analyzed data as tables and graphs. The final chapter, chapter five contains a general discussion, which summarizes the total findings from the research project; limitations encountered during the research, as well as conclusions and recommendations for future research.

Chapter 2. Literature Review

Chapter 2 Literature Review

2.1 Nomenclature and Morphology

Freshwater prawns belong to the family Palaemonidae of the genus Macrobrachium, Bate 1868, with over 200 species (New and Valenti, 2000; New et al., 2010).

The currently accepted taxonomy framework for Macrobrachium rosenbergii is shown below:

Kingdom: Animalia Phylum: Arthropoda Subphylum: Crustacea Class: Malacostraca Order: Decapoda Sub-order: Pleocyemata Infraorder: Caridea Superfamily: Palaemonidae Family: Palaemonidae Subfamily: Palaemoninae Genus: Macrobrachium Species: Macrobrachium rosenbergii

M. rosenbergii are sexually dimorphic with mature males being larger and having longer second pereiopods and a larger head compared to abdomen. Males have highly structured social hierarchy with three distinct morphotype being recognized, each with differing growth rates and behavior (Cohen et al., 1981; Ra’anan and Cohen, 1984; Kuris et al., 1987). Male morphotype are divided into three groups: i) blue claw (BC) comprising approximately 10 percent of the male population and are characterized as having large, blue, spiny, hairy claws and is the most sexually active ii) orange claw

Chapter 2. Literature Review

(OC) comprising around 40 percent of the male population and are characterized as having large, spineless, light-orange claws, not territorial and are sexually incompetent; and iii) small males (SM) with transparent claws which are the smallest of the three and makeup around 50 percent of the male population (Sagi, 1984; Ra’anan and Sagi, 1985). Females have a smaller head and more slender claws along with the first three abdominal pleura that form a brood chamber for incubating eggs until they hatch. Females can also be classified into three types: i) virgin females (VF) not yet spawn; ii) berried females (BF) egg carrying, and iii) spent females (SF) egg released.

M. rosenbergii PLs are completely transparent, revealing all the organs. Juveniles are also transparent, but may show blue stripes (Ling, 1969). In nature, adult are normally distinctively blue in color but occasionally are brownish with orange stripes. Adult males can grow to a length of over 30 centimeters.

2.2 Distribution of M. rosenbergii

The natural distribution of M. rosenbergii extends from Pakistan to southern Vietnam in the east, across SE Asia and south to northern Australia, Papua New Guinea and some Pacific and Indian Ocean Islands (Mather and de Bruyn, 2003). M. rosenbergii has been imported into many other tropical and subtropical areas of the world and is the most favored specie for farming purposes. Two forms of M. rosenbergii have been described independently based on external morphology (Johnson, 1973; de Man, 1879; Lindenfelser, 1984; Wowor and Ng, 2001) and allozymes (Lindenfelser, 1984) across the species natural range; an ‘eastern’ and a ‘western’ form (Mather and de Bruyn, 2003). Furthermore, both forms have been identified as genetically distinct populations (Mather and de Bruyn, 2003; Chand et al. 2005) showing sharp divisions separated by Wallace’s line (Fig. 3). Further, studies (Malecha, 1977, 1987; Hedgecock et al. 1979) carried out on wild populations of M. rosenbergii revealed three geographical ‘races’: an eastern, a western and an Australian ‘race’ (Mather and de Bruyn, 2003).

Chapter 2. Literature Review

Figure 3. Geographical boundary separation between Asia and Australia (Source: http://www.britannica.com)

2.3 Biology

2.3.1 Food Habitat

M. rosenbergii is omnivore, nocturnal and benthic feeder and are cannibalistic during molting. Their diets include aquatic animals, zooplankton, molluscs, crustaceans, algae and organic material, both of animals and vegetables. Food is mostly located by touch with their antennae rather than eye sight. Freshwater prawns are aggressive and distribute themselves evenly across the pond bottom. Unlike fish, prawns are territorial and do not swim great distances to get food therefore, it is important to distribute feed uniformly over the entire pond area. Recent research has shown little production advantages by using high quality, expensive marine shrimp diets, it appears the high protein diet do slightly increase the percentage of large high value animals by almost 5 percent (Tidwell et al., 2002). The amount of feed offered is regulated according to the weight of all prawns in pond, as well as observation on daily feed intake (New, 2002). More feed should be given during first months of stocking and reduced as prawn grows

Chapter 2. Literature Review bigger. Table (3) is a guide for feeding prawn at different sizes which was followed in the present study.

Table 3. Feeding ratio based on body weight of PL (Source: Nandlal and Pickering, 2005).

Prawn size (g) Feeding rate/d (%) Feedings/d

PL 20-50 4-6

2-3 10-15 4

4-adult 5 2-3

2.3.2 Life Cycle and Larval Development

There are four distinct phases (Fig. 4) in the life cycle of the freshwater prawn, namely eggs, larvae, PL and adults. However, time spent by each species of Macrobrachium in the different phases depends on environmental conditions mainly temperature. Sexually mature males mate at any time when females have completed their pre-mating molt. A few hours after mating, eggs are fertilized by semen attached to the exterior of the females. At this stage eggs are bright orange in color, gradually darkening to brownish orange and finally to grey about two to three days prior to hatching. Newly hatched larvae are planktonic and swim actively, upside down, tail first and require brackish water within 48 h or suffer fatality. Larvae undergo 11 distinct stages before metamorphosing into PL in about 35 days. Transformation from newly hatched larvae to PL depends upon quantity and quality of food, temperature, light and a variety of other water quality variables (D’Abramo et al., 2003). PLs abandon the planktonic form and become benthic; they move by crawling and swim rapidly with their dorsal side up, resembling miniature adults and eventually migrating up-stream.

Chapter 2. Literature Review

Figure 4. Life cycle of the giant freshwater prawn M. rosenbergii (larval and pond grow-out stages can vary depending on environmental conditions) (Source: Malecha, 1984).

2.3.3 Hygiene and Diseases

According to Yoganandhan et al. (2005), disease is one of the main factors limiting the survival, growth and production of farmed fish and shellfish. Infectious diseases of viral and bacterial origin cause major production losses in different parts of the world (Yoganandhan et al., 2005). Several diseases of bacterial, fungal and viral affect freshwater prawns. Unlike marine shrimp, diseases have yet to be identified as a major problem affecting production of freshwater prawns. On the other hand, as stocking rate and biomass per unit area increases, the potential for disease related mortality increases. Disease problems have recently become a serious concern to GFP farming, probably due to the intensification of culture and translocation of seed and broodstock. Several diseases have been identified in larvae, juveniles and adults, but many of them are of undetermined aetiology (Pillay, 1990). Good environmental management can effectively control diseases and external fouling; hence proper feeding, frequent water exchange and aeration are important considerations for health management as prawns are subjected to a variety of diseases in ponds (Johnson, 1980). The most frequent is damage to the exoskeleton due to fighting or mishandling, sometimes leading to infection.

Chapter 2. Literature Review

2.3.4 Water Quality Management

In grow-out ponds it is essential to maintain desirable water quality throughout the culture period, there is a gradual accumulation of organic matter and nutrients from feeds, fertilizers, dead organisms in the pond bed. This deposited sediment decomposes to release nutrients into water to stimulate phytoplankton production. However, excessive silt deposition impairs water quality in the pond leading to stress, slow growth, susceptibility to disease, prawn mortality and possible failure of the crop. Therefore, to maintain suitable water quality periodical monitoring is done. The favourable range of water quality in M. rosenbergii culture ponds is in the range: water temperature 28-31oC, water transparency 25-40 cm, pH 7.0-8.5, dissolved oxygen 3-7 mg/L, alkalinity 20-60 mg/L, hardness 30-150 mg/L, non ionized ammonia < 0.3 mg/L and freshwater prawn ponds should be free from hydrogen sulphide. Table (4) is a summary of ideal range good for prawn survival and growth recommended for prawn farming.

Table 4. Water parameters recommended for prawn grow-out (modified from New, 2002).

Parameters Recommended range

Temperature (oC) 28 – 31 pH 7.0 – 8.5

Dissolved oxygen (ppm) 3 - 7 Salinity (ppm) < 4

Alkalinity (ppm) 20 – 60 Transparency (cm) 25 - 40

Total hardness (ppm) 30 – 150

Ammonia NH3-N (ppm) < 0.3

Chapter 2. Literature Review

2.3.5 Growth and Molting

Growth of individuals within a population is often variable involving genetic, social and environmental factors as body size affects reproduction and survival. Growth can be expressed as the increase in length, volume or weight against time (Hartnoll, 1982). Some authors have developed equations to relate data on length and weight for M. rosenbergii raised in tropical and subtropical ponds (Menasveta and Piyatiratitivokul, 1982; Sampaio and Valenti, 1996). In crustaceans, growth is essentially discontinuous process, which involves succession of molts separated by inter-molt period (Mauchline, 1977; Hartnoll, 1982). M. rosenbergii males exhibits heterogeneous growth while females grow at uniform rate. Studies conducted by Meewap et al. (1994) indicated that parental and maternal characteristics have a great effect on the growth of M. rosenbergii. Doyle et al. (1983) demonstrated that selection of early maturing female prawns for hatchery use can improve the genetic base for enhanced growth. Variability in temperature related growth has been observed in populations from different geographic locations (Sarver et al., 1979) and intra specific hybrids of M. rosenbergii races have demonstrated improved growth rates (Dobkin and Bailay, 1979).

2.4 Larviculture of M. rosenbergii

The aquaculture of freshwater prawns can only be developed if reliable supplies of PL’s for stocking of ponds are available. Seed production is often a serious limiting factor in aquaculture operations either because of inadequate numbers or prohibitive cost. Fujimura and Okamoto (1972) developed the first mass culturing methods for M. rosenbergii PL and the techniques are still being modified. A later method the ‘clear- water method’ removed the requirement for phytoplankton and this method is often favored because it gives more efficient survival at higher densities. There are two main hatchery systems: flow-through and recirculation and in the flow-through two types of system exist: clear-water and green water.

Chapter 2. Literature Review

2.4.1 Green Water Culture of Larval M. rosenbergii

The method of Fujimura and Okamoto (1972) is known as ‘green water culture’ because of the use of phytoplankton to improve the water quality and nutrition of larvae. Water changes are minimal in this system compared with the clear-water system. Hatchery operators rely on the density and color of the phytoplankton bloom in the culture tank as an indication of water quality. The green water system works most effectively under low stocking density (10-20 PL/L) and generally gives unpredictable survival. Phytoplankton in the green water system is not consumed directly by the larvae, although it is consumed by zooplankton (e.g. rotifers and Artemia) in larval rearing tanks (LRTs) which are then consumed by the larvae. The main drawback of the green water system is the threat of a phytoplankton crash. If the phytoplankton bloom is very dense its oxygen demand can be very high at night and this can stress the larvae if there is inadequate aeration. In addition, high phytoplankton productivity during daylight hours can give pH values as high as 9.9-10.5. Mass larval mortalities can occur if the pH exceeds 9.5 (New, 2002). Another problem that arises in green water method is that it uses relatively large one tropic level of larvae feeding on their prey. Therefore, this causes a theoretical simplification of the process as it removes the need for the phytoplankton. However, this system is more labor intensive due to the need of cleanliness and the careful control of feeding. The lack of phytoplankton in this system affects the nutrition of the larvae because Artemia that are unfed utilize their yolk reserves. It is considered that the algae provide essential nutrients not present in inert larval feeds or starved Artemia. An advantage of the green water system is that the environmental stresses encountered by the larvae during rearing are less susceptible when they are stocked into grow-out ponds. Ponds often have problems with low aeration and poor water quality. If the larvae are not acclimated to these conditions before introduction to the ponds, survival after stocking can be poor.

Chapter 2. Literature Review

2.4.2 Clear-water Larval Culture

Due to the inherent variability of the green water method, efforts were made to establish a method by which PL’s could be produced without the use of phytoplankton and at substantially higher larval stocking densities. Therefore, it was considered desirable to intensify the method of production. As a result of research at the Centre Oceanologique du Pacifique (Aquacop), the ‘clear-water’ method of larval rearing was established. This method resulted in high mortality rates due to poor water quality caused by the buildup of nitrogenous compounds and the high organic loading present in feed. The problem of deterioration of water quality in static tanks under high stocking density cannot be resolved unless sufficient water is replaced in the tanks on a daily basis. This is because of high feed concentration and the large number of larvae causes fouling of the water within 24 hrs. The high rate of water exchange is unacceptable for a hatchery that is not sited near sea and because of this recirculation systems have been devised. This system allows the reuse of the brackish water by cleaning it after it has left the larval culture tank. Cleaning is achieved by a combination of mechanical filtration and biological decomposition of toxic compounds (e.g. nitrite, ammonia) (D’Abramo et al., 2003). In Fiji a flow through system with clear-water management provides superior results in comparison with other systems. Therefore, a simplified version of this system was developed at NRS and USP’s Marine Studies Laboratory, techniques developed for larval rearing from a combination of both Anuenue (Hawaii) and Aquacop (Tahiti) were used for this research. According to Vereivalu (1989) the clear water technique has been adopted at NRS hatchery with production averaging 60 PL/L. In 1986, 635,000 PL were produced at a cost calculated about FJD7.00 per 1000 PL which is lower than that obtained from any other Macrobrachium spp. hatchery (Vereivalu, 1989).

2.5 Grow-out, Harvesting and Marketing

Freshwater prawns are obtained from rivers or nurseries, for stocking into open waters and other culture systems. Freshwater prawn farming can be done as a single species or

Chapter 2. Literature Review monoculture and with other aquatic species as poly-culture. According to New (2002), freshwater prawn monoculture can be extensive, semi-intensive or intensive.

Extensive culture is rearing in earthen ponds and other enclosures such as reservoirs, irrigation ponds and rice fields which produces less than 500 kg/ha/yr of freshwater prawn. They are stocked with PL or juveniles at 1-4 m2. There is no control of water quality; the growth or mortality is not monitored; supplemental feeding and organic fertilization is rarely applied. This system is not good for commercial prawn culture.

Semi intensive system involves stocking PL or juvenile prawns at 4-20 per m2 in ponds and result in a range of productivity of more than 500 kg/ha/yr and less than 5,000 kg/ha/yr. Fertilization is used and a balanced feed ration is supplied. Predators and competitors are controlled, water quality and prawn health are monitored. The author advised against using intensive system in the tropics because it requires more research, particularly on size management.

Intensive culture refers to freshwater prawn farming in small earthen or concrete ponds provided with high water exchange and continuous aeration, stocked with more than 20 PL per m2 and achieving an output of more than 5,000 kg/ha/yr. Production cost are high and a high degree of management is required, which includes the use of a nutritionally complete feed, the elimination of predators and competitors and strict control over all aspects of water quality.

Semi intensive freshwater prawn grow out in ponds can be managed by a ‘continuous’ or ‘batch’ system, or a combination of the two, ‘combined system’. A variant of the combined system is known as the ‘modified batch system’ (New, 2002). In Fiji, PL are obtained from hatcheries and raised in earthen ponds at semi intensive level mostly in batch management system.

Chapter 2. Literature Review

According to New (2002) semi-intensive stocking rates vary between 4-20 PL/m2 for tropical areas. Vereivalu (1989) reported initial stocking density of 15-20 PL/m2 was utilized for pond trials in Fiji during 1985. Results showed 70-80 percent of prawns were under market size after 6-8 months rearing. In 1987, prawn/tilapia polyculture trials began with stocking density at 1-5 PL/m2 fed with tilapia feed where prawns reached 30- 50 g in over 6 months of culture. Current practice in Fiji is to stock PL directly into ponds with no nursery phase at stocking densities of around 5-10 PL/m2 (A. Singh pers. comm., 2010). This could be one of the reasons why farm productions are much lower than expected. According to Vereivalu (1989) production rates vary among farms and results have not been consistent, largely as a result of differences in input rates. Harvesting procedures are described by New and Singholka (1985); New and Valenti (2000); New (2002); and D’Abramo et al. (2003). Usually, pond reared prawns reach 25-50 g within 4-6 months, depending on the stocking rate, feed quality and quantity, water temperature and D.O. level. The exact time chosen for harvesting is determined by factors like the preferred market size for prawns. In Fiji two types of prawn harvesting are practised: (1) partial harvesting and (2) complete harvesting (K. Vodo, pers. comm. 2011). Prices for both wild and farmed freshwater prawns remain high in Fiji as they are regarded as a delicacy. Farmed as well as wild caught prawns are sold with heads on, mostly frozen and fetch prices of around FJD12 per kilo for small size and FJD18-22 per kg for large size (Pickering and Forbes, 2002). Recently, prices for freshwater prawns have risen to FJD15-20 per kg farm-gate price and FJD25-30 per kg for retail (Pickering and Forbes, 2002). In Fiji there is high demand for larger prawns of 20-30 g or around 30-40 prawns/kg which fetches almost twice as much as smaller prawns. However, there is still good demand for smaller prawns of 10-15 g around 60-100 prawns/kg (Nandlal and Pickering, 2005).

Chapter 3. Comparative Grow-out Trials

CHAPTER 3 Comparative grow-out trials of four GFP strains in earthen ponds.

3.1 Introduction

PLs used in this experiment were obtained from broodstock from three culture stocks introduced under quarantine condition from Indonesia (Research Institute for Freshwater Aquaculture formerly known as RIFFI), Malaysia (Department of Fisheries) and Vietnam (Research Institute for Aquaculture No. 2 RIA2, in Ho Chi Minh City). In April 2008, GFP culture strains from SE Asia were identified by the Australian Center for International Agricultural Research (ACIAR) project team. In October 2008, 1000 PLs were air-freighted from Vietnam, Malaysia and Indonesia to Fiji via Brisbane, Australia. PLs were transported in holding tanks supplied with aeration from Nadi International Airport to Fiji Fisheries Division Naduruloulou Research Station (NRS). Percentage survival upon arrival at NRS was only 21.5 percent for the Indonesian strain, and 45 percent for both the Malaysian and Vietnam strains. Larvae were acclimated in a quarantine facility at NRS (3 imported and 1 Fijian strain) in separate tile-lined cement tanks filled with filtered freshwater, moderate aeration and controlled temperature between 28-30 oC. Larvae from each strain were kept in quarantine for 21 days and fed a locally produced commercial tilapia feed (crude protein 29 percent) with a daily 50 percent water exchange. PLs were quarantined, while four 300 m2 earthen ponds (3 imported and 1 Fijian strain) were prepared for the PLs grow-out to adult stage. All ponds were drained and left to dry before applying agricultural lime [calcium oxide at a rate of 1 kg for every 10 m2]. Liming was done to correct the pH of the soil and to kill undesirable organisms. Inlets and outlets of each pond were covered with fine mosquito mesh to stop entry of predators etc and aquatic animal eggs. After 3 days, the four ponds were filled and depth was maintained at 1.2 m at the outlet side and 0.8 m at the inlet side. After completion of quarantine, a total of 130 juveniles from the Indonesian strain, 250 for Malaysian strain and 300 for Vietnamese strain were stocked in the grow out ponds, in December 2008, to be reared to maturity for PL production. Back-up juveniles from each strain were maintained in the quarantine area in separate tile-lined cement 26

Chapter 3. Comparative Grow-out Trials tanks which were later stocked into separate grow-out ponds. Juveniles were fed with pellet at a daily rate of 50 percent of average body weight (BW) per day for the first month. Feeding rate was gradually reduced to 5 percent of total BW by the end of the fourth month and this level maintained to maturity period. Feed was distributed over the pond twice daily [1/3 am and 2/3 pm]. After five months of culturing, berried females ready to release eggs (grayish color) from the four lines were netted using seine nets and transferred to the hatchery where they were disinfected and acclimated to brackish water in 500 L conical tanks at 3-4 females per tank per strain. The purpose of going through a hatchery phase was to capture a true representation of the genetic diversity present in each culture line from the first generation (G1) individuals grown through adulthood to be used as breeders for the generation of PLs (G2) for strain evaluation trials. Only G2 PLs were used in the strain evaluation trials, to address potential problems of maternal effects from natal environments. The first hatchery cycle was successfully carried out between May and July 2009 and PL’s (see appendix 1, Table 15) stocked in August 2009 at NRS. After two months of rearing larvae in comparative growth trials, first sampling was carried out in October 2009. At this time the experiment had to be stopped since all trial ponds were heavily infested with water weeds which caused high mortalities of up to 99 percent in nearly all ponds. It was decided to repeat the hatchery phase, followed by comparative growth trials with major improvements in the pond management system. The second hatchery cycle was carried out from November to December 2009 and PLs were stocked in January 2010.

3.2 Materials and Methods

3.2.1 Study Site

The tropical islands of Fiji have a temperature range of 18 to 33 oC, with wet and dry zones on the two main islands. Wet season is from November to April and dry season from March to October. Rainfall of 1500 to 6000 mm is normally experienced around the group. The study site NRS is located 7 km north of Nausori town, along Rewa River.

Chapter 3. Comparative Grow-out Trials

Figure 5. M. rosenbergii strain evaluation grow-out ponds (highlighted area) at NRS (Source: http://www.googleearth.com)

3.2.2 Larval Production

In November 2009, a representative sample of females from each of the four strains carrying eggs (grey or orange) were collected randomly using seine nets and carried to the hatchery in buckets. Females were disinfected before putting into the LRTs and acclimated from 0 to 5 ppt in the LRT 12 x 500 L conical rearing tanks for each strain (2 replicates per line) were set-up inside the freshwater prawn hatchery at NRS. From 22 to 29 November 2009, females that had released all eggs were removed from the LRTs and the salinity was increased gradually over the next 1-2 d to 12 ppt. Females were measured for weight and total length after release of eggs before stocking back into their respective ponds (Table 5). The freshwater prawn hatchery located at Naduruloulou (Fig. 5) is inland which requires seawater to be transported from the coast to the hatchery as brackish water is used in hatchery work for freshwater prawn. Larval rearing water was prepared by mixing freshwater and seawater (from Suva Point) in the mixing tank to achieve a salinity of 12 ppt. The mixture was approximately 1 part seawater to 2 parts 28

Chapter 3. Comparative Grow-out Trials freshwater. Water used in the mixing tank was filtered at 5 microns using a biological filter bag as the temperature in mixing tank was maintained within 28-30 oC, using an immersion heater and recirculated in a mechanical filter tank for mixing. Water quality parameters (see appendix 1, Table 13) were maintained within the optimum ranges recommended for a freshwater prawn hatchery by New (2002) and Nandlal and Pickering (2005). Monitoring of temperature and salinity were carried out twice daily at 0900 and 1600 hrs. In the hatchery, larvae were fed with brine shrimp (Artemia), and prepared feed ox-liver and egg custard as they developed. Seven days after hatching larvae were fed with prepared feed and Artemia was given as an afternoon feed. Feed quantity was increased as larvae advanced into later larval stages (see appendix 3, Fig. 31).

Feed preparation was carried out as follows:

Ox-liver – a small amount of fresh liver was cut into cubes and blended. The ground product was transferred to stainless steel sieves of different grading sizes (e.g. 0.84, 0.42 and 0.25 mm) based on larval stage and tap water was used to thoroughly wash the ground ox-liver passing through the sieves.

Squid egg custard – 2 to 3 squids were cut into cubes and blended with 6 eggs and 2 cod liver oil capsules. The blended mixture was poured into a stainless steel bowl and steamed in a rice cooker until a thick custard was formed. The mixture was removed from the steamer and sieved under tap water and fed to larvae. Left over feed was kept in the refrigerator for not more than 2 days (d) after preparation.

Newly hatched larvae were counted 3 times using 1000 ml beakers, and then averaged to calculate larval density in each tank. Healthy larvae swam at the surface of the water, fed actively, had a reddish brown pigmentation and were observed not to be cannibalistic. By d 42 most larvae had metamorphosed into PL. When over 50 percent of larvae had metamorphosed into PL’s, the salinity in the LRTs was gradually decreased to that of freshwater (0 ppt). PL’s were collected using a plankton net then counted individually 29

Chapter 3. Comparative Grow-out Trials and acclimated to freshwater for 3 to 5 d before they were stocked them into the experimental ponds. PL’s used in this study were locally produced in a single hatching cycle from November to December 2009. Hatchery production data are presented in Appendix (Table 15). PL’s were acclimatized to freshwater for 3 to 4 d prior to stocking on 7 January 2010. PL’s (Table 6) were selected randomly and stocked into 12 earthen experimental ponds maintaining a proportional stocking density of 5 ind./m2. Mean stocking weight was determined from a sample of 300 PLs (100 PLs per random sub- sample x 3 repetitions then average was calculated) per strain. PLs were counted individually into separate containers for each strain at a time, blotted free of surface water and bulk weighed (see appendix 1, Table 16).

Table 5: Initial mean weight and length of berried M. rosenbergii female’s representative of the four strains (Vietnam, Indonesia, Malaysia and Fiji) stocked in the ponds on 7 January 2011.

Strain No. of females Weight (g) Length (mm) No. of PL Vietnam 7 67.25 ± 12.37 180.06 ± 0.98 5,093 Indonesia 3 95.08 ± 14.36 204.04 ± 1.06 10,813 Malaysia 2 94.19 ± 7.78 203.21 ± 1.07 3,333 Fiji 7 31.37 ± 11.72 153.26 ± 1.69 3,533

During the second hatchery cycle, a tropical cyclone developed for two days which affected larval production in the hatchery. Survival of larvae was affected for 11 d at NRS was faced with power cuts. Freshwater and seawater quality was very poor (i.e. silt, clay and detritus) due to heavy flooding which affected water exchange in tanks. Over the two hatchery cycles that were conducted for strain evaluation comparison, it was observed that the strains differed in performance even at the hatchery level. The Indonesian strain presented a better survival rate followed by the Vietnamese and Fijian and finally the Malaysian strain which also showed the longest time for larval production in both cycles. The current study was undertaken to differentiate strains at the grow-out level but not at the hatchery level. Therefore, further testing of the strains at the hatchery level will be required to confirm these impressions.

Chapter 3. Comparative Grow-out Trials

3.2.3 Grow-out

Stocking Density – PLs were stocked at the rate of 5/m2 in relation to pond surface area (Table 6) in order to maintain the same stocking densities in each of 12 earthen ponds (see appendix 3, Figs. 18-20).

Table 6: Initial M. rosenbergii PLs weights, stocking density, pond area and strains from four countries stocked in triplicates in 12 earthen ponds.

Stocking Pond area Average weight Strain Pond density (m2) of PL (g) (5PL/m2) Vietnam (R1) 1 113 565 0.02 Vietnam (R2) 8 138 690 0.02 Vietnam(R3) 11 140 700 0.02 Indonesia (R1) 2 116 580 0.02 Indonesia (R2) 7 126 630 0.02 Indonesia (R3) 10 149 745 0.02 Malaysia (R1) 3 105 525 0.01 Malaysia (R2) 6 100 500 0.01 Malaysia (R3) 9 138 690 0.01 Fiji (R1) 4 105 525 0.02 Fiji (R2) 5 121 605 0.02 Fiji (R3) 12 157 785 0.02

Feeding – A formulated feed used by Fiji Fisheries division was used during the experimental period. This commercially produced local tilapia pellet (Crest Feed Mills, Nausori, Fiji) consisted of agricultural by-products and fish meal with a crude protein content of 29 percent (Table 7). The feeding rate (see appendix 1, Table 14) was based on estimated prawn body weight determined by levels employed for freshwater prawn culture in semi intensive ponds recommended by New (2002). Feed was cast over the pond surface with 30 percent of the ration provided in the morning (9 am) and 70 percent in the evening (6 pm).

Chapter 3. Comparative Grow-out Trials

Culture period – Prawns were cultured for 5 months since in Fiji, the preferred market size for prawns is 30 g (heads on) which can be achieved in 4.5 - 5.0 months.

Table 7: Formulated feed composition of pellet diet fed to freshwater prawn M. rosenbergii over the five months (Source: Crest Feed Mill – estimated composition).

Component / Ingredients Percentage Composition (%) Fish meal 30 Mill mix 10 Wheat flour 20 Vitamin and mineral premix 1-2 Molasses 5 Soya bean mill 10

Pond preparation All ponds were filled, drained and left to dry before applying ordinary agricultural lime (calcium oxide at a rate of 1 kg for every 10 m2). Two days after liming, ponds were filled with freshwater up to a depth of 1.5 m at the outlet. Two coconut fronds were added to each pond for shelter purpose and two grass carp were added to control water weed growth inside ponds.

Water quality parameters Water parameters were recorded twice per day at 0900 and 1600 hrs. Water temperature was measured using hand held glass thermometer (0 – 50 oC), D.O. and pH was measured using D.O. meter (SM 600, Milwauke®, Taiwan) and pH meter (SM 101 – Milwauke®, Taiwan).

Chapter 3. Comparative Grow-out Trials

3.2.4 Sampling Design

Sampling of ponds The first sampling was carried out in March at d 63, the second sampling in April at d 105 and final sampling in June at d 143 of culture. In the first and second samplings, ponds were sampled with single seine netting to obtain a sample of 30 individuals per pond to estimate average weight gains (see appendix 3, Figs. 21- 22). Sampled prawns were weighed individually with a digital electronic scale (Sartorius®, ± 0.001 g), and length measured with an digital electronic Vernier caliper (Lufkin®, ± 0.01 mm) (see appendix 3, Figs. 23-24) counted and returned to each respective experimental pond. Feed was adjusted accordingly after each sampling period based on calculated mean weight gain. After 143 days of culture, ponds were drained and prawns harvested by hand and dip nets (see appendix 3, Fig. 25). All harvested prawns were weighed individually, measured and counted to determine survival and average weight, and length gains. Prawns from individual ponds were placed into separate aerated 1000 L fiberglass tanks supplied with freshwater. All prawns from a single strain were scored independently to avoid mixture of replicates. Prawns were classified into either female: berried (egg carrying: BE), spent or shed (open brood chamber: S) and virgin (V) or one of three male morphotypes: blue claw (BC), orange claw (OC), small males (SM) or runts (R) (see appendix 3, Figs. 26-29). Male morphotypes were classed based on color of claws and females were classed based on egg absence or presence.

3.2.5 Measurement of Prawn Growth

Carapace length was measured from the inside of the eye orbit to the posterior margin of carapace (see appendix 3, Fig. 23). Abdomen length measured from the anterior margin of the first segment to the posterior end of the telson (see appendix 3, Fig. 24). Thus, total body length was the added value of both carapace and abdomen lengths, as total

Chapter 3. Comparative Grow-out Trials length include the very fragile rostrum or movable segments in the telson, which increase measurement error. This technique was used to minimize measurement error.

Figure 6. Biometric measurement of M. rosenbergii

3.2.6 Evaluation of Growth Parameters

Prawn culture performance was evaluated over 143 d of culture by determining the following parameters:

Daily Weight Gain (DWG): determines the grams gained per day DWG = Wf (g) – Wi (g) where Wf = mean weight at end t Wi = mean weight at start t = time (d)

Specific Growth Rate (SGR): evaluates percent body wt. gained per day SGR= 100 x (In Wf – Wi) t

Feed Conversion Rate (FCR): determines the amount of feed taken to gain body weight FCR= Dry weight of feed given (g) Live weight gained by prawn

Chapter 3. Comparative Grow-out Trials

At the end of the experiment the following parameters were determined after sampling all the prawn. Survival (%) Sex ratio (male: female) Classification into sexual morphotypes Carapace length (mm) not including rostrum length. Abdomen length (mm) Total length (mm) Weight (g)

3.2.7 Experimental Design and Statistical Analysis

The experiment employed Randomized Block Design (4 x 3 matrix), with 4 treatments and 3 replicates per strains.

Statistical analysis employed ANOVA using Genstat® (VSN International Software, South Africa) for bioscience. To evaluate changes in weight gain over time repeated measures ANOVA was employed. In repeated measures ANOVA, it is the difference or changes in response variable (weight) over time that we are investigating rather than differences in means between groups or treatments. Two-way ANOVA was used to demonstrate the effects of abdomen length, carapace length, total body length and carapace ratio among the four strains and morphotype stages within strains. Significant differences were indicated by ANOVA (P = 0.05); means were separated using the least significance difference test (Zar, 1999). Growth performance and feed conversion ratio were measured in terms of final individual weight (g) and percent survival. Percentage data of weight were converted to arc sin values prior to analysis (Zar, 1999). Results for two way ANOVA and repeated measure ANOVA tests are in Appendix 2.

Chapter 4. Results

Chapter 4.0 Results

4.1 Water Quality

Water quality parameters (Table 8) were within optimum ranges recommended for freshwater prawn grow-out.

Table 8. Water quality parameters recorded in the 12 experimental M. rosenbergii giant freshwater prawn culture ponds at NRS over five months (mean ± se). Data from different treatments for the whole period of experiment were pooled.

Vietnam Indonesia Malaysia Fiji

R1 R2 R3 R1 R2 R3 R1 R2 R3 R1 R2 R3 Temp. (oC) 28.4 28.3 28.5 28.4 28.2 28.6 28.1 27.9 28.5 28.4 28.1 28.6 (min/max) 30.8 30.9 30.8 30.9 30.9 30.9 30.9 30.9 31.0 30.7 30.7 30.9 Mean Temp. 29.6 ± 1.7 29.6 ± 1.7 29.5 ± 1.9 29.5 ± 1.7

D.O. (mg/L) 6.5 5.6 5.8 6.6 5.8 5.8 6.5 5.1 5.9 7.0 5.8 6.4 (min/max) 10.1 9.3 8.8 9.8 9.2 8.8 9.4 9.4 8.8 10.3 9.3 9.7

Mean D.O. 7.7 ± 2.4 8.0 ± 2.3 8.0 ± 2.4 8.1 ± 2.4

pH 7.1 7.0 7.1 7.1 7.1 7.1 7.1 7.0 7.1 7.1 7.0 7.1 (min/max) 7.4 7.4 7.3 7.4 7.3 7.3 7.4 7.4 7.4 7.5 7.4 7.4 Mean pH 7.2 ± 0.2 7.2 ± 0.2 7.2 ± 0.2 7.3 ± 0.2

Mean water temperatures ranged from 27.9 to 31.0 oC in the 12 ponds over the five months of culture (Fig. 7). The highest recorded value was 29.8 oC in pond 9 (Malaysian strain), and the lowest recorded value was 29.4 oC in ponds 5 (Fijian strain) and 6 (Malaysian strain).

Chapter 4. Results

32 31 30 C) ° 29 28 27 Temperature ( Temperature 26 25 1 2 3 4 5 6 7 8 9 10 11 12 Pond

Figure 7. Temperature values measured in 12 experimental earthen ponds cultured over five months for four strains of the giant freshwater prawns, Macrobrachium rosenbergii (mean ± se).

Dissolved oxygen concentration fluctuated between 5.1 and 10.3 mg/L in the 12 ponds over the five months of culture. The overall mean D.O. for the experimental period was 8.0 mg/L. The highest value was 8.6 mg/L slightly higher in pond 4 (Fijian strain) and the lowest recorded value was 7.3 mg/L in pond 6 (Malaysian strain).

Dissolved oxygen (mg/L) Dissolved 2 0 1 2 3 4 5 6 7 8 9 10 11 12 Pond

Figure 8. Dissolved oxygen values measured in 12 experimental earthen ponds over five months for four strains of the giant freshwater prawns, Macrobrachium rosenbergii (mean ± se).

Chapter 4. Results pH values fluctuated between 7.0 and 7.5 in the 12 ponds over the five months of culture. The highest recorded value was 7.5 in ponds 4 and 12 (both Fijian strains) and the lowest recorded value was 7.1 in the rest of the ponds.

7.6 7.5 7.4 7.3 7.2 7.1 pH 7.0 6.9 6.8 6.7 6.6 1 2 3 4 5 6 7 8 9 10 11 12 Pond

Figure 9. pH values measured in the 12 experimental earthen ponds during five months for four strains of the giant freshwater prawns, Macrobrachium rosenbergii (mean ± se).

4.2 Prawn Growth

The body traits for each strain (Vietnam, Indonesia, Malaysia and Fiji) are presented in Table 9. Vietnam strain showed the highest values for body weight gains, longest carapace length, abdomen length and total length over five months of culture. Indonesian strain showed the shortest abdomen and total length whereas Malaysian strain showed the lowest weight gain and shortest carapace length. Fijian strain showed intermediate performance for body traits in comparison with other strains.

Table 9. Least square means of body traits for each strain (Vietnam, Indonesia, Malaysia and Fiji) of the giant freshwater prawn M. rosenbergii cultured over five months in 12 earthen ponds (mean ± s.e).

Strain Weight gain (g) Carapace (mm) Abdomen (mm) T. Length (mm) Vietnam 26.86 ± 2.59 33.40 ± 1.40 64.51 ± 1.68 97.91 ± 2.72 Indonesia 24.66 ± 1.68 31.62 ± 0.06 61.72 ± 1.77 93.34 ± 1.82 Malaysia 23.38 ± 1.18 31.42 ± 0.91 62.01 ± 0.77 93.43 ± 1.64 Fiji 25.89 ± 0.57 31.52 ± 0.17 63.15 ± 0.35 94.68 ± 0.22

Chapter 4. Results

A significant difference was observed in final body weight (Fig. 10) between the different sampling periods (F (3, 24) = 938.57, P = 0.001) but not among the different prawn strains (F (3, 24) = 4.48, P = 0.040). Although, graphically (Fig. 10), the Vietnam strain seemed to out-perform the other three strains; the Fijian strain shows a slightly better growth rate than the Indonesian and Malaysian strains with Malaysian strain showing the slowest growth over the five months culture period although not statistically significant (P = 0.050).

30 Fiji Indonesia Malaysia Vietnam

10 Mean weight (g)

0 0 2 3 5 Time (months)

Figure 10. Mean weight gain in four strains (Vietnam, Indonesia, Malaysia and Fiji) of the giant freshwater prawn Macrobrachium rosenbergii, cultured over five months in 12 earthen ponds (mean ± s.e).

No significant difference were detected among strains for final body weight gains (Fig. 11) (F (3, 48) = 2.36, P = 0.083). Thus, a similar mean weight gain was reached by all four strains over the five months culture period. Vietnam strain showed the highest final body weight gain while Malaysian strain showed the lowest final body weight gain over the culture period.

Chapter 4. Results

Body weight (g) 10

0 Vietnam Indonesia Malaysia Fiji

Figure 11. Body weight of four strains of the freshwater prawns, Macrobrachium rosenbergii, cultured over five months in 12 earthen ponds (mean ± se).

A significant difference was observed in weights (Fig. 12) among different morphotypes stages (F (5, 48) = 431.91, P = 0.001) but not for different prawn strains (F (3, 48) = 2.36, P = 0.083). Vietnam strain showed the highest mean body weight gains for blue claw males, intermediate for orange claw males and highest mean body weight gains for females. Indonesian strain showed the lowest mean body weight gains for blue claw males, highest for orange claw males and the lowest mean body weight gains for females. Malaysian and Fijian strains showed intermediate performance for the different morphotypes stages.

Fiji Indonesia Malaysia Vietnam

Mean body weight (g) 10

0 Blue claw Orange claw Berried females Open females Small males Runts males males

Chapter 4. Results

Figure 12. Body weight of four strains of the giant freshwater prawns, Macrobrachium rosenbergii, classified into either female or male morphotypes cultured over five months in 12 earthen ponds (mean ± se).

Survival rates ranged from 69 to 84 percent among the four strains (Vietnam, Indonesia, Malaysia and Fiji). The highest survival rate was 84 percent for Vietnam strain and lowest was 69 percent for Fijian strain.

100

40 Survival (%)

0 Vietnam Indonesia Malaysia Fiji

Figure 13. Survival of four strains of the giant freshwater prawns Macrobrachium rosenbergii, cultured over five months in 12 earthen ponds (mean ± se).

The final sampling of the four strains (Vietnam, Indonesia, Malaysia and Fiji) allowed for the evaluation of growth parameters over the five months culture period presented in Table 10. Table 10. Summary of stocking density, final body weight, specific growth rate (SGR), survival rate, and feed conversion rates (FCR), sex ratio and percent marketable (>20 g) of the giant freshwater prawn M. rosenbergii cultured over five months in 12 earthen ponds. Sex Market Stocking Final SGR Survival Strain FCR Ratio prawns density wt (g) (%/d) (%) M:F (>20g) (%)

Vietnam 652 26.86 ± 2.59 4.89 84 ± 6.5 2.75 ± 0.41 1:1.0 70 ± 27

Indonesia 652 24.66 ± 1.68 4.84 79 ± 2.3 2.44 ± 0.42 1:1.7 62 ± 10

Chapter 4. Results

Malaysia 572 23.38 ± 1.18 5.28 75 ± 5.3 1.86 ± 0.67 1:1.5 56 ± 34

Fiji 638 25.89 ± 0.57 4.87 69 ± 13.6 2.42 ± 0.64 1:1.3 59 ± 40

Analysis carried out using two way ANOVA and repeated measure ANOVA for body traits for the four strains (Vietnam, Indonesia, Malaysia and Fiji) are presented in Table 11. No significant difference was observed for final body weight gains between strains when analyzed using two way ANOVA. However, a significant difference was observed in final body weight gains between the different sampling periods but not among strains when analysis was carried out using repeated measure ANOVA. Abdomen, carapace and total lengths showed significant differences for both strains and stages when analyzed using two way ANOVA.

Table 11. Summary of outcomes of ANOVA tests carried out for body traits on the giant freshwater prawn M. rosenbergii strains (Vietnam, Indonesia, Malaysia and Fiji). Variable Test used P-value Significant? Weight x Strain 2 way ANOVA 0.083 No Weight x Stage 2 way ANOVA 0.001 Yes Abdomen x Strain 2 way ANOVA 0.004 Yes Abdomen x Stage 2 way ANOVA 0.001 Yes Carapace x Strain 2 way ANOVA 0.001 Yes Carapace x Stage 2 way ANOVA 0.001 Yes Total length x Strain 2 way ANOVA 0.038 Yes Total length x Stage 2 way ANOVA 0.001 Yes Carapace ratio x Strain 2 way ANOVA 0.001 Yes Carapace ratio x Stage 2 way ANOVA 0.001 Yes Weight x Time Repeated measure ANOVA 0.001 Yes Weight x Strain Repeated measure ANOVA 0.040 No

Strains (Vietnam, Indonesia, Malaysia and Fiji) were classified into male and female and the percentage morphotypes are presented in Figure (14). The highest percentage of females was 50 percent for Indonesian strain and the lowest percentage of females was 37 percent for Vietnam strain. The highest percentage of blue and orange claw males was 38 percent for Vietnam strain and the lowest was 26 percent for Malaysian strain. 42

Chapter 4. Results

The highest percentage of small males was 36 percent for Malaysian strain and lowest was 21 percent for Indonesian strain.

Vietnam BC 7% Indonesia BC 11% BF 37% OC 31% BF 50% OC 18%

SM 21% SM 25%

Malaysia BC 8% Fiji BC 11%

OC 18% BF 38% BF 42% OC 22%

SM 36% SM 25%

Figure 14. Percentage morphotype proportions of the giant freshwater prawn M. rosenbergii strains cultured over five months in 12 earthen ponds. Vietnam, Indonesia, Malaysia, and Fiji, (BC = blue claw, OC = orange claw, BF = berried + spent female, SM = small males).

Mean weight of berried females and spent Percentage Berried female at 63 days females (± 2se) at 143 days 40 20 BF SF 15 10 20 5 Mean weight(g) % Berried female female Berried % 0 0 Vietnam Indonesia Malaysia Fiji Vietnam Indonesia Malaysia Fiji

Figure 15. Percentage berried female and mean weight of berried and spent females of the giant freshwater prawn M. rosenbergii strains after 63 and 143 days of culture. Indonesian strain recorded the

Chapter 4. Results highest number of females after 63 days of culture. Vietnam strain recorded the highest mean weight gain in females after 143 days of culture.

Table 12. Growth performance of male and female for strains of the giant freshwater prawn M. rosenbergii cultured over five months. Strain Sex Weight (g) Carapace Abdomen T. Length Carapace/T. (mm) (mm) (mm) length Vietnam M 42.14 ± 6.44 40.12 ± 1.78 72.62 ± 2.15 112.74 ± 3.89 0.36 ± 0.01 F 24.81 ± 2.13 32.67 ± 0.61 65.56 ± 1.11 98.24 ± 1.71 0.33 ± 0.00 Indonesia M 40.30 ± 2.61 39.12 ± 0.98 69.78 ± 3.12 108.89 ± 4.04 0.36 ± 0.01 F 21.34 ± 1.77 30.60 ± 0.22 62.27 ± 1.40 92.87 ± 1.42 0.33 ± 0.01 Malaysia M 41.18 ± 5.65 40.13 ± 1.96 72.32 ± 1.39 112.44 ± 3.02 0.36 ± 0.01 F 22.95 ± 2.14 31.38 ± 0.81 64.03 ± 1.34 95.42 ± 2.03 0.33 ± 0.00 Fiji M 41.81 ± 4.48 39.30 ± 1.44 72.96 ± 2.15 112.26 ± 3.47 0.35 ± 0.01 F 23.72 ± 3.46 30.76 ± 1.30 64.09 ± 2.68 94.86 ± 3.94 0.33 ± 0.01

M F a M F b 60 50 50 40 40 30 30 20 20 Weight (g) (g) Weight 10 10 (mm)Carapace 0 0 Vietnam Indonesia Malaysia Fiji Vietnam Indonesia Malaysia Fiji

M F c M F d 80 150

60 100 40 50 20 Abdomen (mm) Total length length Total (mm) 0 0 Vietnam Indonesia Malaysia Fiji Vietnam Indonesia Malaysia Fiji

Figure 16. Least square means of body traits for male (M) and female (F) of the giant freshwater prawn M. rosenbergii strains (means ± s.e). a) body weight demonstrates large variation between the sexes as males are twice as heavy as females of the same age, b). carapace length demonstrates large variation between the sexes, c). abdomen length also demonstrates large variation between sexes, d). total length demonstrates large variation between sexes of the same age. 44

Chapter 5. Discussion

Chapter 5.0 Discussion

5.1 Water Parameters

In the current strain evaluation experiment, temperatures recorded during the five months culture period ranged from 28-31 oC in the 12 ponds with the mean water temperature being 29.5 oC. This is close to the optimal recommended temperature for GFP culture (New and Valenti, 2000; New et al., 2010). Water quality parameters measured (D.O. and pH) were also within optimum ranges (New 2002) and concentrations of D.O. (Fig. 8) within ponds were within the tolerance range (3-7 mg/L) and did not reach stressful levels (< 2 mg/L). Similarly, pH (Fig. 9) was within the tolerance range (7.0-8.5) of the GFP as recommended by New (2002). Thus culture conditions for the M. rosenbergii strain evaluation experiment with regard to water quality parameters for the grow-out phase were within ideal ranges. This suggests that water quality parameters were unlikely to impact negatively on performance over the five months culture period.

5.2 Comparison of Growth Performance

Growth curves in the present study conform to the von Bertalanffy’s model under batch culture conditions (New and Valenti, 2000; New et al., 2010). While growth rate was initially slow from PL stocking through to the second month (Fig. 10), growth rate then increase. This phenomenon is in agreement with reports by New (2002) as initially stocked PLs or juveniles total weight is low for the first 30 to 40 days as they depend on production of natural food in the pond for feeding (New and Valenti, 2000; New, 2002; New et al., 2010). A similar trend was displayed by the four strains here at the first sampling where no significant difference was detected. However, between the second and third months growth rate increased. During this time PLs change their food preferences as they become more carnivorous, feeding primarily on snails, worms and insects and commercial feed (New, 2002). Therefore, after the second sampling 45

Chapter 5. Discussion significant differences (P = 0.01) in growth rate were observed among the four strains. After the third month until harvest, growth rate decreased again as individuals matured and began converting energy to maintenance and reproductive activities rather than to somatic growth. Statistically growth rates did not differ significantly (P = 0.04) among strains at final sampling but individual variation within strains was high. In general, growth performance of the Vietnam strain was the best but was not significantly different from that of Indonesia, Malaysia or the Fijian strains. Reasons for not detecting significant differences among the four strains could be due to the small number (3) of replicates and the short culture time. In addition, other factors related to environmental effects in the pond including; management practices, handling and/or nutrition could have impacted the results.

Malecha (1980) compared two strains of M. rosenbergii sourced from Malaysia and the Anuenue strain (Hawaii) for growth rate and found no difference in relative size or growth either for strain or sex. A similar result was evident in the current study where four strains were compared including the Fijian strain that is likely to have been impacted more extensively in terms of inbreeding and genetic diversity declines. Two factors that could have affected this outcome include; (1) the initial small broodstock population size introduced from Tahiti and (2) future broodstock sourced from ponds. Since its inauguration the freshwater prawn farming industry has often attributed declines in total grow-out productivity and individual growth rate to inbreeding effects and use of a small gene pool (New et al., 2010). Furthermore, cultured stocks in Taiwan sourced from Hawaii in the early 1990s experienced productivity declines thought to result from inbreeding depression. Weimin and Xianping (2002) stated that inbreeding is unavoidable after over 20 generations of reproduction and culture. According to Thanh et al. (2009), a number of factors can contribute to productivity declines, including high levels of inbreeding due to sourcing broodstock directly from grow-out ponds, and selection of breeders based on readiness to spawn that often involves early maturing, small females. Mather and de Bruyn (2003) stated that use of small females for spawning may lead to a substantial reduction in mean size across generations over time. This practice has being employed by the Ministry of Fisheries and Forest (MFF) 46

Chapter 5. Discussion hatchery operators in Fiji since the introduction of freshwater prawn culture into the country in the 1970s. Therefore, choice of breeders based on their readiness to spawn can lead to genetic quality declines which are reflected in reduction of mean size over time. A similar phenomenon was suspected in the hatchery stock at Anuenue (Hawaii) in the 1980s (New, 1995; Mather and de Bruyn, 2003; New et al., 2010). In contrast, Malecha (1983) argued that inbreeding depression in prawns is not, ipso, facto, inevitable as prawn’s show high fecundity (> 10,000 eggs/female) and experience high larval survival rates in hatcheries (> 50%) compared with in nature (1%) (Malecha, 1984; Cavalli et al., 2001; New et al., 2010). Nhan et al. (2009) compared reproductive performance between M. rosenbergii breeders from four different sources (Vietnam wild, Vietnam pond-cultured, Hawaii pond-cultured and China pond-cultured) and found no difference in terms of their reproductive capacity or relative fecundity. They did report however, variance in offspring quality between the different broodstock sources. A similar observation was made for the four strains in the current study, the Vietnam strain was easier to manage in the hatchery as it had a shorter larval cycle and higher PL survival rates compared with the next best performers (the Indonesian and Fijian strains). Hatchery duration for the Malaysian strain was longer and more difficult and showed the lowest PL survival rate. An explanation for this difference could be that prawns from different geographical areas have been exposed to different environmental conditions, nutrition, and ecological factors, breeding and rearing techniques even though they constitute the same species.

According to Dr. Subha Bhassu (pers. comm., 2011) disease problems in some areas of Malaysia have resulted in significant productivity declines whereby prawn viruses have slowed down growth and metabolism rates and resulted in Thai broodstocks now being sourced for seed production in the country. Bart and Yen (2001) reported that PL survival was much higher when broodstock from Thailand were used instead of those sourced from Vietnam. Furthermore, cross breeding of Thai females with Vietnam males resulted in even higher PL survival rates. Amrit and Yen (2003) argued that the observed difference in offspring quality between domesticated Thailand and Vietnamese breeders may relate to genetic differences due to their different geographical origins and 47

Chapter 5. Discussion respective domestication processes. This highlights the importance of undertaking comparative work on various strains of freshwater prawns and their potential improvement via selection and/or cross breeding. Malecha (1980) argued that high larval survival in prawn hatcheries relative to survival in the wild may have broadened the gene pool through introgression of different sets of compatible genotypes.

5.3 Survival rates

Thanh et al. (2009) stated that truly domesticated strains often perform better because over time; they adapt to culture environments e.g. channel catfish and African catfish, unless they are highly inbred. According to Malecha (1980) the culture environment itself can impose significant selection pressures which can cause an evolutionary change in a population's capacity to perform; this is collectively known as "fitness." According to Nugroho et al. (2008), levels of genetic variation are an important factor when evaluating individual fitness in the short term and will affect survival of the population over the long term. In general, survival rates of the four strains in Fiji were better or similar to that reported for GFP stocked in ponds and ranged from 50 to 80 percent as reported by New and Singholka (1982), New and Valenti (2000), Fujimura and Okamoto (1972), Malecha (1984), New (2002) and New et al. (2010) for prawn monoculture. The Vietnam strain showed the best percentage survival rate compared with the other strains based on lower variation among replicates compared to the Fijian strain (Fig. 13). Indications are that the Vietnam strain maybe more robust and more flexible in dealing with local environmental factors compared with the Indonesian, Malaysian and Fijian strains which in turn enhances relative survival rate and yield. The Indonesian and Malaysian strains showed an intermediate survival rate while the Fijian strain showed the lowest and most variable survival rates over the five months of culture. The relative poor performance in terms of survival shown by the Fijian strain indicates that there are factors reducing productivity, including potentially genetic deterioration in the local hatchery stocks due to inbreeding, environmental conditions, competition and/or pond management.

Chapter 5. Discussion

Doyle (1980) reported that if farmers source breeders from ponds and always source seed from his own hatchery, they may encounter serious inbreeding problems. The same author stated that the estimated decrease in growth and survival rates may be as high as 19 percent after 10 generations, giving an accumulated decrease in productivity of about 34 percent. According to TF farm manager (A. Singh, pers. comm., 2010) the Fijian prawn industry has experienced declines in growth and survival rates of farm produced prawns over recent years. In addition, the Fijian strain over the 3 decades of adaptation seems to have become less aggressive in comparison with the Vietnam, Indonesian and Malaysian strains making them more vulnerable potentially to disease and attack. Hamzah et al. (2008) reported a similar effect on survival rate in three red Tilapia strains in a pond environment in Malaysia whereby the introduced strains were found to be more aggressive compared with the native strain which contributed to higher survival of introduced strains. According to Gjedrem (2005), animals with the highest fitness in the culture environment, will reproduce at a higher level and have a higher survival rate than less fit animals. Survival in the culture environment is critical as relative productivity can depend on the total biomass of the individuals harvested after the culture period. Gjedrem (2005) recommended that selection of broodstock should be from families with the highest survival rates as maintenance of broodstock is an important component of hatchery operations. In principle, the simplest program for genetic stock improvement is to choose superior animals as breeders so that over generation, variation in the original population is translated into improved production (Doyle, 1980). A strong influence of environmental factors on survival rate is indicated by the low level of heritability for this trait in several species with estimates around 0.1 and for freshwater prawn it is < 0.09 (Malecha et al., 1984; New and Valenti, 2000; New et al., 2010). Relative survival rate should be a factor in breeding objectives because the trait determines the total number of fish or prawn available for market, and thus will affect profit from farming (Hamzah et al., 2008).

Under practical production systems, improvement of survival rate can be achieved by modifying environmental factors, affecting growth, such as better management, feeding, diet and water quality (Hamzah et al., 2008). Variation in survival rates at the end of the 49

Chapter 5. Discussion experiment was not unexpected and likely results from the cannibalistic nature of prawn larvae and juveniles, as growth and survival of juveniles are the important traits that determine prawn production. Prawn farmers normally practice two rearing strategies, one strategy is to stock PLs directly into the grow-out ponds. A second method is to stock PLs in nursing tanks or cages for 2-3 months before later transfer of juveniles to grow-out ponds (Karaket et al., 2011). In Fiji farmers practice strategy one and stock PLs directly into grow-out ponds without a nursery phase (M. Dawai, pers. comm., 2010). Several studies have reported that prawn production was relatively low employing the first strategy due to high mortality of PLs resulting from cannibalism in grow-out ponds. In contrast, use of a nursery phase increases the survival of juveniles and can subsequently increase farm production (Karaket et al., 2011). This practice shortens the culture period and reduces the production costs. It should be noted, however, that significant differences in the performance of prawn strains may vary in environments different from those utilized in here. In the present study, the mean percentage of marketable prawns was highest for Vietnam strain (70 percent) and lowest for the Malaysian strain (56 percent) whereas Indonesian and Fijian strain showed intermediate performance. It seems highly likely therefore that ‘fitness’ of the Malaysian strain was poorest compared with the other strains as demonstrated in the hatchery phase and during comparative growth trials. This may have resulted from the relatively small PL size (0.01 g) of the Malaysian strain PLs at stocking relative to the other strains (0.02 g). Utilization of small PLs from the Malaysian strain may have disadvantaged their performance during in early stages of grow-out and they could also be more vulnerable to environmental conditions and thus having a higher risk of death. Based on the results, Vietnam showed the highest proportion of marketable prawns, therefore, this strain promises increased income for farmers without waiting for long term genetic improvement programs to be carried out on the stock.

Gjedrem (2005) stated that natural strains that are well adapted to domestic environments can save many years of within-strain selection. A number of researchers have studied strain differences and detected significant differences (Uraiwan, 1988). To date, few breeding programs aiming at improving growth rates in ponds have been 50

Chapter 5. Discussion conducted on M. rosenbergii (Dobkin and Bailey, 1979; Doyle et al., 1983; Chareontawee et al., 2007 and Thanh et al., 2009). Studies carried out in Vietnam, Indonesia, Thailand, USA (South Florida) and other countries on GFP populations identified strains from different geographical locations that were genetically differentiated and cross breeding of these strains provided better individuals for pond culture (Thanh et al., 2009). In a recent study carried out in India, evaluation of growth performance in a 3 x 3 diallel cross of M. rosenbergii from three geographically distant locations in India (Gujarat, Kerala, and Orissa) reported significant differences for sex, age and harvest weight within and among crosses (Pillai et al., 2010). An earlier study reported that growth rates of crosses between Thai and Malaysian GFP strains performed better than did a pure Malaysian strain (Dobkin and Bailey, 1979). Genetic improvement of freshwater prawn strains is still at an early stage compared with that for many domesticated fish species (Bart and Yen, 2003; Nhan et al., 2009; Thanh et al., 2009 and Karaket et al., 2011). To date, only a few genetically improved strains of prawn have been developed for commercial use (New, 2005). A private company in Thailand has developed a new breed of GFP that has higher meat yield and size uniformity than other farmed strains (Cnaani et al., 2002).

5.4 Genotype – Environment Interactions

Genotype-environment interactions or variation in response of specific genotypes to different aquaculture conditions is another issue to be considered in any strain comparison. Depending on the interaction type, the strain can be classified as “general purpose” stock that performs moderately well in all culture conditions, or “special purpose” stock which exhibits a desirable performance under specific conditions (Gjedrem, 2005). An example of genotype-environment interaction for growth rate was found in common carp (Gjedrem, 2005). The growth rate of three crosses of common carp strains were compared; European X European, Chinese X Chinese and European X Chinese under five different production environments. Results illustrated that growth rate of Chinese carp was better than European carp when poultry manure was used as fertilizer and stocking density was high, while European carp performed better when an 51

Chapter 5. Discussion artificial feed was provided. Crosses also performed better than the pure lines in most cases except at low stocking density. Even though genotype-environment interactions for carp were small and not present in some cases, breeders should not simply generalize, and must test every case. If no interactions are detected, breeders can produce a “general purpose” strain for all farms. On the other hand, if differences are detected “specific purpose” strains are more efficient. Therefore, importing exotic GFP stocks and comparing their performance against the Fijian stock to identify the stock best suited for future development in the Fijian industry was the only option here. Furthermore, when introducing populations from different geographical locations, their performance and fitness under the locally prevailing conditions should be carefully evaluated. In addition, further investigation on genotype by environment interactions are needed as strain selection in a favorable culture environment may not necessarily be a good performer in less favorable conditions. In the current study genotype by environment interactions were not investigated however, in the future further investigation particularly of genotypes in different environmental conditions such as integrated verses non integrated systems, fertilized or not fertilized ponds, different stocking densities, poly-culture and substrates etc should be examined.

5.5 Feed

Apart from physical factors such as temperature, salinity and water quality, feed is one of the single most important factors influencing GFP culture productivity (New and Valenti, 2000). Diets containing about 35-40 percent protein are suitable for growth of freshwater prawn however; many commercially produced feeds for grow-out contain only 24-32 percent protein (Mitra et al., 2005). The observed FCR of 1.9 to 2.8 in the present study was close to the recommended range of 2.5-3.5 in experimental ponds where commercially manufactured feeds are used (New and Valenti, 2000). However, a low FCR value of 1.9 and a high SGR value of 5.3 evident for the Malaysian strain compared with the other strains may indicate that all feed was not used efficiently. According to Gjedrem (2005), behavior of animals in aquaculture farms is influenced by domestication as wild undomesticated animals tend to often be restless and nervous. In 52

Chapter 5. Discussion this type of situation feed will easily be wasted and FCR is likely to be low (Gjedrem, 2005). A low FCR for the Malaysian strain also implies that less feed consumed by the Malaysian strain and high, specific growth rate indicate they are not better net converters of food. Overturf et al. (2003) concluded that in regard to maintenance of genetic variation within domesticated stocks, strains containing the lowest levels of average gene diversity showed the lowest FCR levels and the highest SGR levels in five strains of rainbow trout. High FCR implies that the feed offered was not utilized efficiently by the prawns, so there may be a need to develop special feeds for prawns. In the current study a formulated diet designed for Tilapia with a crude protein level of 29 percent was utilized instead of a diet formulated especially for freshwater prawn with protein level of 30-40 percent. Furthermore, the Vietnam strain showed better flexibility in nutrition over the culture period in the current study as it had a good growth rate with the locally produced feed and under local environmental conditions. In addition, a second component of the ACIAR project was to develop a nutritionally adequate low cost feed specifically for freshwater prawn based on local ingredients that is currently underway. Feed utilization efficiency in fish farming can be improved by both optimizing feed formulations and by improving feed management. Beyond this, further improvement will depend on selective breeding (Gjedrem, 2005). Feed efficiency is a complex trait determined by all traits that influence the expenditure of energy for maintenance and activity and chemical composition required for weight gain and/or production of gonads. Selection for increased growth has been used to indirectly improve feed efficiency (Gjedrem, 2005). Thodesen et al. (cited in Gjedrem, 2005) showed significantly higher relative feed intake, growth and feed efficiency in offspring from Atlantic salmon selected for increased growth for five generations than in offspring of wild unselected salmon. This suggests that the genetic relationship between growth and feed efficiency in Atlantic salmon is non-linear, indicating a decreasing response in feed efficiency with increased growth capacity. Kinghorn (cited in Gjedrem, 2005) concluded that young rainbow trout, that consume more feed, grow faster but are not better net converters of food. Little is known about how much of the applied feed is utilized directly by freshwater prawn, how much is lost or how much is cycled to the prawn through other components of the pond ecosystem (Malecha, 1984). Nhan et al. (2010) concluded that 53

Chapter 5. Discussion effects of larval stocking density and feeding regime can strongly affect larval development, survival and duration of the rearing cycle, as well as larvae quality. Furthermore difference in feeding practices between different strains probably also affect reproductive performance as various environmental and nutritional factors affect the onset of maturation. Wilder et al. (1999) concluded that the connection between nutritional and reproductive functions was unclear but a link existed between diet and hormonal makeup. In addition, feeding position, feeding efficiency, feed quality and time of feeding and stocking density are important aspects that need to be addressed in an effort to design the best feed and feeding management systems.

5.6 Body Traits

In the current study, body traits (abdomen, carapace and total body length), interaction effects were significant among strains and between sexes (P = 0.001) except for weight. Weight differences between the four strains were not statistically significant (P = 0.05). A lack of statistical significance for growth rate appears to be the result of high within- strain variation, specifically resulting from sexually dimorphic growth patterns combined with heterogeneous individual growth (Malecha, 1984). It was clear from the current study that sex had a significant effect on final weight with males exhibiting faster mean growth rates than females. A graphical presentation of the least square means for body traits (Fig. 16) demonstrates that means between strains are almost parallel. Yield of M. rosenbergii depends on its sex and size, as size increases the percentage of tail meat decreases. Females yield a significantly larger proportion of tail meat than do males, independent of size (New and Valenti, 2000). This statement agrees with the demonstrated graphical presentation of abdomen lengths in the two sexes. Likewise body weight demonstrates large variation between the sexes as males on average, are twice as heavy as females of the same age. Malecha (1984) demonstrated that female growth begins to level off sharply much earlier than for males; therefore he suggests the former should be harvested and marketed at an earlier age and smaller size. The current stocking and harvesting system practiced in Fiji works in the opposite direction: males and females are allowed to grow until most reach a desirable market weight. Further, the 54

Chapter 5. Discussion magnitude of differences between sexes here in terms of phenotypes for body traits was generally small, especially for the Vietnamese strain. Males and females of the Vietnamese strain had significantly higher body weight than did other strains. Overall, males and females of the Indonesian strain had significantly lower body weight compared with that of Malaysian and Fijian strains. Despite the sex by strain interaction being statistically significant (P = 0.001), it accounted for only a very small proportion of the variation, furthermore, the magnitude of difference between sexes in terms of phenotypic standard of carapace to total length between males and females was generally small for all strains (0.36 vs. 0.33). The least square means for female body weight varied more widely among strains (Table 9). Thus differences in growth rate that resulted from genetic background in females were not masked by social factors as they may have been in males (Thanh et al., 2009). Malecha (1984) investigated heritability (h2) for growth rate variation in GFP juveniles and reported a significant genetic effect in females (h2 = 0.35 ± 0.15) while h2 was negligible in males. Therefore, it suggests that a breeding program for GFP to improve overall growth performance would best be directed at females only. Genetic factors significantly influence growth of freshwater prawn as indicated by significant heritability estimates (Meewap et al., 1994) and response to selection (Uraiwan, 1998). Therefore, it is possible to improve growth and other traits such as carapace and abdomen length in a conventional genetic improvement program (selection and mating systems). Cross breeding between genetically different strains is also a promising approach and can improve traits through combining desirable alleles from different strains and/or produce hybrid vigor (Meewap et al., 1994). Therefore, hybridizing the Vietnam and Fijian strains in Fiji could broaden the genetic base of the culture population and perhaps offer a better target for a future selection program to improve growth performance.

5.7 Size Variation in Morphotype

Malecha (1984) mentioned that heterogeneous individual growth in GFP is a major constraint on optimum production in growth trials, i.e., single batch harvests contain an array of sizes, all of which must be marketed. As reported by Ra’anan and Cohen (1985) 55

Chapter 5. Discussion freshwater prawns have similar size frequency distribution in populations of both sexes. As individuals mature however, the size distribution becomes quite different for males and females. Both males and females grow however at similar rates until the onset of sexual maturity (15-35 g), when females begin to divert more energy into egg production and incubation and less into growth, so males end up a lot larger than females. Some males become dominant in their social hierarchy however, which reduces growth of other males, the result being large individual size variation (Nandlal and Pickering, 2005). Size variation was evident in the current study as there is significant difference among morphotypes (P = 0.001). Relative frequencies of blue claw, orange claw, small males and females in the four strains in this study indicate that social factors influence weights, abdomen lengths, carapace lengths, total lengths and carapace ratio. Based on overlap of these intervals, the subgroups were pooled by sex and then by strains. No major differences were identified however in relative size or growth rate between sexes in either the cultured or introduced stocks. A comparison of size variation in the current study it was observed that the Vietnamese population have a relatively more homogenous size distribution and evenness of different morphotype stages and sex (Fig. 12), as reflected in their small variance (mean weight (g) ± se). This could be because most prawns are growing at similar rates compared with the Fijian strain that is showing a disproportionate increase in size variation with time as individuals are growing at different rates (Ranjeet and Kurup, 2002). The Indonesian and Malaysian strains were intermediate for size variation. Ranjeet and Kurup (2001) indicated that differential growth can also have a genetic basis; resulting from intrinsic factors such as genetic differences within strains, hatching order or age at metamorphosis (Ranjeet and Kurup, 2002). Studies linking the embryonic development to size heterogeneity in different species of Macrobrachium have been correlated with the differential growth (Ranjeet and Kurup, 2002). It appears likely therefore, that size heterogeneity may be cumulative effect of both intrinsic and extrinsic factors. Moreover, Malecha (1984) stated that heterogeneous growth is due to suppressive effects exerted by larger animals on smaller ones and that this effect may be mediated by a water borne crowding factor and is related to stocking density. In addition, the magnitude of this differential growth greatly depends on a farmer’s management strategies and can be reduced through improved 56

Chapter 5. Discussion nutrition or reduced stocking density. To date not much emphasis has been given to understanding the role of different management strategies for increasing productivity and mean weight of prawns in culture (Ranjeet and Kurup, 2002). Various management approaches have been trialed to minimize heterogeneous growth and have concentrated on selective harvesting and size grading of pond populations.

5.8 Sex Ratio

In the current study similar results were obtained among strains for sex ratio that ranged from 1:1 to 1:1.7 with no significant difference seen. Recommended sex ratio in the breeding pond recommended is a male: female ratio of 1:4 (Doyle, 1980). If we assume that the sex ratio at stocking was 1:1, then the relative mean survival for males within strains was lower than for females in all strains. This is reflected in asymmetrical sex ratios in any strains at harvest that were significantly different from 1:1 except for Vietnam. The rest of the strains were biased towards females especially the Indonesian strain. The Indonesian strain had significantly more females (Fig. 14) in the population compared with other strains. Possibilities are that environmental conditions were more favorable to Indonesian females rather than male development. Higher proportions of females to males have been found in prawn populations in a number of studies when prawns were raised in different geographical localities at a wide range of densities, in earthen ponds and in tanks (Smith et al., 1981; Sandifer et al., 1985; Karplus et al., 1986; D’Abramo et al., 1989; Siddiqui et al., 1997). Three mechanisms have been suggested by Smith et al. (1981) as possibly regulating the relative frequencies of females in prawn populations. Firstly, environmental conditions may affect sex determination and favor the development of females. Secondly, females may already out number males at stocking. Thirdly, selective male mortality may occur. Siddiqui et al. (1997) reported an equal sex balance in the progeny of two normal mating, whereas Malecha (1984) reported higher frequency of females in the progeny of five crosses between normal males and females. No virgin females were reported in all strains this was because virgin females are only reported in all female populations and not in mixed sex populations (Siddiqui et al., 1997). The Indonesian strain (Fig. 15) was the earliest 57

Chapter 5. Discussion strain to reach sexual maturity here at day 63 and had higher proportions of females at smaller size. The Malaysian strain was the slowest to mature and did so at relatively smaller size. When comparing the Fijian and Vietnamese strains it was found that Vietnamese strain were still getting larger in size and reaching maturity at a larger size compared with the other strains. Farmers usually prefer however, more males than females as males fetch higher price. As the farmer produces individuals for an immediate market, preference for more male or an equal sex ratio is usually desirable than higher proportions of female. According to one of the largest prawn farmers in Fiji, (A. Singh pers. comm., 2010) ‘consumer preference for larger size is greatly increasing in the country’ and as a producer he prefers larger size due to the high selling price they fetch. In addition, government or universities who manage broodstock are in general, primarily concerned with maintaining high genetic diversity of stocks that are supplied to farmers in order to maintain low inbreeding rates. Therefore, to minimize the inbreeding rate in culture these institutes need to maintain equal number of spawners as the base population from the best strain.

5.9 General Discussion

Freshwater prawn culture has been practiced in Fiji for more than three decades. Since the advent of successful PL production in the 1980s, broodstock have been kept in captivity but their levels of management have varied. Breeding records including the size of the founding population and the number of broodstock used each generation are not known. Issues of inbreeding level and genetic deterioration of local stocks have been raised by both prawn farmers and government officials as the probable cause of declining farm production. As a result, government officials requested an evaluation of local broodstock and introduction of exotic stocks for evaluation from countries that have better culture stocks, i.e., Asian countries to replace or upgrade their own stocks. Reduced genetic diversity has been observed in several hatchery stocks of fish and crustaceans, e.g., Japanese flounder (Sekino et al., 2002), Nile tilapia (Brummett et al., 2004), channel catfish (Simmons et al., 2006), Kuruma prawn (Luan et al., 2006) and tiger prawn (Xu et al., 2001). In most cases, impacts of selection and poor hatchery 58

Chapter 5. Discussion practices were the prime factors causing reductions in genetic variation in culture populations (Chareontawee et al., 2007). The freshwater prawn farming industry has attributed declines in total grow-out productivity and individual growth rate to inbreeding and the degeneration of the cultured stocks caused by a small gene pool (i.e. Anuenue based stock). Inbreeding depression is manifested as a general loss of fitness including lower growth, survival and fecundity and increased incidence of physical deformities (Uraiwan, 1988). Inbreeding rate is inversely proportional to effective population size (Ne) meaning the lower the Ne the more likely mating of close relatives will occur. In order to avoid inbreeding or loss of genetic variation through mating of close relatives, high Ne should be maintained by (1) mating being restricted to members of the same line, (2) generation are distinct and do not overlap and (3) number of breeding individuals in each line are the same, this will require a balanced sex ratio of broodstock in all generations. For GFP, the common practices of using gravid females from ponds to produce PL’s can lead to a reductions in Ne and an increase in inbreeding (Chareontawee et al., 2007). Uraiwan (1988) pointed out that inbreeding in fish reduced productivity; however, inbred populations can still produce good offspring even though they have lost some diversity. Inbreeding also affects allele frequencies by increasing the number of homozygotes, thus changing genotype distributions within populations. In this study, I found evidence for slightly better productivity of some introduced exotic strains compared with the existing Fijian stock. This difference could have resulted from both the small population size of the original stock and ongoing mating of close relatives over time. The practice of acquiring broodstocks of different genetic backgrounds is a common practice in hatcheries. It was difficult to determine the genetic relationship among broodstocks because the pedigree and broodstock records do not exist for the Fijian strain. However, a parallel study on the genetic diversity analysis of the same strains showed that the Fijian strain had high levels of genetic diversity (see appendix 2, Fig. 17) (P. Mather, unpublished data). This shows that it is unlikely that inbreeding depression is responsible for any declines in the performance of the Fijian stock. Therefore, further investigation of genetic diversity levels in hatchery stocks of M. rosenbergii using microsatellite markers could provide information about the effectiveness of past genetic management of hatchery populations, detect population 59

Chapter 5. Discussion differentiation and define current levels of genetic diversity (Chareontawee et al., 2007). Chareontawee et al. (2007) demonstrated that local hatchery stocks had more genetic variation than was expected based on a comparison with some wild populations. All strains exhibited similar and relatively high levels of growth and survival traits. While some level of inbreeding depression cannot be ruled out for the Fijian stock, in contrast the Malaysian strain showed the lowest levels of genetic diversity (P. Mather, unpublished data) among the four strains. The presence of high genetic variation in the local populations was probably due to the fact that over the domestication process, the gene pool of the Fijian strain had remained sufficiently broad and low productivity may result primarily from environmental factors that need investigation. In addition, to prevent the chance of inbreeding levels increasing in future generations, hatchery managers should start to keep breeding records and pedigree information on the culture strain. Strain evaluation provides a basis for selecting the best individuals across populations to form the founder stock of a synthetic population (Gjedrem, 2005). Based on the magnitude of differences in additive, heterotic and reciprocal effects, crossing among geographically discrete populations of freshwater prawn can assist formulation of robust, genetically diverse synthetic populations. However, experience shows that selecting animals for high growth rate and other traits in several generations, will favor animals, which are better adapted to the farm environment that are, calmer and less afraid of people as well as being less aggressive (Gjedrem, 2005). This population could then be subjected to a systematic selection program based on additive genetic variation. However, in the interim, the current results suggest that the Vietnamese strain provides the best choice for the local Fijian freshwater prawn industry because (1) survival was better than Fiji (2) growth rate was higher than Fiji (3) the evenness of different morphotypes and sex stages in the population (4) genetic diversity was high (5) and the Vietnam strain has only been domesticated for a few generations compared with the Fijian strain which has been domesticated for more than 100 generations in the present environment. This means the Vietnam strain is still adjusting to the environmental conditions available in Fiji and hence overtime, it should continue to improve in productivity whereas the Fijian strain has over the years displayed optimal performance in quality and may have already exceeded ability to improve further. In addition, a 60

Chapter 5. Discussion breeding program is likely to stimulate industry development and sustainability of freshwater prawn industry in Fiji. However, results of the study emphasize that other factors such as domestication management, hatchery practices, nutrition and environmental conditions are important factors apart from inbreeding and genetic diversity that should be investigated further. The present study has provided baseline information and a platform for development of the giant freshwater prawn culture industry in Fiji and the Pacific more widely.

Conclusion

6.0 Conclusion

Strain evaluation experiments generally involve several steps from choice of strains, population sampling, preparation of testing environments, determination of sample sizes, and implementation of the experiment, collection of data, statistical analysis and interpretation of results. Results here indicate that there were no significant differences (P = 0.083) in growth performance or survival among the giant freshwater prawn strains from Vietnam, Indonesia, Malaysia and Fiji tested here when environmental factors were suitable for growth. However, the Vietnam strain demonstrated the overall best performance. Therefore, from the results it is evident that Vietnam strain performed the best for a combination of phenotypic traits (growth, survival, sex ratio, percentage marketable prawns, etc.) of economical importance to the industry.

Over the hatchery phase carried out here the Malaysian strain showed a longer larval development time with lower survival rates than the other strains. It is common hatchery practice to discard slow developing larvae and to select strains that show faster larval development as observed for the Vietnam and Indonesian strains but more rigorous experiments on difference in larval development rates among the four strains will need to be conducted.

This study has demonstrated that there are good prospects for setting up sustainable breeding programs for resource poor prawn farming as farm productivity can be enhanced based on the strain selection carried out here. Because poverty alleviation and food security are primary goals in the developing world, the initiation and implementation of inexpensive breeding programs will ensure that the genetically improved materials are accessible to rural poor prawn farmers. High response can be achieved within a few generations using either the Vietnam or Indonesian strains, and because of short generation times prawn farmers can reap the benefits of stock improvement programs without delay. Appropriate breeding goals for future breeding schemes need to be setup with involvement of local farmers to ensure that the selected strain meets the requirement of local farmers. Full benefits of the selected strain will be 62

Conclusion realized by improving husbandry practices that ensure water quality and nutrition is suitable for maximum growth. It is important that aquaculture officers, farmers and government hatchery managers are trained on how to keep records because although record-keeping is a tool for monitoring progress, to-date it has not been a common practice. This is a necessary step for the maintenance of the selected domesticated strain. In conclusion, this study provides baseline for establishing an improved hatchery stock for freshwater prawn aquaculture for the Fijian region and Pacific region. The high levels of genetic variation in local hatchery stocks suggested that genetic factors were probably unlikely to be the cause of low production of this species. A proper management program for local hatchery stocks is needed to preserve genetic diversity for further development of prawn aquaculture across the region.

Recommendations

7.0 Recommendations:

1. Culture period for the current study should be extended to 6 - 8 months instead of five months to investigate opportunities to value add to harvest. 2. Experimentation with different seasons to identify the most productive strain. 3. More replicates per strain to minimize error and variance in better identification of strain performance over culture period. 4. Experimentation of strains under different production environments to identify the most productive strain in G x E interactions. 5. Record keeping of breeding and pedigree information by breeders and hatchery managers to prevent inbreeding in later generations. 6. Investigation of other potential problems that may have negative effects on productivity. 7. A further step should be to form a base population combining characteristics via cross breeding of the most productive strains. 8. Identification of superior strains which perform best in the hatchery. 9. Use of quality seed by larval rearing up to juvenile stage for stocking of grow out ponds. 10. Exchange of broodstock among hatcheries to reduce inbreeding impacts.

Literature Cited

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Aquacop, 1977. Macrobrachium rosenbergii culture in Polynesia: progress in developing a mass intensive larval technique in clear-water. Workshop of World Mariculture Society.

Avault, J.W. 1996 Fundamentals of Aquaculture – A step by step guide to commercial aquaculture. AVA Publishing Company Inc. U.S.A.

Bart, A.N. and P.T., Yen. 2001. Improved performance of domesticated giant prawn Macrobrachium rosenbergii in Vietnam. In: Abstracts of Aquaculture 2001. 21-25 January 2001. Lake Buena Vista. FL. USA. World Aquaculture Society. Baton Rouge. LA. USA. 15pp.

Brummett, R.E., Angoni, D.E. and V., Pouomogne. 2004. On-farm and on-station comparisons of wild and domesticated Cameroonian populations of Oreochromis niloticus. Aquaculture 242: 157–164.

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Chand, V., de Bruyn, M. and P.B., Mather. 2005. Microsatellite loci in the eastern form of the giant freshwater prawn (Macrobrachium rosenbergii) Molecular Ecology Notes 5: 308-310.

Chareontawee, K., Poompuang, S., Nakorn-Nu, U. and W., Kamonrat. 2007. Genetic diversity of hatchery stocks of giant freshwater prawn (M. rosenbergii) in Thailand. Aquaculture 271: 121-129.

Cnaani, A., Ron, M., Seroussi, E. and G., Hulata. 2002. Genetic analysis of immunological traits in Tilapia. Naga. ICLARM Quarterly 25 (2): 40-42.

Cohen, D., Ra’anan, Z. and T., Brody. 1981. Population profile development and morphotypic differentiation in the giant freshwater prawn Macrobrachium rosenbergii (de Man). Journal of the World Mariculture Society 12: 231–243.

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Appendix

Appendix 1

Table 13. Mean water quality parameters collected over 47 days of larval rearing of freshwater prawn in second hatchery operation.

Vietnam Indonesia Malaysia Fiji Am Pm Am Pm Am Pm Am Pm

Temp. 27.5 ± 27.6 ± 27.3 ± 27.5 ± 27.5 ± 27.7 ± 28.0 ± 28.1 ± (oC) 1.9 1.3 1.6 1.1 2.0 1.4 2.1 1.5

Salinity 10.8 ± 10.8 ± 10.6 10.5 ± 11.1 11.1 ± 11.2 ± 11.2 ± (ppt) 2.4 2.4 ±2.5 2.3 ±1.1 0.9 1.4 0.9

Table 14. Freshwater prawn M. rosenbergii feed rate used for the comparative grow-out trials over five months as recommended by New (2002).

Rearing Period (days) Feeding rate (%) Jan 7 – March 11 (63) 50 March 11 – April 26 (109) 15 April 26 – June 3 (146) 5

Table 15. Initial weight of berried M. rosenbergii female’s representative of the four strains (Vietnam, Indonesia Malaysia and Fiji) used in first hatchery phase and number of PLs produced.

Strain No. of females No. of PL Vietnam 18 19,000 Indonesia 5 39,000 Malaysia 11 1,783 Fiji 23 5,450

Appendix

Table 16. Initial weight for representative sample (n=100 in triplicate per strain) of post larvae reared for 47 days in hatchery cycle.

Weight (g) Strain 0.016 ± 0.003 Vietnam 0.018 ± 0.002 Indonesia 0.013 ± 0.004 Malaysia 0.018 ± 0.001 Fiji

Appendix

Appendix 2 Results of homogeneity of variance for the data tested with two way and repeated measure ANOVA.

Table 17. Repeated measure analysis of variance of body weight gain over five months culture period in earthen ponds.

Source of variation d.f. s.s. m.s. v.r. F pr. Strain 3 39.611 13.204 2.36 0.083 Stage 5 12096.121 2419.224 431.91 <0.001 Strain.stage 15 100.593 6.706 1.20 0.306 Residual 48 268.859 5.601 Total 71 12505.184

Table 18. Two way analysis of variance of abdomen length with strain and morphotype condition over five months culture period in earthen ponds.

Source of variation d.f. s.s. m.s. v.r. F pr. Strain 3 49.143 16.381 5.00 0.004 Stage 5 7331.768 1466.354 447.45 <0.001 Strain.stage 15 70.620 4.708 1.44 0.169 Residual 48 157.302 3.277 Total 71 7608.832

Table 19. Two way analysis of variance of carapace length over strain and morphotype condition.

Source of variation d.f. s.s. m.s. v.r. F pr. Strain 3 22.328 7.443 7.34 <0.001 Stage 5 3435.946 687.189 678.14 <0.001 Strain.stage 15 26.500 1.767 1.74 0.074 Residual 48 48.640 1.013 Total 71 3533.414

Appendix

Table 20. Two way analysis of variance of the total length with strain and morphotype condition. Source of variation d.f. s.s. m.s. v.r. F pr. Strain 3 147.33 49.11 3.03 0.038 Stage 5 20985.37 4197.07 258.63 <0.001 Strain.stage 15 301.63 20.11 1.24 0.277 Residual 48 778.96 16.23 Total 71 22213.29

Table 21. Two way analysis of variance of carapace ratio with strain and morphotype condition. Source of variation d.f. s.s. m.s. v.r. F pr. Strain 3 0.00120556 0.00040185 10.33 <0.001 Stage 5 0.01830000 0.00366000 94.11 <0.001 Strain.stage 15 0.00057778 0.00003852 0.99 0.480 Residual 48 0.00186667 0.00003889 Total 71 0.02195000

Mean allelic richness 14 12 10 8 6 4 Allellic Richness 2 0 Vietnam Indonesia Malaysia Fiji

Figure 17. Genetic diversity analysis carried out on the four strains using microsatellite technique (Source: P. Mather, unpublished data)

Appendix

Appendix 3

Figure 18. Grow out ponds (Photo: S Singh, 2010)

Figure 19. Grow out ponds (Photo: S Singh, 2010)

Appendix

Figure 20. Stocking grow-out pond (Photo: S Singh, 2010)

Figure 21. Sampling grow-out ponds (Photo: S Singh, 2010)

Appendix

Figure 22. Measuring weight of prawns during sampling (Photo: S Singh, 2010)

Figure 23. Measuring carapace length (Photo: S Singh, 2010)

Appendix

Figure 24. Measuring abdomen length (Photo: S Singh, 2010)

Figure 25. Harvesting grow-out pond (Photo: S Singh, 2010)

Appendix

Figure 26. Sorting of harvested prawns (Photo: S Singh, 2010)

Figure 27. Blue claw male and berried female (Photo: S Singh, 2010)

Appendix

Figure 28. Orange claw male (Photo: S Singh, 2010)

Figure 29. Small male and runts (Photo: S Singh, 2010)

Appendix

Figure 30. Packed prawns ready for selling (Photo: S Singh, 2010)

Figure 31. Larval stages of M. rosenbergii (Source: New and Valenti, 2000)