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FACULTEIT LANDBOUWKUNDIGE EN TOEGEPASTE BIOLOGISCHE
WETENSCHAPPEN
Academiejaar 2004-2005
OPTIMIZATION OF MUD CRAB (SCYLLA PARAMAMOSAIN) LARVICULTURE IN VIETNAM
OPTIMALISATIE VAN DE LARVICULTUUR VAN DE TROPISCHE KRABSOORT SCYLLA PARAMAMOSAIN IN VIETNAM
by/door
Truong Trong Nghia
Thesis submitted in fulfillment of the requirements for the degree of Doctor (Ph. D.) in Applied Biological Sciences
Proefschrift voorgedragen tot het bekomen van de graad van Doctor in de Toegepaste Biologische Wetenschappen
On the authority of Op gezag van
Rector : Prof. dr. A. DE LEENHEER
Decaan : Promotor:
Prof. dr. ir. H. VAN LANGENHOVE Prof. dr. P. SORGELOOS
Auteur en promotor geven de toelating dit doctoraatswerk voor consultatie beschikbaar te srellen en delen ervan te copiëren voor persoonlijk gebruik. Elk ander gebruik valt onder de beperkingen van het auteursrecht, in het bijzonder met betrekking tot de verplichting uitdrukkelijk de bron te vermelden bij het aanhalen van de resultaten van dit werk. The author and the promoter give the authorization to consult and to copy parts of this work for personal use only. Every other use is subject to copyright laws. Permission to reproduce any material contained in this work should be obtained from the authour. Ghent, October 2004 De promotor/Promoter De auteur/Author Prof. dr. Patrick Sorgeloos Truong Trong Nghia
Acknowledgements My deepest sincere gratitude to my promoter and Professor dr. Patrick Sorgeloos for his scientific
orientation and assistantce, especially his patience in correcting the first and final thesis drafts during his already busy time.
I think highly of Dr. Patrick Lavens who came up with the initial plan for my research before he
left ARC for INVE Technologies.
I am very grateful to my “second promoter” Mathieu Wille for his proper suggestions on experiment design and scientific discussion, especially for his devoted and thoughtful revision and recommendations in the preparation and completion of all chapters of the thesis. I also appreciate
An Van Der Eecken for her contribution to my thesis correction.
I am especially thankful to the members of the reading committee, Prof. Peter Bossier (Ghent University, Belgium), Dr. Lewis Le Vay (University of Wales Bangor, the UK), Dr. Geoff Allan
(NSW Department of Primary Industries, Australia), Prof. Koen Dewettinck and Prof. Frans Ollevier (Leuven University, Belgium) for their critical reviews and extremely valuable
suggestions to improve this thesis.
I deeply thank the administrative support headed by Magda Vanhooren and her assistants Dorina Tack and Alex Pieters for arranging my accommodation and money; Marc Verstraeghen
and Tom Baelemans for purchasing computer wares and experimental facilities; Katerina Gamrotova for helping to access references and especially Els Vanden Bergh for her
kindness in guiding the paperwork for my PhD annual registration and defense.
I greatly appreciate the encouragement and support from my former superiors Prof. Nguyen Kim Quang (Ex-Vice Rector of Can Tho University), Prof. dr. Tran Phuoc Duong
(Ex-Rector of Can Tho Unversity), Prof. dr. Tran Thuong Tuan (Ex-Rector of Can Tho University) and Dr. Vu Do Quynh (Ex-Director of Shrimp Artemia R&D Institute, Can Tho University) and
the current Rectorate of Can Tho University for the fulfillment of the thesis.
I am greatly indebted to all teachers, farmers and friends who have transferred unconditionally their valuable knowledge and experience in aquaculture since I have involved in this field.
The experiments during seven years were possible thanks to the very valuable cooperation received
from the research assistants Tran Cong Binh, Nguyen Van Danh, Nguyen Minh Dat, Tran Thi Dep, Huynh Thi Ngoc Hien, Tran Tan Huy, Nguyen Hong Loc, Ngo Le Ngoc Luong, Pham Thi Tuyet Ngan, Tran Suong Ngoc, To Cong Tam, Hoang Phuoc Thanh, Ngo Thi Thu
Thao, Nguyen Thi Thanh Thao, Cao Phuoc Tho, Nguyen Thi Hong Van, Quach The Vinh and Vu Ngoc Ut; especially Geert Vandewiele for analizing the HUFA samples
and the Belgian MSc student Stijn Vandendriessche.
I would like to express my warmest feelings to all my friends and my colleagues in various institutions and universities, Can Tho University and its College of Aquaculture and Fisheries, who
always were concerned about my PhD completion.
My PhD program was financed through scholarship and research grants from the International Foundation for Science (IFS), Binh Chau Company (established by Mr. Trinh Vinh Binh), the Flemish Inter-University Council (Vl.I.R.-IUC) and the European Commission (INCO-DC).
My family would be very happy with my success and this is a gift for them.
I hope I did not forget anyone, but just in case… thank you.
List of abbreviations Σ Total ANOVA Analysis of variance ARA Arachidonic acid BIARC Bribie Island Aquaculture Research Centre in Australia C1 First crab stage C2 Second crab stage DAH Day(s) after hatch DHA Docosahexaenoic acid DIS Dry Immune Selco® (INVE Aquaculture, Belgium) EC5 Effective concentration at 5 % endpoint EFA Essential fatty acid EPA Eicosapentaenoic acid FAME Fatty acid methyl ester FAO Food and agriculture organization GRIM Gondol Research Institute for Mariculture in Indonesia HSD Honest significant difference HUFA Highly unsaturated fatty acid ICES International council for the exploration of the sea IFS International Foundation for Science INCO - DC International cooperation for developing countries LC50 Lethal concentration at 50 % endpoint LSI Larval stage index M Megalopa(e) MDS Moult death syndrome MR1 First metamorphosis rate MR2 Second metamorphosis rate PNR Point of no return PNR50 Point of no return at 50 % endpoint PUFA Poly unsaturated fatty acids PVC Polyvinyl chloride RIA3 Research Institute for Aquaculture No III in Vietnam TAN Total ammonia nitrogen VASEP Vietnam Association of Seafood Exporters and Producers Vl.I.R - IUC Vlaamse Interuniversitaire Raad (Flemish Interuniversity Council) - Institutional University Co-operation WSSV White spot syndrome virus Z1 - Z5 Zoeal stages 1 - 5
Table of contents CHAPTER 1............................................................................................................................1 Introduction ...........................................................................................................................1 1. World production and demand of high-value crustaceans ..................................................1 2. Production of high-value crustaceans in Vietnam...............................................................2 3. Mud crab culture..................................................................................................................3 4. Reclassification of mud crab species...................................................................................4 5. Importance of mud crab larviculture in Vietnam ................................................................6 6. Aims and outline of the thesis .............................................................................................7 CHAPTER 2..........................................................................................................................11 Current status of mud crab (Scylla spp.) hatchery technology .......................................11 Abstract..................................................................................................................................11 1. Introduction .......................................................................................................................12 2. Broodstock.........................................................................................................................13
Sourcing and maturation ...................................................................................................13 Spawning (egg extrusion) ..................................................................................................16 Incubation and hatching ....................................................................................................17
3. Larval rearing ....................................................................................................................20 Selection and stocking .......................................................................................................20 Water quality and parameters ...........................................................................................20 Culture systems..................................................................................................................22 Feeding and nutrition ........................................................................................................24
4. Nursery ..............................................................................................................................27 5. Bottlenecks to commercial production ..............................................................................29 6. Discussion..........................................................................................................................31 CHAPTER 3..........................................................................................................................33 Reproductive performance of captive mud crab (Scylla paramamosain) broodstock in
Vietnam.............................................................................................................................33Abstract..................................................................................................................................33 1. Introduction .......................................................................................................................34 2. Materials and methods.......................................................................................................35
2.1. Broodstock ..................................................................................................................35 Broodstock source .........................................................................................................35 Rearing systems and culture conditions ........................................................................35 Broodstock management................................................................................................36
2.2. Egg incubation............................................................................................................37 2.3. Reproductive performance .........................................................................................37 2.4. Statistical analysis ......................................................................................................39
3. Results ...............................................................................................................................40 3.1. Effect of selected management and environmental parameters on reproductive performance.......................................................................................................................40
Eyestalk ablation ...........................................................................................................40 Rearing system...............................................................................................................40 Broodstock source .........................................................................................................41 Month and seasonal cycle..............................................................................................41 Female weight................................................................................................................43
Contents
ii
Time to spawn ............................................................................................................... 44 3.2. Artificial incubation of eggs and egg diameter during incubation ............................ 44
4. Discussion ......................................................................................................................... 44 4.1. Effect of selected management and environmental parameters on reproductive performance ...................................................................................................................... 44
Eyestalk ablation ........................................................................................................... 44 Types of rearing system................................................................................................. 45 Broodstock source ......................................................................................................... 47 Month and seasonal cycle ............................................................................................. 47 Female weight ............................................................................................................... 50 Time to spawn ............................................................................................................... 51
4.2. Artificial incubation of eggs and egg diameter in function of incubation time ......... 52 5. Conclusions and suggestions............................................................................................. 52 CHAPTER 4 ......................................................................................................................... 61 Optimal feeding schedule for mud crab (Scylla paramamosain) larvae......................... 61Abstract ................................................................................................................................. 61 1. Introduction ....................................................................................................................... 62 2. Materials and methods ...................................................................................................... 63
2.1. Broodstock rearing..................................................................................................... 63 2.2. Live feed culture and enrichment ............................................................................... 64 2.3. Larval rearing: objectives, experimental design and techniques .............................. 65
Experiment 1 ................................................................................................................. 65 Experiment 2 ................................................................................................................. 66 Experiment 3 ................................................................................................................. 67 Experiment 4 ................................................................................................................. 67 Experiment 5 ................................................................................................................. 68 Experiment 6 ................................................................................................................. 68 Experiment 7 ................................................................................................................. 69 Experiment 8 ................................................................................................................. 69
2.4. Evaluation criteria ..................................................................................................... 69 2.5. Statistical analysis...................................................................................................... 70
3. Results ............................................................................................................................... 70 4. Discussion ......................................................................................................................... 73
4.1. Ability of S. paramamosain zoeae to catch instar-1 Artemia..................................... 74 4.2. Suitable first feed........................................................................................................ 75 4.3. Alternative Artemia forms as first feed ...................................................................... 78 4.4. Feeding schedule........................................................................................................ 80
5. Conclusions and suggestions............................................................................................. 81 CHAPTER 5 ......................................................................................................................... 89 Influence of the content of highly unsaturated fatty acids in the live feed on
larviculture success of mud crab (Scylla paramamosain) ............................................ 89Abstract ................................................................................................................................. 89 1. Introduction ....................................................................................................................... 90 2. Materials and methods ...................................................................................................... 91
2.1. Source of larvae ......................................................................................................... 91 2.2. Larval rearing ............................................................................................................ 92
Larval rearing systems and procedures ........................................................................ 92 Live feed culture and enrichment .................................................................................. 93
Contents iii
Feeding ..........................................................................................................................94 2.3. Experimental design ...................................................................................................94 2.4. Evaluation criteria......................................................................................................95
Fatty acid composition ..................................................................................................95 Larval performance .......................................................................................................96
2.5. Statistical analysis ......................................................................................................97 3. Results ...............................................................................................................................98
3.1. Fatty acid composition of live feed and crab larvae (experiment 3)..........................98 3.2. Zoeal survival .............................................................................................................99 3.3. Larval development rate during the zoeal stages .......................................................99
Experiment 1..................................................................................................................99 Experiment 2................................................................................................................100 Experiment 3................................................................................................................100 Experiment 4................................................................................................................100
3.4. Metamorphosis .........................................................................................................101 Experiment 3................................................................................................................101 Experiment 4................................................................................................................102
3.5. Correlation between the fatty acid composition of the live feed and the crab larvae, and larval development rate and metamorphosis success...............................................103
LSI in relation to the fatty acid composition of the live feed .......................................103 LSI in relation to the fatty acid composition of the crab larvae..................................103 Metamorphosis success in relation to the fatty acid composition of the live feed and the crab larvae...................................................................................................................103
4. Discussion........................................................................................................................104 4.1. Fatty acid composition of live feed and crab larvae ................................................104 4.2. Survival in the zoeal stages ......................................................................................104 4.3. Larval development rate during the zoeal stages .....................................................106 4.4. Metamorphosis .........................................................................................................109
Metamorphosis rate .....................................................................................................109 Timing and duration of metamorphosis.......................................................................110
4.5. Survival of Z1 to megalopa and the first crab (M/Z1 and C1/Z1 survival rates).....111 5. Conclusions and suggestions ...........................................................................................113 CHAPTER 6........................................................................................................................125 Improved larval rearing techniques for mud crab (Scylla paramamosain) .................125Abstract................................................................................................................................125 1. Introduction .....................................................................................................................126 2. Materials and methods.....................................................................................................127
2.1. Source of larvae........................................................................................................127 2.2. Food and feeding ......................................................................................................128
Micro-algae culture .....................................................................................................128 Rotifer culture and enrichment....................................................................................128 Artemia culture and enrichment..................................................................................128 Feeding ........................................................................................................................129
2.3. Larval rearing experiments: objectives and experimental design............................129 Experiment 1................................................................................................................130 Experiment 2................................................................................................................130 Experiment 3................................................................................................................131 Experiments 4, 5 and 6 ................................................................................................131 Experiments 7 and 8 ....................................................................................................131
Contents
iv
2.4. Evaluation criteria ................................................................................................... 132 2.5. Statistical analysis.................................................................................................... 132
3. Results ............................................................................................................................. 133 Experiment 1 ................................................................................................................... 133 Experiment 2 ................................................................................................................... 133 Experiment 3 ................................................................................................................... 133 Experiment 4 ................................................................................................................... 134 Experiment 5 ................................................................................................................... 134 Experiment 6 ................................................................................................................... 134 Experiment 7 ................................................................................................................... 135 Experiment 8 ................................................................................................................... 135
4. Discussion ....................................................................................................................... 135 4.1. Rearing system ......................................................................................................... 135
Recirculation ............................................................................................................... 135 Role of supplemented micro-algae.............................................................................. 136 Choice of system.......................................................................................................... 137
4.2. Other zootechnics..................................................................................................... 138 Z1 stocking density ...................................................................................................... 138 Rotifer density for feeding early larval stages (Z1-Z2 stages).................................... 138 Artemia for feeding later larval stages (from Z3 onwards) ........................................ 139 Prophylactic chemicals ............................................................................................... 141 Cannnibalism .............................................................................................................. 142
5. Conclusions and suggestions........................................................................................... 143 CHAPTER 7 ....................................................................................................................... 149 General discussion............................................................................................................. 149 1. Challenges and perspectives for mud crab larviculture .................................................. 149
Feasibility of mud crab larviculture compared to other crustaceans............................. 149 Challenges for mud crab larviculture ............................................................................. 150 Perspectives for larviculture of mud crabs ..................................................................... 151
2. Broodstock management (Chapter 3).............................................................................. 152 3. Optimal feeding for the larvae (Chapter 4) ..................................................................... 154
Rotifers and Artemia as suitable live feed ...................................................................... 154 Early zoeae of S. paramamosain might be more rotifer-dependent compared to other Scylla species .................................................................................................................. 154 Alternatives for rotifers ................................................................................................... 155
4. Live feed quality (Chapter 5) .......................................................................................... 156 Role of essential fatty acids in crustaceans..................................................................... 156 Effects of DHA, EPA and ARA on mud crab larvae ....................................................... 157
5. Rearing systems and other zootechnics (Chapter 6) ....................................................... 158 Selecting an efficient rearing system............................................................................... 158 Other zootechnics............................................................................................................ 161
6. Overall conclusions and suggestions for further research............................................... 162 References .......................................................................................................................... 165 Summary ............................................................................................................................ 179 Curriculum vitae ............................................................................................................... 189
CHAPTER 1
Introduction
1. World production and demand of high-value crustaceans
According to FAO (2002), reported global capture fisheries in 2000 returned to the
level of the early 1990s, reaching about 94.8 million tonnes. Unlike capture fisheries,
aquaculture production has continued to increase markedly (35.6 million tonnes) with an
annual growth rate ranging from 5.3 - 7.1 % over the two last decades. It is believed that
aquaculture potential still exists in many areas and for many species. More than half of
global aquaculture production originated from marine or brackish coastal waters. High-
value crustaceans and finfish predominate in brackish water, and molluscs and aquatic
plants in marine waters. Although brackish water production represented only 4.6 % of total
global aquaculture production by weight in 2000, it comprised 15.7 % of total production by
value.
Crustaceans are amongst the most highly valued foods, but of the 26,000 species of
crustaceans (Ruppert and Barnes, 1994) only penaeid shrimps, mitten crab (Eriocheir
sinensis), freshwater prawns (Macrobrachium spp.) and red swamp crawfish (Procambarus
clarkii) are being produced on an industrial scale (Wickins and Lee, 2002). The availability
of crustaceans per capita more than tripled from 0.4 to 1.4 kg (between 1961 and 1999),
largely because of the production of shrimps and prawns from aquaculture practices. Shrimp
is already the most traded seafood product internationally, and about 26 % of total
production now comes from aquaculture (1.1 million tonnes in 2000). Similarly, trade in
crab species has increased with growing aquaculture production (140,300 tonnes in 2000)
(FAO, 2002). Mud crabs (Scylla spp.) are considered luxury seafood items due to their large
size and delicate flavor, and therefore have high market value and are in great demand
(Dorairaj and Roy, 1996; Triño and Rodriguez, 2002). They are highly sought after in East
Asia (particularly in Hong Kong, Japan, Taiwan and Singapore), where live crabs
(especially gravid females) command premium prices (Agbayani, 2001; Keenan, 1999a).
There is also a growing market in the USA for frozen, soft-shelled mud crab and the
demand for mud crab meat for value added products is expanding internationally (Cholik,
1999; Keenan, 1999a; Tan, 1999; Wickins and Lee, 2002).
CHAPTER 1 – Introduction
2
Statistics on mud crab production are much more fragmentary and variable than for
shrimp because the species are often fished and cultured artisanally and therefore not
regularly reported. Depending on the source, reported mud crab production figures range
from a few hundred to several thousands tonnes, mainly from countries in the Indo-Pacific
region (Agbayani, 2001; Camacho and Aypa, 2001; Chaitanawisuti and Krittsanapuntu,
1998; Cholik, 1999; Fortes, 1999; Liong, 1992). The official global catch of mud crabs in
2001 was less than 17,000 tonnes (FAO, 2002). World capture and culture production of the
genus Scylla were 14,841 and 5,883 tonnes, respectively (FAO, 1998). Capture production
is predicted to decline because worldwide exploitation of mud crabs is aiming at all size-
classes and is increasing constantly (Le Vay, 2001; Le Vay et al., 2001). As a result,
declining crab landings and smaller maximum sizes have been reported over the last two
decades (Angell, 1992).
For the period up to 2030, gross trends project that, for developed countries,
consumption patterns will reflect demand for, and imports of, high-cost/high-value species;
and in developing countries, trade flows will reflect the exportation of high-cost/high-value
species and the importation of low-cost/low-value species (FAO, 2002).
As capture production of mud crabs has been declining and the demand for high-value
species in developed countries is predicted on the rise (FAO, 2002), culture of mud crabs is
expected to increase. Especially since (in contrast to other crustaceans and even other crab
species) aquaculture production to date only makes up a small proportion of the availability
of this high-cost/high-value species.
2. Production of high-value crustaceans in Vietnam
According to the most recent report of the Vietnamese Ministry of Fisheries (MOFI,
2003), between 1990 and 2002, capture fisheries and aquaculture doubled (709,000 -
1,434,800 tonnes) and respectively tripled (310,000 - 976,100 tonnes) their output, resulting
in a ten-fold increase in export value. Although world seafood consumption in 2002
remained high, the market is subjected to harsh competition that required Vietnamese
seafood exporters to struggle continuously to protect their tenth position on the world
market. In 2002, Vietnamese seafood export reached 459,000 tonnes, valued over 2 billion
US$. Export was was composed of four main groups, namely shrimp, fish, squid and others
CHAPTER 1 – Introduction 3
(bivalves, marine crabs, seaweed) valued proportionally 47, 22, 12 and 19 %. Proportions in
weight of these groups were 25, 16, 29 and 30 %, respectively.
During the period 1950 - 2000, marine crab production has increased sixteen-fold
(2,000 to 32,000 tonnes) in Vietnam (FAO, 2002). The highest production was recorded in
1998 (48,000 tonnes), which represents about 4 % of the highest reported global crab
production figure in 2000.
The Mekong Delta is the most important region in Vietnam for both capture fisheries
and aquaculture (including mud crabs), accounting for 43 and 67 % of the nation’s total
production, respectively and 57 % of the total export values in 2003 (Huy, 2004). Central
Vietnam is less suited for mud-crab farming because it lacks extensive pond areas, which
are plentiful in the two large delta regions of the Red River (North Vietnam) and Mekong
River (South Vietnam). Culture potential in North Vietnam however, is limited due to its
temperate climate.
Data on mud crab production in Vietnam are also variable and not trustworthily
reported for the same reasons (artisanal fishing and culture) as for the rest of the world. In
1993, reported culture production of Scylla was 3,800 tonnes (Dau, 1998). The total mud
crab production in 1995 was estimated at 4,500 to 5,500 tonnes (IFEP, 1996). A 1995
survey of the 6 eastern coastal provinces in the Mekong Delta estimated a total mud crab
production in that region of about 1,644 tonnes (Tuan et al., 1996). In 1999, the mud crab
production in Ca Mau province (the largest fisheries producer accounting for 7.8 % of the
country total) amounted to 5,000 tonnes, of which 20 % came from farming (Xuan, 2001). It
is therefore seems, mud crab production in Vietnam has followed the trend of increasing
global production and trade.
3. Mud crab culture
Mud crabs are attractive candidates for culture because in their post-larval stages they
are hardy to fluctuations in water quality and temperature, relatively resistant to disease and
grow quickly on a wide variety of diets (Williams and Primavera, 2001).
Scylla spp. feed on a variety of food items. Juveniles, which are more mobile, feed on
prawns, smaller crabs, fish and other small invertebrates (Joel and Sanjeevaraj, 1986).
Larger crabs are primarily carnivorous, eating benthic molluscs and crustaceans (Hill 1979;
Joel and Sanjeevaraj, 1986; Paterson and Whitfield, 1997), but are also opportunistic
CHAPTER 1 – Introduction
4
omnivores (Warner, 1977) and will eat a wide variety of animal protein and even vegetable
matter such as submerged aquatic weeds (Hill, 1979), filamentous algae (Williams and
Primavera, 2001), detritus (Hill, 1979) and cooked maize (Rodríguez et al., 2003).
Cholik (1999) classified three types of mud crab culture in ponds based on the final
product, i.e. grow-out from juvenile to consumption size, fattening and gravid female
production. Triño and Rodríguez (2002) reported pen culture of mud crab in tidal flats with
mangrove trees is a newly profitable culture model. In its simplest form, mud crabs are
cultured by fishers in order to add value to poorer quality crabs. Newly-moulted mud crabs
have flaccid, watery flesh and thus a low market value and females with immature ovaries
are worth less than gravid females. Fishers stock “empty” or “thin” crabs and immature
females in bamboo pots, cages or penned enclosures where they are fed trash fish and other
“waste” material for 15 - 40 days until fattened (300 - 800 g) or ripe (in the case of females)
and thus more valuable in the marketplace (Cholik and Hanafi, 1992; Dat, 1999a; Tan
1999). This is highly profitable and has evolved into more sophisticated farming where wild
caught juveniles are bought from fishers and stocked into ponds or enclosures and cultured
until they reach market size (Chong, 1993; Tan, 1999).
In Vietnam, mud-crab farming occurs along the entire coast, especially in areas where
there is abundance of wild populations as a source of seed stock (i.e. in mangrove areas)
(Dat, 1999a). Felix et al. (1995) described the various crab culture types practiced in
Vietnam (“moulting”, “soft-shell” and “mature” female crab culture). Keenan (1999a)
indicated that in the Mekong Delta, one of the culture models which produces very high
numbers of crab is the extensive culture in mixed mangrove aquasilviculture systems. With
this model, no supplemental feed is added and the crabs forage across the forest floor for
natural food. Tung (1995) reported that mud crab culture can deliver high profits within a
couple of months, with limited risk of disease and low inputs compared to shrimp culture.
4. Reclassification of mud crab species
In the original descriptions, mud crabs were identified as belonging to the genus
Scylla, but made up of different species (Keenan, 1999b). By observing color and
morphological features (color of the carapace, polygonal pigmented patterns, the
anterolateral teeth of the carapace, the “H” mark on the carapace, the length of chelipeds,
and size attained), the mud crabs in the Philippines were classified into three species and
CHAPTER 1 – Introduction 5
one variety, i.e. S. olivacea, S. tranquebarica, S. serrata and S. serrata var. paramamosain
(Estampador, 1949; cited by Keenan, 1999b). This classification was supported by Serene
(1952; cited by Keenan, 1999b) based on a study which examined spination and color of
Scylla populations in Nha Trang, Central Vietnam.
Recently, new samples (from near Hong Kong, the Mekong Delta, Vietnam and near
Semarang, Central Java, Indonesia) were found to be closely interrelated, but distinctly
different based on morphology, DNA sequencing and allozyme electrophoresis data from
the other three species, indicating they all belonged to a fourth species of Scylla, S.
paramamosain. Furthermore, the absence of heterozygotes (i.e. hybrids) of the different
species provides strong evidence that there is no genetic exchange between them (Keenan,
1999b).
In Vietnam two types of mud crabs have been distinguished: the large-sized or
greenish crab (S. paramamosain) and the small-sized or reddish crab (S. olivacea). The
former has the widest distribution and is preferred for culture and consumption. Logically,
the large-sized species (S. paramamosain) has been selected as a priority for research.
The reclassification into four species (Keenan et al., 1998; Keenan, 1999b) also
imposed the need to review all previous publication carefully and created the need to
differentiate research for all four species within the genus as there could be significant
species-dependant differences in requirements (e.g. environmental and nutritional
requirements).
Little research has been conducted on S. paramamosain (Dat, 1999b and Nghia et al.,
2001b in Vietnam; Djunaidah et al., 2001a in Indonesia; Zeng and Li, 1999 in China)
compared to S. serrata most probably because the latter species has a much wider
distribution (Davis, 2003; Keenan et al., 1998; Le Vay, 2001). Most research centers dealing
with mud crab are also located in the regions where S. serrata is the dominating species
(Baylon and Failaman, 1999 and Quinitio et al., 1999 in the Philippines; Brick, 1974 in
Hawaii, USA; Davis et al., 2001 and Davis, 2003 in South Africa; Mann et al., 1999b and
Williams et al., 1998 in Australia). In addition, the larger size and the higher market value
of S. serrata (Carpenter and Niem, 1998) compared to other Scylla species, have fascinated
the culturists and therefore was the preferred species (even in areas where several species
are co-existing like in the Philippines.
CHAPTER 1 – Introduction
6
5. Importance of mud crab larviculture in Vietnam
During the past two decades, there has been a rapid expansion of traditional shrimp
culture in the Mekong Delta, Vietnam. Unfortunately, this expansion has been at the
expense of mangrove clearance at a rate of approximately 5,000 ha year-1 to less than half
their original 280,000 ha in 1990 (Hong and San, 1993).
Disease outbreaks in 1993 - 1994 led to a dramatic decline in shrimp yields, with farm
incomes falling to 10 % of the previous year (Johnston and Keenan, 1999). This was the
first time that white spot syndrome virus (WSSV) appeared in Vietnam. There was a need
for diversification of cultured species besides shrimp. Mud crab has been the first priority
because it is the only other species that has been cultured traditionally in the coastal area
besides shrimp. Expansion of mud crab culture requires a reliable supply of quality seed
stock. The availability of wild seed has however considerably declined due to the over-
fishing of natural resources at all size classes (Le Vay, 2001; Le Vay et al., 2001) and the
destruction of mangrove forests being the natural nursery for many marine species including
mud crabs. When the research described in this thesis started, artificial seed production in
hatcheries had not commenced.
In conclusion, the need for development of mud crab larviculture in Vietnam is
justified for the following reasons: (i) mud crab is a high-value species with an increasing
demand on global markets, (ii) mud crab farming is a readily applicable alternative for
shrimp culture which is suffering serious disease problems, (iii) hatchery technology is
necessary to overcome the shortage of wild seed due to overfishing and habitat destruction,
(iv) mudcrab is a hardy species that can be cultured using simple and traditional practices
requiring low initial investment but generating considerable profit and (v) Scylla
paramamosain can be considered a “new species”, especially after the reclassification of the
genus Scylla, for which only very limited information exists.
The first research on mud crab larviculture in Vietnam dates back to 1993 - 1995, but
it was only several years later that the results of this study was published (Dat, 1999b).
However, back then, Vietnamese researchers were too much isolated from their
international colleagues working on the same species, and could not benefit from the
knowledge exchanged at the first specialized workshop on larval rearing of Scylla in Broom,
Australia in 1995.
CHAPTER 1 – Introduction 7
The first experiments for this study started in 1996 thanks to the financial support
from the International Foundation for Science (IFS). After some preliminary success under
the IFS grant, we had the opportunity to join in an EU sponsored INCO research project
with partners from Belgium, Indonesia and the UK aiming to optimise and standardise
larviculture of S. paramamosain for stock enhancement in the Mekong Delta. With the
support of the Laboratory of Aquaculture & Artemia Reference Center (Ghent University,
Belgium), through this project, our knowledge of crustacean larviculture has improved and
research efforts strengthened. Research findings of this programme were presented at the
international workshop on “Mud crab rearing, ecology and fisheries” held in Can Tho at the
beginning of 2001 (see www.dec.ctu.edu.vn/sardi/AacrabCWare/index.htm). This event
strengthened the network of mud crab researchers and even extended it with new partners
from Australia, China, Thailand and the Philippines.
6. Aims and outline of the thesis
The aim of this thesis is to develop technologies that could contribute to the
standardization and optimization of the larviculture of Scylla paramamosain in Vietnam.
Nutrition, zootechnics and disease control are the three main areas of research, which
have led to commercial hatchery production of marine fish and crustacean larvae (Sorgeloos
and Léger, 1992). The three aspects are interconnected to some extent and developing
hatchery technology for new species is not possible unless all three are addressed.
There has been a great deal of progress in marine larval rearing technology ever since
the pioneering years of the 1960’s (Howell et al., 1998; Shelbourne, 1964). Many of the
more technical aspects developed in the past can be directly applied to new species. Only
minor modifications are usually required to achieve acceptable survival rates, but if mass
mortality persists, more specific research becomes necessary.
Crab larvae need a proper environment to feed and develop. In culture conditions, that
environment is the rearing tank, filled with high quality water and conditioned by a number
of controlling factors (e.g. aeration, filtration, prophylactic chemicals …). All these together
make up the culture zootechnics. When the medium deteriorates by some causes (e.g.
insufficient water exchange, unsuitable feed, overfeeding, too high larval densities …) then
the risk of disease appears. Therefore, nutrition and zootechnics need to be considered at the
same time and are the research fields that have to be addressed first. Next but not less
CHAPTER 1 – Introduction
8
important are disease studies, especially on the interference of bacteria on larval
performance. However this study requires more specialised equipment and staff and was
beyond the scope of this thesis. For these reasons, the content of this Ph.D. thesis is limited
to the main aspects of nutrition and zootechnics. The thesis outline can be summarized as
follows:
- CHAPTER 1 (Introduction, this chapter) outlines the need for and importance of mud crab
larviculture in Vietnam based on figures of global and local production of Scylla crabs in
relation to the total production of high-value seafood (shrimp and other crab groups). The
specific circumstances in Vietnam, particularly in the Mekong Delta are also considered to
verify the need for artificial reproduction of Scylla paramamosain.
- CHAPTER 2 (Current status of mud crab Scylla spp. hatchery technology) reviews the
status of mud crab larviculture. A review of the literature is normally presented as an
introduction to the subject of a thesis. But because mud crab aquaculture is still an emerging
industry, particularly the hatchery phase of production, very little peer reviewed literature
exists. Research projects have been conducted in Australia, China, India, Indonesia, Japan,
Malaysia, the Philippines and Vietnam over the past decade, but most of this information
has remained in-house, was not published in English or has appeared as papers or abstracts
in conference proceedings. Because a more empirical approach is commonly taken while
establishing new hatchery technology, the most pertinent information is only available as
personal observations and unpublished results collected by the various scientists active in
the field. This was also the case with our project and the complementary Vl.I.R. Own
Initiative project between Rhodes University (South Africa) and the Laboratory of
Aquaculture. At the initiative of Prof. Patrick Sorgeloos and after consultation of scientists
from Australia, Belgium, the Philippines and the UK, it was decided to prepare a
collaborative paper which would describe the “state of the art” of mud crab larviculture
technology.
- CHAPTER 3 (Reproductive performance of captive mud crab Scylla paramamosain
broodstock in Vietnam) covers the effect of various culture parameters on some
reproductive characteristics. Broodstock availability and management are the first concerns
for those who wish to develop a new species for aquaculture. Environmental parameters,
CHAPTER 1 – Introduction 9
broodstock management conditions and reproductive characteristics were recorded as a
basis for improving broodstock culture with the ultimate purpose to fully domesticate and
control seed production for this species.
- CHAPTER 4 (Optimal feeding schedule for mud crab Scylla paramamosain larvae)
identifies the most suitable prey type and size (rotifers or Artemia) for the early stages of
mud crabs because initial feed for larvae is a critical research aspect that has to be solved
first. The earliest appropriate time to shift from rotifers to Artemia was proposed since
rotifer culture is very laborious and not well mastered by most hatchery managers in
Vietnam. The possibilities are reviewed to replace rotifers in early stages by various forms
of processed Artemia awaiting the availability of suitable micro-bound diets for mud crab.
- CHAPTER 5 (Influence of the content of highly unsaturated fatty acids in the live feed on
larviculture success of mud crab Scylla paramamosain) describes the effects of different
standard HUFA (highly unsaturated fatty acid) live feed enrichment emulsions on survival
and growth of crab larvae. In captivity, rotifers and Artemia nauplii support growth and
survival of mud crab larvae, but this simplified diet is not ideal. As for other species, the
phenomenon of moult death syndrome (MDS), i.e. high mortality during or after the moult
from Z5 to megalopa, has been observed. Poor nutrition, even if confined to the early larval
stages, has been suggested as a cause for MDS. Inferior nutrition may also be a factor
contributing to the highly variable survival and the high susceptibility to disease often
recorded in mud crab larviculture.
- CHAPTER 6 (Improved larval rearing techniques for mud crab Scylla paramamosain)
compares six different rearing systems varying in the level of micro-algae supplementation
and the form of water exchange. In areas where clean and full strength seawater is limited,
as in the Mekong Delta, the combination of “green-water” and recirculation is
recommended. Other important zootechnics (zoea 1 stocking density, live feed density,
prophylactic chemicals and cannibalism) were also discussed.
- CHAPTER 7 (General discussion) reviews the results of the experiments and observations
presented in the technical chapters (Chapters 3 - 6) in order to value achievements and
pinpoint unsolved issues of this study. Based on the general discussion, some suggestions
for further research are proposed.
CHAPTER 2 Current status of mud crab (Scylla spp.) hatchery
technology
Davis, J.A.*1,2, Nghia, T.T.2,3, Wille, M.2, Mann, D.4, Quinitio, E.T.5, Williams, G.6, Fushimi, H.7, Hecht, T.1, Shelley, C.6, Churchill, G.J.1 and Sorgeloos, P.2
1 Department of Ichthyology and Fisheries Science, Rhodes University, South Africa. 2 Laboratory of Aquaculture & Artemia Reference Center, Ghent University, Ghent, Belgium. 3 College of Aquaculture and Fisheries, Can Tho University, Can Tho City, Vietnam. 4 Bribie Island Aquaculture Research Center, Bribie Island, Queensland, Australia. 5 Aquaculture Department Southeast Asian Fisheries Development Center, Tigbauan, Iloilo, the Philippines. 6 Darwin Aquaculture Center, Department of Primary Industry and Fisheries, Channel Island, Darwin, Australia. 7 Laboratory of Aquaculture and Stock Enhancement, Department of Marine Biotechnology, Fukuyama University, Ohama, Innoshima, Hiroshima, Japan. Abstract
A major bottleneck to the expansion of mud crab (Scylla spp.) aquaculture is a lack of
hatchery produced seed. Although research on egg production and larval rearing techniques has been undertaken for the past 30 years (and intensively for the last decade), little information is available in the primary literature. Most of the technical information has been published in the “grey” literature and in research reports and is largely unavailable to the wider scientific community. This paper attempts to collate and summarize the published and unpublished data together with observations made, and techniques used by scientists currently involved in mud crab hatchery research. The paper describes techniques for broodstock sourcing, maturation, spawning, egg incubation and hatching. Larval rearing techniques are outlined with regard to stocking, water quality requirements, culture systems, feeding techniques and nutrition. Rearing of megalopae to the juvenile stage is also described. Bottlenecks to commercial production are discussed and possible solutions proposed.
Manuscript submitted to Aquaculture
CHAPTER 2 – Current status
12
1. Introduction
The four species of Scylla; S. serrata, S. tranquebarica, S. paramamosain and S.
olivacea are large, primarily carnivorous portunid crabs that are found in coastal and
estuarine waters throughout the tropical and subtropical Indo-Pacific region (Keenan et al.,
1998). Growth is rapid and the life cycle can be closed within a year. The post-larval stages
are highly resistant to disease and can tolerate a wide range of environmental conditions.
Mud crabs in all their marketed forms have a high value (Agbayani, 2001; Wickins and Lee,
2002) and are suitable for aquaculture (Williams and Primavera, 2001). Currently mud crab
aquaculture is reliant on juveniles or adults caught from the wild (Le Vay, 2001). Despite a
long history of culture, records of which date back to 1890 (Shen and Lai, 1994), world
production is low. In 2001 slightly more than 10,000 tonnes were produced (FAO, 2002).
The expansion of the industry is currently limited by the lack of hatchery-produced seed
(Williams and Primavera, 2001).
Research on the breeding of mud crabs started approximately 40 years ago and several
early publications describe captive spawning and larval rearing (Brick, 1974; Du Plessis,
1971; Hill, 1974; Ong, 1964; Ong, 1966). In Japan, research on hatchery production of seed
for restocking purposes has been ongoing since 1979 (Fukunaga and Uzumaki, 1982;
Hamasaki, 2002; Hasegawa, 1989; Horiuti and Yamamoto, 1987). In the last decade there
has been a substantial increase in research, fueled in part by the disease problems in the
shrimp industry and partly by declining mud crab fisheries in some parts of South East Asia
(Le Vay, 2001). The results of this research have been presented in workshops and forums
in Western Australia (1995), Northern Territories, Australia (1997), the Philippines (1998)
and in Vietnam (2001), amongst others. Although some of the results have been published
in the primary literature the majority of information on mud crab hatchery technology and
aquaculture appears in the “grey” literature and in unpublished research reports. This paper
reviews the available literature from all sources, summarizes the current hatchery and
rearing techniques, identifies research problems and provides comment on current research
trends. This will inform those currently involved, or those wishing to become involved in
mud crab hatchery production.
CHAPTER 2 – Current status
13
2. Broodstock
Sourcing and maturation
Broodstock are most commonly sourced from the wild. They are either caught
specifically for this purpose (Mann et al., 1999a), are bought from markets (Dat, 1999a;
Quinitio et al., 2001) or directly from fishers (Dat, 1999a; Marichamy and Rajapackiam,
2001). In areas where commercial culture is practiced, mature breeders are sourced from
production ponds (Millamena and Quinitio, 2000). The life cycle can be closed within 9
months (Quinitio et al., 2001).
Mud crabs readily mate in captivity. This occurs while the female is in a soft-shell
condition following molting. However, adult females caught from the wild have usually
mated (Robertson and Kruger, 1994) and carry spermatophores for extended periods,
producing viable eggs up to 6 months after capture without the need for mating (Nghia et
al., 2001a).
Before introducing wild broodstock into the hatchery, the crabs are commonly
scrubbed to remove mud, encrusting algae, infestations and detritus. Individual crabs are
usually marked for identification by engraving (Churchill, 2003; Djunaidah et al., 2003;
Millamena and Quinitio, 2000) or by gluing an identification tag to the shell. They are then
disinfected in a strong formalin bath (50 - 100 µl l-1) for periods ranging from 1 hour
(Millamena and Bangcaya, 2001) to overnight (Mann et al., 1999b). Shell disease is
commonly observed, especially amongst older crabs that have long intermoult periods
(Robertson, 1987). The disease seldom causes mortality and regular cleaning of the shell
can minimize its effects (Lavilla-Pitogo et al., 2001). Various parasites have also been
reported, none of which present serious threats to broodstock or egg production (Lavilla-
Pitogo et al., 2001).
Neither the size nor type of vessel seems to affect maturation or spawning (egg
extrusion). Brood crabs have been successfully maintained individually in small bins (60 -
300 l) or communally in large tanks ranging from 1 - 12 m3 (Baylon et al., 2001a; Dat,
1999b; Hamasaki, 2002; Mann et al., 1999a; Millamena and Quinitio, 2000; Williams et al.,
1998). Successful spawning has been achieved at water depths of 25 - 150 cm (Dat, 1999b;
Djunaidah et al., 2001b; Millamena and Bangcaya, 2001). The average stocking densities in
maturation systems is approximately 1.5 crabs m-2 (Mann et al., 1999a, 1999b) but
CHAPTER 2 – Current status
14
broodstock can be stocked up to 5 m-2 (Djunaidah et al., 2001b). Broodstock are often kept
in separate tanks or cages which eliminate the risk of cannibalism and allows for controlled
feeding.
Shelters are usually provided to reduce stress and prevent cannibalism (Djunaidah et
al., 2003; Hamasaki, 2002; Millamena and Quinitio, 2000; Millamena and Bangcaya, 2001).
Shelters should be easy to remove so that brood crabs can be observed and the tank cleaned.
Mud crabs require a substratum for spawning. The crab digs a depression in the
substratum over which the abdominal flap is extended and into which the eggs are extruded
(Rusdi et al., 1994). The pleopods then create a current that assists the attachment of eggs to
the tertiary setae. Absence of a substratum results in poor egg attachment and potential loss
of the batch. Spawning of S. paramamosain is improved by providing a mud substratum
rather than sand (Djunaidah et al., 2001b). It has been observed that the Zoothamnium spp.
load on the eggs is reduced when the crabs spawn on a mud substrate, which may be a
consequence of turbidity (Nguyen Co Thach, Research Institute of Aquaculture III, Nha
Trang, Vietnam, pers. comm.). However, sand substratum is more commonly used since it
does not affect water quality and allows for the use of under-gravel filters (Churchill, 2003).
The entire bottom of the tank can be covered with spawning substratum (Williams et al.,
1998; Millamena and Quinitio, 2000). However, it is more common to contain the
substratum in a tray (Mann et al., 1999a) allowing the rest of the tank to be cleaned more
easily. Substratum depths of 50, 80 and 200 mm have been used successfully (Baylon et al.,
2001a; Djunaidah et al., 2001b; Millamena and Bangcaya, 2001).
Females also mature their ovaries and extrude eggs in earthen ponds or netted pens.
There is some indication in Vietnam that a higher percentage of crabs spawn in ponds,
possibly due to reduced stress. The diet available to broodstock in ponds is more varied and
probably closer to the one they would encounter in the wild. However, managing
broodstock in ponds is more difficult than in tanks and the eggs from broodstock spawned in
ponds are occasionally heavily infested with parasites (Quinitio and Parado-Estepa, 2003).
In smaller broodstock maturation systems the water is recirculated, while some larger
systems operate on a flow-through basis (Hamasaki, 2002; Marichamy and Rajapackiam,
2001; Millamena and Bangcaya, 2001; Quinitio et al., 2001) or on partial re-circulation
(Baylon et al., 2001a; Mann et al., 1999a; 1999b; Millamena and Quinitio, 2000; Millamena
and Bangcaya, 2001, Williams et al., 1998).
CHAPTER 2 – Current status
15
Full strength seawater (30 - 35 g l-1) is normally used for captive maturation and
spawning of S. serrata in the hatchery (Mann et al., 1999a). Lower salinities (20 - 25 g l-1)
can be used for S. tranquebarica, S. olivacea and S. paramamosain (Ng, 1998). However,
full strength seawater is still commonly used for maturing and spawning all four species.
Broodstock are generally kept between 25 °C and 35 °C. A lower critical temperature
for egg development of 18 °C has been identified (Hamasaki, 2002; Heasman and Fielder,
1983) but successful ovulation and spawning have been observed at water temperatures
ranging from 20 - 30 °C (Davis et al., 2003; Mann et al., 1999a; Millamena and Bangcaya,
2001). S. serrata broodstock extrude significantly larger eggs with higher hatch rates during
cooler months (18 - 22 °C) Mann et al. (1999a) suggesting that lower temperatures than
normal should be applied.
Mud crabs are nocturnal and normally inhabit turbid estuaries (Dat, 1999a; Barnes et
al., 2002; Hill, 1978). For this reason broodstock crabs are maintained under low light
conditions (Mann et al., 1999a; Quinitio et al., 2001; Williams et al., 1998), although
darkness is not required for spawning (Nghia et al., 2001a). Spawning tanks are normally
securely covered in order to prevent the crabs from escaping.
Broodstock are normally fed fresh or frozen foods including penaeid shrimp, squid,
fish, bivalves such as clams and mussel, gastropod snails and annelid worms (Dat, 1999a;
Hamasaki, 2002; Mann et al., 1999a; Millamena and Bangcaya, 2001; Quinitio et al., 2001).
Enriched Artemia biomass has also been used as food, presented as agar bound pellets
(Djunaidah et al., 2003). The natural diet of mud crabs consists mostly of benthic molluscs
and crustaceans (Hill, 1979) and it is generally agreed that these organisms must be present
in the diet. The source and health status of feed organisms, particularly crustaceans, need to
be carefully assessed as they can act as vectors for disease as WSSV (Chen et al., 2000).
Formulated diets developed for penaeid broodstock have been fed to mud crab
broodstock to investigate the effects on fecundity and spawning. Best results have been
achieved when formulated feeds have been used as a supplement to fresh or frozen feeds
(Djunaidah et al., 2003; Millamena and Quinitio, 2000; Millamena and Bangcaya, 2001).
Formulated diets are currently not used extensively.
Broodstock is usually fed on an ad libitum basis (Mann et al., 1999a; Williams et al.,
1999b), but when rationed, crabs are fed at rates ranging from 3 - 5 % (Dat, 1999b), 6 - 10
% (Millamena and Bangcaya, 2001) to 20 % body weight day-1 (Baylon and Failaman,
2001). Owing to their nocturnal feeding habits (Barnes et al., 2002; Hill, 1978) food is
CHAPTER 2 – Current status
16
normally provided in the evening, but feeding twice (Dat, 1999a) or three times daily
(Millamena and Bangcaya, 2001) allows for more careful monitoring of food consumption
and better maintenance of water quality.
Li et al. (1999) highlighted the importance of n-6 and n-3 highly unsaturated fatty
acids (HUFA) in broodstock diets. Levels of HUFA and protein in newly-hatched larvae can
be manipulated by altering the levels in the diet fed to the broodstock (Djunaidah et al.,
2003). No studies have yet been undertaken to test the effect of HUFA levels and ratios in
broodstock diets on egg quality or larval performance.
Spawning (egg extrusion)
Mud crabs adapt well to artificial conditions and spawn readily in captivity without
intervention. Over 85 % of wild caught crabs brought into the laboratory or hatchery
normally spawn, mostly within 40 days of stocking (Davis et al., 2003; Mann et al., 1999a;
Williams et al., 1998). The status of the ovary can be assessed by biopsy (Mann et al.,
1999a; Millamena and Bangcaya, 2001) or by pushing down on the first abdominal segment
which exposes the ovary under the carapace (Djunaidah et al., 2003; Quinitio and Parado-
Estepa, 2003). Mature ovaries are bright yellow to deep orange in colour and fill the body
cavity (Quinitio and Parado-Estepa, 2003).
Natural spawning is the norm in most hatcheries. However, eyestalk ablation can be
used to shorten the pre-spawning period. It is only necessary to ablate one of the eyestalks
(Baylon and Failaman, 1997; Baylon and Failaman, 2001; Baylon et al., 2001a; Dat, 1999b;
Djunaidah et al., 1998; Mann et al., 1999a; Marichamy and Rajapackiam, 2001; Millamena
and Quinitio, 2000; Nghia et al., 2001a; Quinitio et al., 2001). Prior to eyestalk ablation,
brood crabs are anaesthetized in an aerated chloroform bath (1 - 3 µl l-1). Hot pincers are
used to crimp the eyestalk and cauterize the wound. Ablation does not seem to stress the
crabs as there is no reduction in survival, fecundity or fertilization rate (Millamena and
Bangcaya, 2001; Nghia et al., 2001a). However, there is little and conflicting information on
the effect of ablation on egg and larval quality (Millamena and Quinitio, 2000). Mann et al.
(1999a) reported that ablated crabs produced larger eggs with better hatch rates while
Millamena and Bangcaya (2001) recorded lower fertilization and hatch rates of eggs from
ablated females.
CHAPTER 2 – Current status
17
Captive mud crabs produce viable eggs year round. Some authors have reported
seasonal changes and peaks in spawning activity (Davis et al., 2003; Hamasaki 2002; Mann
et al., 1999a; Marichamy and Rajapackiam 2001; Nghia et al., 2001a) especially in areas
that have extremes in seasonal water temperatures or salinity (Li et al., 1999; Le Vay, 2001).
However, these are usually not distinct and spawning patterns seem to vary between
locations (Hai et al., 2001; Nghia et al., 2001a). Mud crabs are capable of spawning up to
three times from a single mating (Dat 1999a; Marichamy and Rajapackiam, 2001; Quinitio
et al., 2001). Rematuration of the ovary in captive crabs occurs about one month after
spawning (Hai et al., 2001; Djunaidah et al., 2003; Marichamy and Rajapackiam, 2001).
Although some authors report no reduction in fecundity or fertilization rate with repeated
spawning (Millamena and Quinitio, 2000), Dat (1999b) recorded a substantial decrease in
egg production. The effect of repeated spawning on egg and larval quality has not been
quantified. Female S. serrata are capable of more than one post pubertal moult (Robertson
and Kruger, 1994) and spermatophores are retained after molting.
Scylla are highly fecund and typically produce more than 1 million eggs batch-1. Egg
production ranges widely between species, locations and individual crabs. S. serrata seems
to be more fecund than other Scylla species (Dat, 1999a; Djunaidah et al., 2001b; Hai et al.,
2001; Jyamanna and Jinadasa, 1993; Mann et al., 1999a; Marichamy and Rajapackiam,
1992; Millamena and Bangcaya, 2001; Quinitio et al., 2001; Srinivasagam et al., 2000;
Zainodin, 1992). Batches of 50,000 are considered small and the largest batch recorded in
captivity is 10 million eggs (Davis et al., 2003). Egg number is not a limiting factor to
juvenile production. Broodstock matured in captivity are more fecund and produce eggs
with a higher rate of fertilization than wild caught crabs. This is apparently due to enhanced
environmental conditions and nutrition (Millamena and Bangcaya, 2001; Quinitio et al.,
2001). Broodstock domestication is a high priority area of research.
Incubation and hatching
Brood crabs which have spawned are conspicuous. The large, bright yellow to orange
egg mass (“sponge” or “berry”) is carried prominently under the abdominal flap. Berried
brood crabs are transferred to incubators as soon as possible after spawning. Flat bottomed
glass aquaria of 60 1, plastic containers of 100 l or larger fiberglass tanks of approximately
CHAPTER 2 – Current status
18
300 - 500 l (Hamasaki, 2002; Mann et al., 1999a; Millamena and Quinitio, 2000; Millamena
and Bangcaya, 2001) and 1000 l (Williams et al., 1998) have been used as incubators.
Water in the incubators is usually sterilized with UV light and exchanged either on a
flow-through basis or recirculated through biofilters (Hamasaki, 2002; Mann et al., 1999a;
Williams et al., 1998). Because of the need for a stable environment during incubation,
recirculation is preferred. To maintain water quality and to prevent contamination of the
eggs, berried crabs are not fed. Mild aeration is provided and incubators are siphoned clean
daily (Hamasaki, 2002; Mann et al., 1999b).
Poor attachment of the eggs can result in the gradual loss of a brood during
incubation. The causes of poorly attached eggs are not well understood, but may include
poor initial attachment of the eggs, poor egg quality, or parasitic and fungal infections (Hai
et al., 2001; Quinitio et al., 2001). When eggs are lost during incubation, the hatch rate of
the remaining eggs is generally low (Dat, 1999b). Where space is limiting, brood crabs with
poorly attached eggs are removed to make space for crabs carrying well attached eggs
(Churchill, 2003; Dat 1999b; Davis et al., 2003). Dropped eggs can be incubated. However
hatch rates are highly variable and fungal infections and eggs adhering to the sides of
containers has so far made this practice impractical (Hai et al., 2001; Churchill, 2003).
The eggs can be infected with a variety of parasitic worms, fungus (Haliphthoros,
Sirolpidium, Atkinsiella and Lagenidium spp.) and ciliates (Zoothamnium spp.) (Churchill,
2003; Hamasaki and Hatai, 1993a). Parasitic infections generally occur if water quality is
not adequately maintained and may result in poor embryogenesis or egg loss as a result of
brood crabs tearing at the egg mass. The vulnerability of eggs to fungal infection decreases
as their development progresses (Hamasaki and Hatai, 1993a). Berried brood crabs can be
bathed in formalin and/or malachite green as a prophylactic, or can be treated if infestations
occur. There is evidence that formalin (25 µl l-1) is toxic to eggs up to one day after
spawning, although older eggs can tolerate high doses of both formalin (50 - 150 µl l-1) and
malachite green (50 µl l-1) (Churchill, 2003; Davis et al., 2003; Hamasaki and Hatai, 1993b;
Kaji et al., 1991). Heavy aeration is applied during the bathing process. Antifungal agents
such as Treflan®1 (44 % trifuralin) (0.05 - 0.1 µl l-1) or formalin (25 µl l-1) can be added to
the incubation water resulting in a reduced fungal load of the eggs and preventing
transmission of the fungus from the egg surface to the larvae at hatch (Kaji et al., 1991;
Quinitio et al., 2001). Preventative measures, such as maintaining a hygienic incubation
1 Mention of a branded product does not mean endorsement by the authors
CHAPTER 2 – Current status
19
environment and good water flow in the tank are also effective and are preferable to anti-
microbial chemicals.
At 27 - 28 °C first cleavage occurs 5 - 8 hours after extrusion (Quinitio and Parado-
Estepa, 2003). The developing embryo is visible under a dissecting microscope from day 4
after spawning (Djunaidah et al., 2003). Embryonic development can occur successfully
between 20 and 30 °C but temperatures above 26 °C are preferred (Li et al., 1999; Mann et
al., 1999a; Quinitio et al., 2001). The incubation period is strongly temperature dependent.
For S. serrata, the relationship is best represented by the equation y = 6,030 - 407 x + 7.2 x2
where y = incubation period and x = temperature (Churchill, 2003) Embryonic development
is infinitely protracted below 17 °C (Heasman and Fielder, 1983). Depending on
temperature, mud crab eggs usually hatch within 9 - 12 days after extrusion (Dat, 1999b;
Baylon and Failaman, 2001; Quinitio et al., 2001). Time of hatch can be predicted by
monitoring development of the embryo. Vigorous limb movement, a beating heart and well
developed eye spots with a purplish patch are all indications of imminent hatching (Figure
1). Hatching can also be predicted by measuring egg diameter, which increases during
incubation (Mann et al., 1999b; Nghia et al., 2001a). For S. serrata the relationship is best
represented by the polynomial equation y = 299 + 2 x + x2 where y = egg diameter (µm) and
x = incubation period in days (Churchill, 2003).
Hatching usually occurs in the morning (Hai et al., 2001; Hamasaki, 2002). Brood
crabs are often transferred to a separate tank 1 - 2 days prior to hatching (Hamasaki, 2002;
Mann et al., 1999a, 1999b). Hatching tanks tend to be larger than 500 l, providing sufficient
space for the hatched larvae to disperse and to delay deterioration of water quality. The
water in the hatching tank is usually full strength seawater (30 - 35 g l-1) that has been
filtered and treated to reduce the microbial load. A small number of larvae (100s to a few
1000s) normally hatch one day before the main hatch. Hatching is normally a spontaneous
and rapid event with 90 % of the larvae hatching within 10 minutes. The larvae hatch as pre-
zoeae, a non-swimming phase that typically moults into the first zoeal stage within 10
minutes post-hatch (Figure 1). The persistence of pre-zoeae after the normal initial moult
time has elapsed is used as an indicator of poor batch quality (Dat, 1999b; Hai et al., 2001,
Mann et al., 1999a).
CHAPTER 2 – Current status
20
3. Larval rearing
Selection and stocking
Once the eggs have hatched, the brood crab is removed from the hatching tank. Hatch
success is generally high, ranging from 80 - 90 %. Larvae are negatively buoyant and swim
vigorously in order to maintain their position in the water column. In the Philippines, larvae
are left in the hatching tank for approximately one hour after hatching, providing sufficient
time for “poorer quality” larvae to sink to the bottom so that “better quality” larvae at the
surface can be used for rearing (Quinitio and Parado-Estepa, 2003). The value of this
practice needs to be weighed against the fact that Z1 larvae are highly susceptible to fungal
infections both from a variety of sources including the egg envelope (Hamasaki and Hatai,
1993a) and in a typical hatching tank of 1 m3, bacterial numbers rise rapidly within an hour
of hatching (Mann et al., 1999b). This suggests that larvae be removed soon after hatching
(10 - 15 minutes). Larvae are sometimes rinsed with clean seawater before transferring them
to rearing tanks to reduce the bacterial load (Mann et al., 1999b). To transfer the larvae to
rearing tanks they are scooped from the water surface (Baylon and Failaman, 1997; Baylon
et al., 2001a; Djunaidah et al., 2003, Mann et al., 1999a). Nets cannot be used because the
long spines of the larvae become entangled in the mesh and are damaged. Larvae have been
reared at densities ranging from 10 - 200 larvae l-1 (Baylon and Failaman, 1999; Dat, 1999b;
Djunaidah et al., 1998; Quinitio et al., 1999; Quinitio et al., 2001; Williams et al., 1998)
although densities of 30 - 60 l-1 are more commonly used. No correlation has been found
between stocking density and survival although in the Philippines vibriosis has been
associated with stocking densities higher than 100 larvae l-1.
Water quality and parameters
The larvae require high quality water, free of potential pathogens, predators or
parasites. Seawater is commonly filtered to 1 µm and then either chlorinated overnight and
dechlorinated with sodium thiosulphate (Mann et al., 1999b; Parado-Estepa and Quinitio,
1998; Quinitio et al., 2001; Williams et al., 1998; Williams et al., 1999b;), ozonated and
then recirculated through a biofilter, inoculated with nitrifying bacteria and settled for
several days before use (Baylon and Failaman, 1999; Williams et al., 2002) and/or re-
CHAPTER 2 – Current status
21
filtered through activated carbon and sterilized with UV light (Dat, 1999b). Water in the
rearing vessels is also allowed to stabilize for several days before introduction of the larvae
(Mann et al., 1999b; Parado-Estepa and Quinitio, 1998) and background algae are
sometimes added for apparent antimicrobial qualities. Water pretreatment has been found to
significantly improve survival through to megalopa (M) (Mann, 1999b; Williams et al.,
2002).
Early stage S. serrata have a lower temperature tolerance of 12 °C (Hill, 1974).
Larvae have been successfully reared at 25 - 30 °C, but temperatures in the upper range (29
- 30 °C) shorten development time (Dat, 1999b; Li et al., 1999; Mann et al., 2001; Quinitio
et al., 1999; Quinitio et al., 2001). Larvae are extremely sensitive to abrupt changes in
temperature. Temperatures are maintained within 1 °C from hatch to harvest of megalopae.
Larvae (particularly during the early stages) are vulnerable to temperature gradients
generated by immersion heaters of the type used in shrimp hatcheries (Mann et al., 1999b).
If temperatures must be increased, low capacity aquarium heaters should be used or heating
can be applied indirectly by incubating rearing vessels in heated baths or with remote
heaters in the sump of recirculating systems. Heating is unnecessary in large-scale vessels
under tropical conditions, as long as diurnal temperature variation is not extreme.
At temperatures above 25 °C S. serrata Z1 have lower and upper 24 hour salinity
LC50s of 17.5 g l-1 (Hill, 1974) and 40 g l-1 respectively (Churchill, 2003). Scylla Z1 - Z4 are
usually reared in seawater ranging from 30 - 35 g l-1, depending on locality (Djunaidah et
al., 1998; Mann et al., 2001; Quinitio et al., 2001) although in Japan, S. tranquebarica are
reared at 25 g l-1 in order to control fungal infections.
Reducing the salinity at Z5 to 20 - 24 g l-1 for Z5 S. tranquebarica and 20 g l-1 for S.
olivacea significantly improves metamorphosis to megalopa (Baylon and Failaman, 2001;
Quinitio et al., 2001) and reducing salinity from 30 to 25 g l-1 has been found to trigger
metamorphosis of Z5 S. paramamosain (Dat, 1999b). Seawater is therefore gradually
diluted (1 - 2 g l-1 daily) at the end of the Z5 stage or at the beginning of the megalopa stage
for these three species. S. serrata does not seem to require reduced salinity at
metamorphosis and the larvae are commonly reared from hatch through to the first crab
stage (C1) in full strength seawater (Baylon et al., 2001b; Cowan, 1984; Mann et al., 2001).
Early (Z1 and Z2) larvae are strongly photopositive and light is often used to keep
early larvae close to the water surface. The larvae are visual predators, particularly in the
latter stages of development (Z3 - M), and high light intensities (1800 - 4000 lux) are
CHAPTER 2 – Current status
22
typically applied (Mann et al., 2001). Survival and development after the Z3 stage are
significantly compromised in the absence of light or under low (50 lux) light intensities.
Where possible, larvae are reared under natural light (Takeuchi et al., 2000; Williams et al.,
1998).
Larvae seem to require a dark phase (Djunaidah, et al., 1998), but no significant effect
on survival was recorded between 12 and 18 hour photoperiods (Nghia et al., 2001b).
Photoperiods of at least 12 hours are generally provided (Mann et al., 2001; Quinitio et al.,
2001).
S. serrata larvae can tolerate relatively high levels of nitrogenous waste. A 24 hour
LC50 of 39.7 ± 2.0 mg l-1 total ammonia nitrogen (TAN) at pH 8.2 was determined for S.
serrata Z1 in South Africa (Churchill, 2003), while in Australia a level of 62 mg l-1 was
determined (Ravi Fotedar, Aquatic Science Research Unit, Muresk Institute, Curtin
University, Western Australia, pers. comm.). The Australian study also determined a 24
hour LC50 for later instars of approximately 50 mg l-1. Larval growth rate was reduced by 5
% after 96 hours in comparison to a control (96 hour EC5) at 5 - 7 mg l-1 TAN. TAN is
generally maintained below 1 mg l-1 in hatchery runs. S. serrata Z1 have a 96 hour LC50 of
3 mg l-1 ammonia (NH3-N) (Quinitio and Parado-Estepa, 2001). All zoeal stages have a 96
hour LC50 of 80 mg l-1 nitrite (NO2-N) (Mary Lyn Seneriches-Abiera, Mindanao State
University, General Santos City, the Philippines, pers. comm.). Little research on ammonia
tolerance has been conducted for the larvae of the other species although S. paramamosain
zoeae survive concentrations of 5 mg l-1 NH3-N in recirculating systems in Vietnam.
No research has been conducted on the tolerance of Scylla larvae to extremes in pH
and oxygen concentration.
Culture systems
For highly controlled experimental conditions, the vessels used for larval rearing are
small, ranging from 100 ml to 5 l in capacity (Baylon and Failaman, 1999; Baylon et al.,
2001a; Mann et al., 2001; Quinitio et al., 1999; Williams et al., 1999a; Zeng, 1998; Zeng
and Li 1999). Highly predictable survival (60 - 90 % up to megalopa) can be achieved in
these systems, particularly in the presence of antibiotics.
Larger cylindro-conical, fiberglass tanks are used for investigating zootechnical
aspects or for nutritional studies. Under pilot and commercial scale conditions larvae are
CHAPTER 2 – Current status
23
reared in plastic, fiberglass or reinforced concrete tanks ranging from 1 to 200 m3 capacity
(Dat, 1999b; Fukunaga and Uzumaki, 1982; Hamasaki et al., 2002b; Millamena and
Bangcaya, 2001; Quinitio et al., 2001; Williams et al., 1999b). Tank colour does not appear
to be an important factor in mud crab larval rearing. A range of tank colours has been used
successfully.
Water is exchanged either on a constant flow-through basis, or by draining or
siphoning 50 - 85 % of the tank volume daily and replacing it with clean seawater, or by
recirculation through a biofilter (100 % every 2 - 3 hours) (Nghia et al., 2001b). Under
green-water culture conditions water is not exchanged for the first three days. Thereafter,
water exchange is slowly increased from 10 - 20 % day-1 for Z2 - Z3 to between 40 and 50
% day-1 at the end of the rearing cycle (Z4 - M) (Mann et al., 1999b; Quinitio et al., 2001).
In Japan a mesocosm system is used for culturing larvae in larger tanks (> 10 m3). The tanks
are partially filled with green-water at Z1 (20 - 25 % volume). The tank is filled with clean
seawater during the course of the Z2 - Z3 stages and during the Z4 and M stages water is
exchanged on flow-through basis (Hamasaki et al., 2002b).
Dead larvae and uneaten food that accumulate on the tank bottom are generally
siphoned out of rearing vessels daily (Baylon and Failaman, 2001; Quinitio et al., 2001) and
care must be taken to avoid siphoning out larvae which have sunk to the bottom of the
container. A biofilm develops on the sides of the tank during culture. Williams et al. (1998)
achieved significantly higher survival in 5-l bowls when the biofilm was removed daily.
Cleaning the biofilm from the tank sides can however release large amounts of bacterial
flock into the water column and cleaning must be done by careful vacuuming or the tank
must first be drained down before cleaning.
Larvae ingest micro-algae by chance when swallowing water. Although the presence
of micro-algae in the water prolongs the survival of Z1, they cannot moult to Z2 unless the
diet is supplemented with zooplankton (Brick, 1974). Several genera of microalgae
including Tetraselmis, Skeletonema, Chlorella, Nannochloropsis, Chaetoceros and
Isochrysis have been added to the rearing water during larval rearing at densities ranging
from 5 104 to 5 105 cells ml-1 (Dat, 1999b; Djunaidah, et al., 1998; Mann et al., 1999b; Mann
et al., 2001; Quinitio et al., 2001; Williams et al., 1999b; Zeng and Li, 1999) in order to
“condition” the water and to serve as food for rotifers and Artemia. Species with high
HUFA levels such as N. oculata and I. galbana are added in order to continually enrich
rotifers and Artemia. The effect of background algae on larval survival and growth is
CHAPTER 2 – Current status
24
however not clear. The current trend in mud crab larval rearing is to use algae at least during
the rotifer feeding stages and for megalopae.
In conclusion, it appears that the current hatchery systems around the world are more
a reflection of available facilities (e.g. tank sizes and live food production capacity) than
best practice and are indicative that a range of approaches to mud crab larviculture can be
successful. An ideal system for mud crab larval rearing has not yet been perfected.
Feeding and nutrition
Larvae are fed as soon as possible after transfer into the rearing vessels. High protease
activity in newly-hatched zoeae indicates their ability to digest food immediately after hatch
(Li et al., 1999). Delaying feeding for up to 24 hours after hatching has no significant effect
on survival (Lumasag and Quinitio, 1998). However the effects of such starvation on the
later stages are not known. Starving larvae for longer than 48 hours induces high mortality
despite resumption of normal feeding (Djunaidah et al., 2003; Lumasag and Quinitio, 1998).
The point of no return (PNR) for newly-hatched larvae has been estimated at 30 hours and
96 hours for S. paramamosain and S. serrata, respectively (Li et al., 1998; 1999). In an
earlier study Mann and Parlato (1995) found that S. serrata Z1 could survive for up to 142
hours without feeding but were not able to moult to Z2. Temperature significantly
influences the PNR. Newly-hatched S. serrata have a PNR50 of 57.6 hours at 28 ºC and 91
hours at 24 ºC (Lumasag and Quinitio, 1998).
Z1 and Z2 are usually fed on rotifers (Brachionus spp.) (Li et al., 1999; Mann et al.,
1999b; Takeuchi et al., 2000, Zeng and Li, 1999). Larvae fed rotifers benefit from the
supplementation of Artemia nauplii as early as Z1 but Z2 utilize Artemia more efficiently
(Li et al., 1998). If Artemia are withheld beyond Z3, growth and survival are compromised
(Li et al., 1998; Suprayudi et al., 2002a; Takeuchi et al., 2000; Zeng and Li 1999).
Although larvae can be reared on Artemia nauplii from hatch, survival is usually
enhanced by the addition of rotifers to the diet (Baylon and Failaman, 1999; Ong, 1966).
Larvae are commonly reared on rotifers during Z1 and Z2 while Artemia are usually
introduced at Z3 (Li et al., 1999; Mann et al., 1999b; Nghia et al., 2001b; Takeuchi et al.,
2000; Quinitio et al., 2001). However, in large systems (10 - 100 m3 tanks) that cannot be
flushed regularly, feeding with Artemia is delayed to Z4 (Hamasaki et al., 2002b). Although
Artemia are typically provided as newly-hatched nauplii, Z3 are large enough to consume
CHAPTER 2 – Current status
25
metanauplii, allowing for the delivery of supplementary nutrients via bio-encapsulation (see
below). On-grown Artemia (5 days old to adult) provide a larger sized prey item and are fed
to Z5 and megalopae (Mann et al., 1999b; Quinitio and Parado-Estepa, 2003).
Z1 are functionally passive feeders relying on chance encounters with their food
(Heasman and Fielder, 1983). Laboratory based studies have indicated that high densities of
rotifers (30 - 80 ml-1) significantly enhance survival (Djunaidah, et al., 1998; Suprayudi et
al., 2002a; Zeng and Li, 1999). However, in mass production systems practical
considerations such as maintenance of water quality and rotifer production capacity
sometimes dictate that lower densities (10 - 20 ml-1) are used (Baylon et al., 2001b; Mann et
al., 1999b; Quinitio et al., 2001; Williams et al., 1998). Artemia are generally provided at
0.5 - 10 ml-1 (Baylon et al., 2001a; Mann et al., 1999b; Mann et al., 2001; Quinitio et al.,
2001; Williams et al., 1999b; Zeng and Li 1999;) although when larvae are densely stocked
(100 larvae l-1) Artemia densities of up to 20 ml-1 are used (Nghia et al., 2001b). The
optimum density of Artemia has not been investigated and it appears that a practical
approach is commonly taken with Artemia density based on the stocking density of larvae,
the culture system used and the available budget.
Mud crab larvae accept inert food. Formulated feeds adapted from shrimp larval diets
(Quinitio et al., 1999) and dried Artemia flakes (Nghia et al., 2001b) have been used with
some success. Neither is an effective replacement for live food, but they would appear to
have some potential as supplements. In the Philippines, it is common practice to supplement
live food with 2 mg l-1 day-1 formulated feeds in large tanks (> 10 m3) from Z1 to Z5
(Quinitio et al., 2001).
Inferior nutrition may be a factor contributing to the highly variable survival and the
high susceptibility to disease often recorded in mud crab larviculture. Poor nutrition, even if
confined to the early larval stages, has been suggested as a cause for the phenomenon of
moult death syndrome (MDS) - high mortality during or after the moult from Z5 to M
(Hamasaki et al., 2002a, b; Li et al., 1999; Mann et al., 2001; Marichamy and Rajapackiam,
2001; Quinitio et al., 2001; Suprayudi et al., 2002b; Zeng and Li, 1999).
It is generally accepted that larvae eat zooplankton in the wild which are nutritionally
superior to rotifers and Artemia. Copepods commonly caught from the wild such as Acartia
tsuensis and Pseudodiaptomus spp. have been used as live feeds (Toledo et al., 1998) but
ensuring a regular supply is difficult as they cannot be cultured consistently at high densities
(Delbare et al., 1996). The nutritional quality of rotifers and Artemia can be improved by
CHAPTER 2 – Current status
26
enriching them with nutrients in a process known as bioencapsulation (Coutteau et al., 1997;
Kanazawa and Koshio, 1994; Rees et al., 1994; Wouters et al., 1997). The effect of
enriching the live food with essential fatty acids (EFAs) contained in algae, yeasts and
formulated emulsions on survival and growth of mud crab larvae has been tested.
Suprayudi et al. (2002b) recorded significantly improved survival of S. serrata larvae
after boosting the total n-3 highly unsaturated fatty acid (Σn-3 HUFA) content of rotifers
from 3 - 5 mg g-1 to 7.6 - 8 mg g-1. However, Hamasaki et al. (2002a) reported abnormal
development of S. serrata Z5 leading to high mortality at metamorphosis to megalopa as a
result of feeding the larvae rotifers containing Σn-3 HUFA levels above 6 mg g-1. Whereas
Suprayudi (2002b) recorded high mortality through the moult to megalopa and first crab
after feeding rotifers boosted to 31 mg g-1 Σn-3 HUFA, in Vietnam enriching rotifers with
emulsions containing 30 % Σn-3 HUFA enhanced growth of S. paramamosain larvae. The
contradictory results of these studies indicate that the requirement for Σn-3 HUFA may
differ between batches or species. They also indicate that specific n-3 HUFAs may be more
important than the absolute levels of n-3 HUFA.
Suprayudi et al. (2002a) found that the low levels of eicosapentaenoic acid (20:5 n-3)
(EPA) (3 mg g-1) and docosahexaenoic acid (22:6 n-3) (DHA) (1 mg g-1) found in Artemia
are sufficient for good survival through the moults to M and C1 for S. serrata. These results
were supported by Mann et al. (2001) who found that boosting levels of EPA (39 mg g-1)
and DHA (15 mg g-1) in Artemia did not lead to significant improvements in survival.
Kobayashi et al. (2000) even suggested that levels of 16 - 35 mg g-1 EPA and 17 - 29 mg g-1
DHA in Artemia were excessive, compromising survival of S. tranquebarica. However,
Kobayashi et al., (2000) found that enriching Artemia with EPA at 13 mg g-1 while
maintaining DHA at trace levels enhanced survival indicating that the ratio of DHA/EPA in
Artemia may be more important than absolute levels. There are indications that the
DHA/EPA ratio in the rotifers is also important. Kobayashi et al. (2000) found that a
relatively low DHA/EPA ratio in rotifers (0.07) produced significantly better survival than
higher ratios (0.8 - 0.9) for S. tranquebarica larvae. This is supported by Suprayudi (2002b)
who recorded best survival through first metamorphosis for S. serrata when feeding rotifers
containing a relatively low DHA/EPA ratio of 0.3. However, in Vietnam, zoeal growth and
first metamorphosis of S. paramamosain larvae were significantly enhanced when the
DHA/EPA ratios in the enriching emulsions were high (0.6 - 4) (Vandendriessche, 2003).
CHAPTER 2 – Current status
27
This is another indication of possible differences in fatty acid requirements between the
different Scylla species.
High arachidonic acid ARA/EPA ratios (0.23) in unenriched as opposed to enriched
(0.02 - 0.05) Artemia significantly enhanced larval survival of S. tranquebarica (Kobayashi
et al., 2000), but no other studies on the ARA requirements of the larvae has been
conducted.
The results of the experiments conducted on the enrichment of rotifers and Artemia
with EFAs have generally been contradictory and our understanding of the requirements of
larvae for fatty acids is scant. The experiments described above represent work on four
different species at different laboratories using different larval rearing techniques. Most of
the studies have concentrated on the enrichment of either rotifers or Artemia, whereas work
in South Africa and Vietnam has revealed that both need to be enriched with EFAs to
significantly improve larval performance. It also appears that the larvae may require
different quantities and ratios of EFAs at different stages of development. Despite limited
information, several inferences concerning the fatty acid requirements of larvae can be
made. Firstly, although HUFA rich algae can maintain the nutritional value of previously
boosted live food and may supply other essential nutrients, the addition of algae to the
rearing tank does not adequately enrich live food with the necessary EFA levels. Live food
needs to be artificially boosted before being fed to the larvae. Secondly, Σn-3 HUFA levels
ranging from 8-10 mg g-1 in live food may be beneficial, but larvae seem unable to tolerate
excessive levels in the diet. Thirdly, EPA and DHA enrichment of the rotifers is required,
but the absolute amounts do not seem to be as important as the DHA/EPA ratio.
4. Nursery
Cannibalism is a common problem and can account for a large percentage of mortality
(30 - 50 %) at both first and second metamorphosis (Dat, 1999b; Quinitio et al., 2001;
Suprayudi et al., 2002a). Asynchronous moulting exacerbates the problem (Quinitio et al.,
2001). Moulting synchronicity can be improved by enriching the live food (Takeuchi et al.,
1999). Megalopae are commonly transferred to separate rearing systems which reduces the
rate of cannibalism. Megalopae change from planktonic to benthic orientation at 4 - 5 days
after first metamorphosis and are typically transferred prior to this change. Metamorphosis
to C1 occurs 7 - 10 days after metamorphosis to M (Baylon and Failaman, 1999; Dat,
CHAPTER 2 – Current status
28
1999b). S. serrata megalopae tolerate handling (Quinitio and Parado-Estepa, 2000),
however megalopae of S. paramamosain appear to be more delicate and it is common
practice in Vietnam to leave S. paramamosain megalopae in the culture tanks until they
have moulted to C1.
Megalopae are reared through to crab stages 4 - 7 (crablet) in flat-bottomed tanks
ranging from 8 l (Baylon and Failaman, 1999) to 2000 - 8000 l (Williams et al., 1998; Dat,
1999b). Cannibalism continues to be a problem throughout the nursery stage though it can
be partially averted by placing additional substrata and shelters into the rearing vessels
(Mann et al., 1999b; Marasigan, 1998; Quinitio et al., 2001). Coconut palm fronds,
seaweeds (e.g. Gracilaria bailinae) and a variety of plastic meshes and pipes have been
used for this purpose.
Megalopae are also reared in earthen ponds (50 m2 filled to a depth of 80 - 100 cm)
and prepared to promote a dense plankton bloom prior to stocking (Marasigan, 1998;
Rodríguez et al., 1998). Crablet growth is faster in ponds than in tanks and this is
presumably due to the presence of naturally occurring live feeds, although controlling
mortality in ponds is more difficult (Rodríguez et al., 2001). Management of nursery ponds
can be simplified by culturing megalopae and crablets in suspended cages or “hapa” nets (1
mm mesh size and 1 × 1 × 1.5 m deep) (Marasigan, 1998; Rodríguez et al., 2001).
Megalopae are stocked at rates of 1 to 150 l-1 in indoor tanks (Baylon and Failaman, 1999;
Dat, 1999b; Quinitio et al., 2001) and 0.1 to 125 m-2 in outdoor ponds (Marasigan, 1998;
Rodríguez et al., 1998). The density at which megalopae are stocked, depends on when the
crablets are to be harvested. If crablets are harvested at C1/C2, they can be stocked at
densities exceeding 300 m-2. However, survival to later crab stages is compromised at such
high densities (Rodríguez et al., 1998).
Megalopae are fed a variety of minced fresh and frozen feeds (Hamasaki et al., 2002b;
Quinitio and Parado-Estepa, 2003; Williams et al., 1999b) and particulate diets formulated
for penaeid prawn post larvae. Supplementation of inert diets with Artemia nauplii or adults
significantly increases survival to C1 (Baylon and Failaman, 1999; Heasman and Fielder,
1983; Mann et al., 1999b; Marasigan, 1998; Quinitio et al., 2001; Williams et al., 1999b).
CHAPTER 2 – Current status
29
5. Bottlenecks to commercial production
Larvae have been produced on a commercial scale in several countries. Rearing trials
conducted on S. serrata in China (Li et al., 1999) and more recently (2003) in the
Philippines and Australia have produced thousands of C1 in commercial shrimp hatcheries
with survival ranging from 3.2 - 10 %. In Japan 1.5 million hatchery-produced S.
paramamosain and S. serrata juveniles were released in 1999 for restocking purposes
(Hamasaki, 2002). Although survival rates have improved markedly over the last decade,
there are still several bottlenecks that prevent widespread commercial seed production.
Larvae are vulnerable to a variety of diseases and parasites. Under suboptimal
conditions, Zoothamnium, Vorticella and other sessile ciliates can attach to the larval
integument (Dat, 1999b); fungi of the genera Haliphthoros, Lagedinium and Atkinsiella can
be transmitted to newly-hatched larvae, which are highly susceptible to infection (Hamasaki
and Hatai, 1993a; Kaji et al., 1991; Quinitio et al., 2001). White spot syndrome virus
(WSSV) (Chen et al., 2000) can be introduced.
Bacterial disease is considered to be one of the most important bottlenecks to
commercial hatchery production. The best evidence for the role of bacterial pathogens in the
hatchery is that regardless of other factors, the only treatment known to significantly reduce
mortality is treating the rearing water with antibiotics (Parado-Estepa and Quinitio, 1998;
Mann, 2001). Potential pathogens including Vibrio spp. (Mann et al., 1999b), luminescent
Vibrio, filamentous bacteria (Takeuchi et al., 2000) and others have been identified in larval
cultures where they can rapidly rise to levels generally considered to be deleterious to
crustacean larvae. If not controlled, bacterial numbers can increase by 2 log units on
successive days (Quinitio et al., 2001). Information on how bacteria affect the larvae is
limited. Histological studies have thus far not determined the aetiology or mechanism of
mortality in mud crab larvae (Mann et al., 2001). Additionally there has been no consistent
correlation between larval performance and the structure of the bacterial community or the
presence or absence of particular bacterial strains. The bacterial community of larval
cultures seems to be highly volatile among culture vessels and batches as well as during a
culture cycle in a single vessel (Mann et al., 2001). Mass cultures do not show the same
improved response to hygiene protocols as laboratory scale cultures, possibly due to
differing microbial environments. The common thread is that antibiotics consistently lead to
improved production. Although regular application of antibiotics is not a desirable practice
CHAPTER 2 – Current status
30
for reasons of sustainability, it has frequently been used for experimentation as it is often the
only means of ensuring the survival of sufficient larvae from which data can be gathered.
Antibiotics are either used as a prophylactic in small scale experiments: e.g. 20 mg l-1
streptomycin (Takeuchi et al., 2000) or 2 mg l-1 Sodium nifurstyrenate (Hamasaki et al.,
2002b) or as a bath (100 mg l-1 Oxytetracycline) for Z1 larvae before being stocked into
pilot scale systems (Baylon and Failaman, 2001).
Alternatives to antibiotics such as formalin and probiotic preparations (bacterial
strains which inhibit known pathogens) have been applied. Mud crab larvae can tolerate up
to 25 µl l-1 of formalin (Kaji et al., 1991) and a 24 hour LC50 for S. serrata Z1 has been
estimated at 37 µl l-1 (Churchill, 2003). Regular dosing with 20 µl l-1 formalin every 2 days
is used to prevent fungal (e.g. Haliphthoros spp.) and bacterial infection of larvae in
Vietnam. Although of less potential risk than antibiotics, microbes can eventually build up
resistance to formalin especially if applied at regular, low doses. Results for larval rearing
trials using probiotic bacteria (and their products) have been inconclusive thus far.
Probiotics have been effective in enhancing seed production for other portunid species
(Nogami and Maeda, 1992) and the technology is receiving a great deal of attention (Irianto
and Austin, 2002; Verschuere et al., 2000). With continued research, probiotics and
immunostimulants may provide good alternatives to antibiotics for mud crab larval rearing
in the future (Lavilla-Pitogo et al., 2002).
A characteristic of mud crab larval culture is the highly variable survival. Successive
batches in a hatchery can produce 80 % survival to C1 or total mortality before first
metamorphosis. Several authors have suggested that variable survival is caused by
differences in egg and larval quality (e.g. Mann et al., 1999a; Millamena and Bangcaya,
2001). Differences in batch quality may also be a factor contributing to differences in results
and apparent contradictions between studies (Zeng and Li, 1999). Egg colour has been used
to estimate egg quality, but no correlation has been found between initial egg colour and the
quality of the eggs or larvae (Churchill, 2003). The quality of newly-hatched larvae has
been tested by subjecting them to stressors such as starvation (Djunaidah et al., 2003) or
high concentrations of salinity, ammonia and formalin (Churchill, 2003). Using stress tests
Churchill (2003) identified female size and DHA, EPA and Σn-3 content of the eggs as
possible determinants of egg and larval quality. Because rearing techniques have not yet
been standardized, validating the accuracy of a stress test is difficult. There is also an
apparent large variability in larval quality within batches so that practical interpretation of
CHAPTER 2 – Current status
31
stress test results is difficult. A reliable test for evaluating the quality of Z1 mud crab needs
to be developed. Ultimately the domestication of broodstock and the development of a
standardized broodstock diet could reduce variability in larval quality at hatch.
6. Discussion
The four Scylla species spawn readily in captivity. Millions of eggs with a high hatch
rate can be produced, year round from a relatively small number of wild-caught or pond-
reared broodstock. Scrupulous hygiene during gonad maturation, egg extrusion and egg
incubation is required to reduce infection of the eggs by pathogens which can reduce egg
viability and be transmitted to the larvae. The typical high variability in survival may be
partly due to variable egg and larval quality. Reliable criteria to determine the quality of
newly-hatched larvae should be developed. Once the life cycle can be closed reliably, egg
and larval quality could be enhanced by providing a domesticated broodstock with a
formulated diet.
Mud crab larvae are sensitive to captive conditions and high mortalities at all stages of
development are common. Causes of mortality include poor water quality, incorrect or
fluctuating environmental conditions, cannibalism, feeding and nutritional deficiencies,
parasitic and fungal infections and viral and bacterial disease. In some instances mortality
has been reduced during early larval stages, but high mortalities are still commonly recorded
towards the end of the rearing period. As the rearing process progresses, water quality
deteriorates. Accumulated faeces, uneaten food and dead larvae provide substratum for the
proliferation of pathogenic bacteria. Although strict hygiene protocols, pretreatment of
seawater and high rates of water exchange can mitigate the problem, antibiotics, formalin
and other anti-bacterial prophylactics are still the only way to obtain consistent survival.
Manipulation of the bacterial community through the application of probiotics may provide
a solution (Lavilla-Pitogo et al., 2002). The pathogens associated with mud crab larviculture
need to be identified and the mode of infection determined.
Mass mortality at metamorphosis to megalopa and C1 due to MDS has been linked to
nutritional deficiencies and excessive levels of antifungal or antibiotic agents. Even early
nutritional deficiencies can manifest in MDS at the end of the rearing period. Zoeae can be
reared on rotifers and Artemia and reducing the bacterial load, optimizing feeding densities
and improving nutritional quality through bioencapsulation could reduce MDS and improve
CHAPTER 2 – Current status
32
resistance to stress and disease (Merchie et al., 1997; Mourente and Rodríguez, 1997). A
formulated diet could help to identify the specific nutritional requirements of the larvae and
aid the development of immuno-stimulants. A domesticated broodstock could allow genetic
selection for resistance to disease (Bachère et al., 1995).
Although some research groups have some success in the mass production of mud
crab juveniles, there is still much scope for further research and development before seed
production for aquaculture becomes economically viable and widely adopted.
Figure 1. Life cycle of mud crab. Photo: David Mann; rearranged from Williams et al., 1999b.
CHAPTER 3
Reproductive performance of captive mud crab (Scylla paramamosain) broodstock in Vietnam
Nghia, T.T.*1, Wille, M.2 and Sorgeloos, P.2
1 College of Aquaculture and Fisheries, Can Tho University Email: [email protected] 2 Laboratory of Aquaculture & Artemia Reference Center, Ghent University, Belgium Email: [email protected]
Abstract From 1996 to 2002, reproductive performance of 786 wild S. paramamosain breeders
(355 ± 80 g), purchased from local markets, was recorded. The effect of a number of selected broodstock characteristics, as well as management and environmental conditions on reproductive success was evaluated. Eyestalk ablation improved spawning success, but did not alter the latency period between purchase and spawning. No negative effects of ablation on broodstock survival nor egg quality were found. Breeders collected from an inshore region with higher and more stable average salinity levels tended to perform slightly better than those collected from a region with lower and varying salinity. Females in the range of 300 to 500 g had the best overall reproductive efficiency and are preferred as breeders. Although detached eggs could be incubated artificially, egg incubation by the females themselves was the best practice. The period from March to July was most efficient for broodstock rearing and might correspond to the natural spawning season of S. paramamosain in South Vietnam. September to February could be the period for gonad maturation in the wild. With eyestalk ablation, the most favorable period for artificial reproduction could be extended from February to August.
It was noticed that shading the broodstock tanks was not necessary if shelters for hiding were available. Rearing broodstock in earthen ponds was more efficient; however, management of a pond proved more complicated than tank systems. In terms of complete domestication, broodstock rearing in tanks therefore provides a more practical alternative, provided larger tanks and more suitable substrate are used.
Spawning activity decreased with prolonged time in captivity. Egg quality criteria such as fertilization rate and egg diameter did however not vary in function of time in captivity.
Overall, controlled reproduction of wild mature broodstock females of S. paramamosain for research and pilot production is not problematic, especially with the practice of eyestalk ablation. Although individual females had high fecundities and fertilization rates, spawning and hatching success were however not very high. In this respect, it is hypothesized that broodstock captured offshore might be better.
Further research should be undertaken to completely domesticate the species and further document maturation and fertilization characteristics in captive conditions. Therefore dietary requirements and suitable rearing conditions should be investigated. Life history studies in the wild could provide very useful information in this respect.
CHAPTER 3 – Broodstock
34
1. Introduction
Inadequate seed supply is a major constraint for further development of mud crab
farming (Millamena and Bangcaya, 2001). Over-exploitation, environmental pollution and
mangrove destruction have led to declining wild populations (Heasman and Fielder, 1983;
Hill, 1984). As a consequence a renewed interest in mud crab farming has emerged. As in
most areas where larviculture of mud crab Scylla spp. is conducted, mature wild females are
however still relatively readily available, the source of eggs continues to rely on gonadal
maturation and spawning of wild-caught females in captivity (Mann et al., 1999a). In order
not to impose extra pressure on the already heavily exploited stocks, broodstock
domestication would be a convenient and sound alternative to wild spawners for a more
reliable supply of seed (Millamena and Bangcaya, 2001), moreover creating new
opportunities for disease prevention, selective breeding, etc.
Due to the migratory behavior of female mud crabs in the wild (Hill, 1994),
knowledge of spawning, brooding and hatching of eggs under natural conditions is limited
(Mann et al., 1999a). This probably also explains why far less research has been conducted
on broodstock management techniques than on larval rearing. Several studies have however
indicated that variable egg quality is one of the most critical factors underlying the variable
success of controlled seed production (Churchill, 2003; Davis, 2003).
The number of studies on broodstock rearing of Scylla spp. is limited and mainly
focused on the effects of diet and feeding regimen on ovarian maturation, spawning and
hatching rates (Lin et al., 1994; Millamina and Bangcaya, 2001; Zeng, 1987; Zeng et al.,
1991) and on effects of eyestalk ablation and season on the performance of eggs and larvae
(Mann, 1999a). However, many parameters or conditions for broodstock management in
captivity are still not yet understood thoroughly. Moreover reproductive characteristics
might differ significantly between the four species of the Scylla genus and need to be further
investigated. Experience and knowledge on one species of the genus Scylla could however
also prove useful for the other three species of the Scylla genus (Keenan et al., 1998;
Keenan, 1999b).
In this study, reproductive performance of captive S. paramamosain is described.
Although no real factorial experiments were conceived in this study, the reproductive
characteristics of broodstock animals used over a period of six years to produce larvae for
CHAPTER 3 – Broodstock
35
larval rearing purposes, were recorded and later on analyzed for the effect of a number of
variables.
In addition, the results of a number of side experiments are reported. Artificial
incubation of shedded eggs was attempted and the evolution of egg diameter during
incubation was documented.
2. Materials and methods
2.1. Broodstock
Broodstock source
Over the years (from 1996 to 2002) a variable number of female crabs was purchased
each month from local markets and transported to the hatchery for research on larval rearing
techniques. They had been caught from either the coast of East South Vietnam or the east
coast of West South Vietnam (or the Mekong Delta) where the monthly salinity levels range
around 30 ± 2 and 25 ± 5 g l-1 respectively (Figure 1). In total, 786 gravid crabs (355 ± 80 g,
ranging from 183 - 700 g) were collected.
Rearing systems and culture conditions
Three different types of rearing systems were used to accommodate the brood crabs. A
first system consisted of 20 individual plastic rearing containers of 70-l (0.7 × 0.4 × 0.25 m).
The tanks were placed indoors and darkened completely by black covers. The 20 tanks
were connected to a central biofilter of 700 liter (50 % of the total volume of the rearing
tanks). In a second system, the crabs were individually housed in ten 100-l (1 × 0.4 × 0.25
m) compartments of a 2 × 2 × 0.5 m cement tank. Two of such cement rearing units were
connected to a third cement tank which served as biofiter. The tanks received natural
daylight but were protected by a roof. In a last system, crabs were reared communally in a
60-m3 (40 × 3 × 0.5 m) earthen pond.
Both tank systems were operated in recirculating mode with approximately 100 %
water exchange every 2 - 3 hours. Once a month, approximately 80 % of the water was
renewed. A 5-cm sand layer was provided on the tank bottom to allow proper attachment of
CHAPTER 3 – Broodstock
36
spawned eggs to the tertiary setae of the female’s abdominal flap. In the cement tanks, a
ceramic tile was placed on the bottom of each compartment as shelter for the animals.
Rearing water for the tank systems (30 ± 1 g l-1) was diluted from brine (90 - 110 g l-1) with
tap water and chlorinated before use. Water temperature was not controlled (Table 1). The
concentration of NH4+, NO2
- and NO3+ during culture ranged between 0 - 0.3, 0 - 0.1 and 0 -
5 mg l-1, respectively. Every other day, a dose of 20 µl l-1 formalin was applied for the
whole system.
For the pond system, rearing water was renewed completely every 5 days upon
checking the breeders for spawning. A dose of 50 µl l-1 formalin was applied directly to the
pond after new seawater was filled. Water temperature in the earthen pond varied by the
season (29.6 ± 1.4 °C). Freshwater from a well was pumped to the pond to control the
salinity at 30 ± 4 g l-1. As the pond bottom was covered by a 5-cm mud layer and water level
was high, no extra shelters were provided. The concentration of NH4+, NO2
- and NO3+ of
rearing water ranged similarly as in the tank system.
Broodstock management
Prior to stocking in the hatchery, the crabs were bathed in a 100 µl l-1 formalin
solution for 1 hour to kill potential pathogens.
Depending on the need for larvae for larval rearing purposes, part of the animals was
unilaterally eyestalk ablated to enhance spawning. Before ablation, animals were
anaesthetized in a 1 - 3 g l-1 chloroform solution, with slight aeration. After 20 to 30
minutes, the crab became motionless. The animal was removed from the water and the
eyestalk was scorched by a hot pair of pincers. Then the part of eyestalk above the scorched
location was cut by a pair of scissors. This method also disinfected the wound. After
ablation, the crab was put in fresh seawater with strong aeration. The animal normally
recovered from anaesthesia after 5 - 10 minutes.
Each crab was fed a daily ration of 10 - 15 g of fresh marine squid, bivalve and shrimp
on alternate days.
CHAPTER 3 – Broodstock
37
2.2. Egg incubation
After spawning, the berried crab was bathed in 100 µl l-1 formalin solution for 1 hour
and transferred to a 70-l plastic tank connected to a biofilter for incubation. Daily
management consisted of siphoning out waste material and shedded eggs from the tank
bottom and controlling temperature (30 ± 1 °C), salinity (30 ± 1 g l-1) and ammonia and
nitrite levels (similar to broodstock rearing tanks). Every other day, the crab was bathed in a
50 µl l-1 formalin solution for 1 hour to prevent infestation of the eggs with fungi and
bacteria. One to two days prior to hatching, the female was moved to a 500-l fibreglass tank
for hatching. During egg incubation, the crabs were not fed.
For the pond system, water was drained every 5 days to check for berried females.
Incubation was then carried out as described above.
In some cases, the spawned eggs did not attach to the abdomen. In an attempt to make
use of these shedded/detached eggs, an artificial incubation experiment was carried out in
700-ml glass cones. Eggs were incubated at 50 eggs ml-1 with rather strong aeration.
Temperature was controlled at 28 °C. Two treatments in triplicate were set up, with or
without daily application of 30 µl l-1 formalin. Formalin addition was done upon the daily
90 % water exchange.
2.3. Reproductive performance
From 1996 to 2002, in total, reproductive performance of 786 brood crabs was
recorded. In order to standardise measurements, reproductive performance was followed for
only 60 days (763 females). To calculate survival time in captivity, part of the animals (182
females) where however further maintained beyond 60 days. Twenty three females spawned
after more than 60 days after purchase; these data are however not included for calculation
of reproductive parameters.
Reproductive performance was evaluated through the following parameters:
- time to spawn (days) = the latency period between stocking the female and spawning
- ablation-spawn time (days) = the latency period from eyestalk ablation and spawning of
the female
CHAPTER 3 – Broodstock
38
- survival time in captivity (days) = period from stocking to mortality of the crab
- spawning success = % of the females that spawning
- hatching success = % of the females that produced viable larvae to the total number of
spawning females
- reproductive efficiency = % of the females that produced viable larvae of the total number
of females (= spawning success × hatching success)
- fertilization rate = % of eggs exhibiting an eyespot on day 5 - 6 after spawning to the total
number of sampled eggs (average of triplicate samples of 30 eggs)
- egg diameter (µm) = average diameter of 30 eggs, measured under a microscope
- total zoea 1 production (103 Z1) = number of Z1 larvae produced by a female in a single
spawning event (estimated volumetrically)
- relative zoea 1 fecundity (103 Z1 g-1) = total number of viable Z1 per spawning event over
female weight (g).
In order to develop best management practices, reproductive parameters were later on
treated statistically to determine the effect of a number of factors related to broodstock
management and environmental conditions: eyestalk ablation (intact versus ablated
females); rearing system (plastic versus cement tanks, earthen pond versus cement tanks);
broodstock source (low- versus high-salinity region); month of the year; monsoon season
[rainy (May-October) versus dry (November-April) season]; and temperature-based season,
[spring-summer (March-August) versus autumn-winter (September-February) season]; and
individual female weight and time to spawn. Table 2 gives an overview of broodstock
acquisition in function of broodstock source, month of the year, as well as the percentage of
animals that was ablated in each period.
As mentioned earlier, for studying most of these factors no real experiments were
conceived. Depending on the factor under investigation, the complete or only part of the
data set was used (see Table 2). For investigating the effect of eyestalk ablation on
reproductive performance, data from all 763 females were used, consisting of 358 intact and
405 ablated females. For the effect of broodstock source, only the data from 2001 (241
females) were used, consisting of 116 and 125 females from the high- and low- salinity
region, respectively. To assess the influence of season (month of the year), female weight
and time to spawn again data from all 763 females were used.
CHAPTER 3 – Broodstock
39
However, also a number of planned comparisons were set up. In 2000, every month
approximately 20 animals were bought from the Mekong Delta and divided equally over a
cement and plastic tank system in order to compare reproductive performance in both
systems. Simlarly, 50 females were sourced from the high-salinty region and divided over
both systems for comparison. In total, data of 272 females were used for this study. In
another experiment, 117 females purchased from the Mekong Delta were divided over an
earthen pond (stocked at 6 - 12 m2 female-1) and cement tanks (0.4 m2 female-1) in order to
compare reproductive performance in both systems more straightforward.
2.4. Statistical analysis
One-way analysis of variance (ANOVA) was used to compare data. Homogeneity of
variance was tested with the Levene statistic (P or α value was set at 0.05). If no significant
differences were detected between the variances, the data were submitted to a one-way
ANOVA. The Tukey HSD post-hoc analysis was used to detect differences between means
and to indicate areas of significant difference. If significant differences were detected
between variances, data were transformed using the arcsine-square root (for percentage, i.e.
fertilization rate) or logarithmic transformations (for other other data, i.e. time to spawn,
ablation to spawn time, survival time in captivity, egg diameter, total zoea 1 production and
relative zoea 1 fecundity) (Sokal and Rohlf, 1995). The two-tailed Fisher exact test
(modified from the contingency table method) was used to compare ratios (expressed in
percent, i.e. spawning success, hatching success, reproductive efficiency and other
parameters). All data are presented as mean ± standard deviation when using the Tukey test
or as a ratio/percentage without standard deviation when the Fisher exact test was used. The
Pearson’s correlation coefficient was used to examine the correlation between selected
factors and reproductive characteristics. P was set at both 0.05 and 0.01. Whenever
differences are significant at P < 0.01, this is also indicated. All analyses were performed
using the statistical program STATISTICA 6.0.
CHAPTER 3 – Broodstock
40
3. Results
3.1. Effect of selected management and environmental parameters on reproductive
performance
Eyestalk ablation
Table 3 compares the reproductive performance of ablated and intact females.
Although ablated animals spawned on average 19 days after ablation, overall time to spawn
was similar between both groups (27 - 28 days). Spawning success and (due to a similar
hatching success) also reproductive efficiency were almost double in the ablated group
compared to intact females (35 versus 20 % and 11 versus 6 %) (both significantly different
at P < 0.01). Fertilization rate and relative Z1 fecundity on the other hand were unaffected.
Interestingly, also survival time in captivity was not affected by the ablation procedure.
Rearing system
- Plastic versus cement tank system
Table 3 also presents the reproductive performance of females reared in 2 different
tank systems. Both the spawning success and the reproductive efficiency in the cement
tanks were significantly higher than those in the plastic tanks (30 and 16 %, P < 0.01 and 6
and 1 %, P < 0.05 for cement and plastic tank systems respectively). Although not
statistically significant, also the hatching success was considerably higher in the cement
tank system (19 % as opposed to only 5 % in plastic tanks). Other reproductive
characteristics were not significantly different.
- Cement tank system versus earthen pond
The reproductive performance of broodstock reared in cement tanks and the earthen
pond is presented in Table 4. A higher spawning success and a lower percentage of females
that did not spawn were observed in the pond (both significantly different at P < 0.01). The
CHAPTER 3 – Broodstock
41
percentage of dead or missing females on the other hand, tended to be higher in ponds
compared to the tank system.
Where the percentage of females that successfully produced viable larvae seemed to
be slightly higher in tanks, a lower percentage of females shedding eggs during incubation
was found in the pond (not significantly different).
When combining spawning success and hatching success, the reproductive efficiency
in the pond was significantly higher (P < 0.05) compared to that of the tanks (31 and 15 %
respectively).
Broodstock source
Broodstock source (high versus low salinity regions) did not affect reproductive
performance significantly (Table 3). Females from high salinity regions however tended to
have however a higher (not significant) spawning and hatching success resulting in a higher
overall reproductive efficiency (13 versus 6 % for high and low salinity respectively). Time
to spawn and the latency period between ablation and spawning of the females from the
high-salinity region also tended to be slightly shorter than those of females collected from
low-salinity water.
Month and seasonal cycle
- Month
Tables 5 and 6 present the reproductive performance in relation to the month of
stocking of the females. In Table 5, data for intact and ablated females are presented
separately to detect interaction between both factors. As sample size for these split data is
for certain months small, data were also pooled for all females in Table 6. It should be noted
that the data were also affected by the broodstock source that varied from month to month.
Time to spawn showed no significant differences for both intact, ablated and all
females. The extreme data for the intact group in August (4 days) and December (60 days)
are attributable to the small sample size for these months (Table 5). Based on Table 6,
slightly shorter (not significant) time to spawn values were found from April through
August (on average 22 - 24 days). The shortest time to spawn was observed for females
CHAPTER 3 – Broodstock
42
collected in November (19 days); this value however only represents ablated females (see
Table 5).
With the exception of November, the latency period between ablation and spawning
(Table 6) seemed also shorter (though not significantly) from April to August. In January
and December, it took a considerably longer time to trigger the crabs to spawn (27 and 34
days, respectively). A statistically significant correlation was found between both time to
spawn and ablation-spawn time of the broodstock (y) and the average monthly temperature
(x, see Table 1) (r2 = 0.35, P < 0.05 and 0.64, P < 0.01 respectively, Table 6). For ablation-
spawn time, this relationship could best be described by the equation y (days) = 257.4 - 8.4
x (°C).
Based on the spawning success of the intact group (Table 5), it was apparent that the
spawning activity was higher in females collected from March to July (ranging from 23 to
41 %) compared to those collected during the other months (0 - 17 %). Eyestalk ablation
widened the period with high spawning success almost throughout the year (ranging from
18 to 52 % in January to November). The pooled data presented in Table 6 show that the
spawning success tended to increase from January to July (from 24 to 41 %) with a peak in
June - July (40 - 41 %) and thereafter decreased again towards the lowest value in
December (11 %).
The hatching success revealed a similar tendency as the spawning success. This was
especially apparent for the intact females, i.e. only the intact females collected in March to
August produced viable larvae (Table 5). Also from the pooled data presented in Table 6, it
is clear the hatching success tended to be higher for crabs collected in February to August
(29 - 55 %) and less or no hatching occurred in those collected from September to January
(0 - 27 %). With the exception of December, ablated females on the other hand managed to
successfully hatch eggs (18 - 50 %) year round (Table 5).
Similarly, February to August tended to result in the highest reproductive efficiency
(10 - 14 %) compared to 0 - 7 % in the remaining months) (Table 6).
Except the spawning success, most reproductive characteristics correlated
significantly (P < 0.05) with the month temperature (Table 6).
CHAPTER 3 – Broodstock
43
- Monsoon season
In Table 7, reproductive performance is regrouped according to the monsoon seasons.
None of the reproductive characteristics evaluated however differed significantly between
the rainy and dry season.
- Temperature-based season
Table 7 also groups reproductive performance of the crab females according to the
temperature-based seasons [warm (spring-summer) and cool (autumn-winter) seasons] (see
Table 1). Similarly to the effect of the month, the reproductive ouput increased significantly
in the warmer spring-summer (March to August), i.e. shorter time to spawn (P < 0.05) and
ablation-spawn time (P < 0.01); and higher spawning success and reproductive efficiency
(both with P < 0.01). Also the hatching success, the fertilization rate and the relative Z1
fecundity tended to be higher in the warmer season.
Female weight
Table 8 does not show any significant differences in reproductive performance
between the 3 weight classes. Females of 300 - 500 g tended to have the highest
reproductive efficiency, fertilization rate and total number of Z1 produced. The largest
weight class tended to result in the highest spawning success but much lower hatching
success. Also relative Z1 fecundity seemed much lower for the last group (1,800 Z1 g-1
female). Females in the weight classes 100 - 300 g and 300 - 500 g had similar relative Z1
fecundities of approximately 2,800 Z1 g-1.
The correlations between the female weight and some selected reproductive
characteristics are presented in Table 9. The egg diameter did not vary by the female weight
(r2 = 0.01, A). In the small weight class (236 - 415 g), the egg fertilization rate increased
significantly (P < 0.01) when female weight increased (r2 = 0.52, B); whereas in the larger
weight class (425 - 552 g), no correlation was found (r2 = 0.01, C). Logically, relative Z1
fecundity was correlated significantly (P < 0.01) with fertilization rate (r2 = 0.46, E). Time
to spawn on the other hand had no effect on fertilization rate (r2 = 0.00, D).
CHAPTER 3 – Broodstock
44
Time to spawn
Table 10 presents reproductive performance and egg quality criteria of females that
spawned either between 0 - 15, 16 - 30 or 31 - 60 days after stocking in the hatchery. No
significant differences were found for any of the criteria tested. 30 % of the spawning events
occurred in the first 15 days and 65 % in the first month. Only 35 % of the spawning events
took place in the second month of captivity. Egg fertilization rate did not vary in function of
time in captivity and varied around 60 %. Although not significant, the relative number of
viable Z1 produced seemed to be higher in the groups that spawned between 16 and 30 days
in captivity.
3.2. Artificial incubation of eggs and egg diameter during incubation
When incubating shed eggs artificially (data not shown in the tables), the hatching rate
in the treatment using formalin was significantly higher (P < 0.01) than for untreated eggs
(55 ± 1 and 25 ± 2 %, respectively).
In Table 11, egg diameter is presented in function of incubation time. The diameter of
viable eggs increased steadily during incubation from 287 µm immediately after spawning
to 387 µm upon hatching. The diameter of eggs that proved non-viable increased only
slightly during incubation however and from day 3 onwards, a significant difference (P <
0.01) could be noticed with the viable ones.
4. Discussion
4.1. Effect of selected management and environmental parameters on reproductive
performance
Eyestalk ablation
Worldwide commercial maturation of female penaeid shrimp relies almost exclusively
on the technique of unilateral eyestalk ablation (Browdy, 1992; Fingerman, 1997). Eyestalks
are the endocrine center for regulating many physiological mechanisms, such as moulting,
metabolism, sugar balance, heart rate, pigmentation, and gonad maturation. Therefore,
CHAPTER 3 – Broodstock
45
unilateral eyestalk ablation affects all aspects of shrimp physiology (Quackenbush, 1986).
As they belong to the crustacean group like shrimp, also for Scylla species the technique has
been applied ever since the start of artificial reproduction of the species.
Although time to spawn was not enhanced, eyestalk ablation did not result in any
negative effects on the crab broodstock (e.g. survival time in captivity) or egg quality
parameters (fertilization rate and egg hatchability). Ablation on the other hand significantly
improved the spawning success and hence, increased the reproductive efficiency. Ablated
females could also produce newly-hatched zoeae almost year-round, whereas the intact
females were only able to spawn in some months of the year (March - August). Most studies
have agreed that eyestalk ablation improves reproductive performance. Mann et al. (1999a)
observed no adverse effects of eyestalk ablation on egg and larval production in S. serrata.
These authors however found that eggs of ablated crabs were on average larger than those of
intact crabs and the proportion of non-viable eggs and larvae was lower for ablated crabs. In
our study, we also found a slightly higher average egg diameter and fertilization rate for
ablated females, but the results were not significantly different. Millamena and Bangcaya
(2001) found that ablated females were more fecund and presented higher survival,
suggesting that ablation did not bring undesirable stress to the crabs. However, the authors
observed that the intact females had better egg fertilization rates with higher total number of
zoea produced. In our study, the relative Z1 fecundity seemed marginally lower in the
ablated group. It therefore seems that, as in shrimp, eyestalk ablation makes maturation and
spawning of mud crab more predictable, but may have associated problems like
deterioration in spawn quality and quantity over time (Emmerson, 1980; Primavera, 1985;
Tsukimura and Kamemoto, 1991), or, depending on the conditions, lead to conflicting
results on spawn size, hatching success and other variables (Browdy, 1992).
Rearing system
The two main differences between the plastic and cement tank systems were the water
volume and the light conditions. A review of mud crab broodstock rearing techniques
showed that females kept either individually in small bins (60 - 300 l) or communally in
large tanks ranging from 1 to 12 m3 (Baylon et al., 2001a; Dat, 1999b; Hamasaki, 2002;
Mann et al. 1999a; Millamena and Quinitio, 2000; Williams et al., 1998) have all been
successfully used. Davis (2003) concluded that neither the size nor the type of the rearing
CHAPTER 3 – Broodstock
46
container seems to affect maturation. Therefore, the slight difference in rearing water
volume of the two container types (70 and 100 l) as such is unlikely to be the main factor
that resulted in the difference in reproductive performance. Mud crabs are nocturnal and
inhabit turbid estuarine water (Barnes et al., 2002; Dat, 1999a; Hill, 1978). Moreover they
are known to be very cannibalistic and territorial. Therefore, shelters are usually provided to
reduce stress and prevent cannibalism (Djunaidah et al., 2003; Hamasaki, 2002; Millamena
and Quinitio, 2000; Millamena and Bangcaya, 2001). The cement tanks in our study were
not shaded but a shelter was supplied for each crab. Possibly the natural daylight and
photoperiod, combined with the slightly more spacious (and hence less stressful)
accommodation in the cement tanks resulted in the significantly higher spawning success
and reproductive efficiency compared to the completely darkened plastic tanks. Davis
(2003) observed a kind of “spawning syncronicity” between breeders. He suggested that
pheromones released by spawning crabs or developing eggs might induce other crabs to
spawn. Although the rearing water was also shared between crabs in the plastic tank system
through the central biofilter, it could be that the more direct contact between crabs in the
cement tank system was another factor that positively influenced spawning success.
However, it is difficult to conclude which factor(s) have positively enhanced the
spawning in the cement tank system. To this point, it can only be concluded that covering
the broodstock containers completely is not necessary if shelters are available.
In the earthen pond, the females were subjected to less stress than in the tanks due to
the larger water volume, a more frequent supply of new seawater, a lower stocking density
and more natural rearing conditions. These favourable conditions enhanced the spawning
success (and consequently overall reproductive performance) significantly in the earthen
pond compared to cement tanks. A larger pond bottom with more suitable places for hiding
and spawning might also support better spawning with proper egg attachment to the
female’s abdominal flap (i.e. fewer females with detached eggs in the pond). However,
rearing the broodstock in ponds also had some disadvantages. This was clear from the high
number of escapees and dead crabs in the earthen ponds. Rearing the crabs communally
obviously increases cannibalism. Also the hatching success was lower in ponds than in
tanks. The higher infestation rate of eggs with parasites could be attributable for this. Also
Quinitio and Parado-Estepa (2003) observed that eggs from broodstock spawned in ponds
were occasionally heavily infested with parasites. This problem was probably aggravated by
CHAPTER 3 – Broodstock
47
the fact that spawned females from tanks were treated with prophylactics shortly after
spawning; while for ponds, some females were only collected several days after spawning.
Management of a pond is also much more complicated than tanks and does not allow tight
control of all parameters (e.g. water exchange, evaluation of feeding regime, hygiene
control). It moreover requires more labour.
In terms of complete domestication, broodstock rearing in tanks is therefore preferred,
provided larger water volumes and suitable substrate conditions are used as a compromise.
Broodstock source
Breeders from both regions proved useful to obtain viable larvae as none of the
reproductive characteristics were significantly different. However, broodstock from the
high-salinity region tended to be slightly better overall. This difference could result from the
more stable and higher average monthly salinities on the coast of East South Vietnam
compared to the east coast of the Mekong Delta that is influenced by the large Mekong
river. It is hypothesized that because of the lower salinity in the Mekong Delta compared to
East South Vietnam, crabs caught in the former region are less mature and therefore might
be less suitable as broodstock. Offshore migration of Scylla females for spawning was
reported by several authors (Arriola, 1940; Hill, 1975; Hill, 1994; Hyland et al., 1984; Le
Vay et al., 2001; Ong, 1966; Poovichiranon, 1992; Tongdee, 2001). Also in the Mekong
Delta, mature females of S. paramamosain seem to move from estuarine mangroves into the
subtidal fishery as reported by Le Vay (2001). It therefore seems possible that females
collected in low salinity waters are on average less mature as more mature ones move out
and therefore, there is less chance to catch advanced mature crabs near the coast.
Month and seasonal cycle
- Month
From May to August 2002 and 2003, abundant recruitment of small crablets (0.5 - 1
cm carapace width) on the coastal mudflat of South Vietnam was reported (VASEP, 2003).
Considering that the spawning season should date back about 1 - 2 months, the natural
spawning season for S. paramamosain should then be from March to July. This is in
CHAPTER 3 – Broodstock
48
agreement with the high spawning success of the intact females (not affected by eyestalk
ablation) collected in March - July observed in this study. There was also a tendency that the
time needed by females to spawn after induction by eyestalk ablation was shorter from April
to July. Also Dat (1999b) designated April and May as the peak spawning season due to
high spawning rates observed in that period. Overall, it could be considered that the main
spawning season of S. paramamosain mud crab in South Vietnam extends from March to
July.
However, spawning females could be obtained year-round in this study. Observations
on maturation and spawning of Scylla species show that for nearly all populations,
reproduction is continuous throughout the year, with some seasonal peaks (Le Vay, 2001).
Reproductive activity of S. serrata occurs year-round at low latitudes and seasonally at
higher latitudes (Heasman, 1980; Quinn and Kojis, 1987). In China, two spawning peaks
were reported for S. paramamosain (Li et al., 1999).
Although our data indicate readiness to spawn to be highest from March to July, Le
Vay (2001), in a study on population dynamics of S. paramamosain in Vietnam, recorded
mature females throughout the year, with a peak in September - October. Dat (1992) on the
other hand found a maturation peak in December - February. As maturation and spawning
are separate processes in crustaceans, which can be considerably shifted in time, these
observations do not necessarily contradict our results.
For aquaculture purposes, the combination of high spawning and hatching success of
females collected in February - August resulted in an overall higher reproductive efficiency
(10 - 14 %) than for the other months (0 - 7 %). Hatchery production of mud crab would
therefore be easier in this period.
- Monsoon season
Reproductive performance was not very different in the rainy and dry season. Several
papers have however indicated that reproduction of Scylla crabs peaked in the rainy season.
For example, the female to male ratio of S. paramamosain sampled inshore in the Mekong
Delta was much higher in the dry season, which would confirm the offshore migration of
females for spawning in the rainy season (Ut, 2003). Also in tropical populations of S.
serrata, a higher incidence of mature females appears to be associated with seasonal high
rainfall (Heasman et al., 1985). Offshore spawning migration of S. serrata was also found to
CHAPTER 3 – Broodstock
49
peak during the rainy season (Arriola, 1940; Brick, 1974; Hill, 1975). It has been suggested
that in tropical estuaries the period of peak spawning generally coincides with periods of
high nutrient input associated with monsoonal or cyclonic rainfall (Heasman, 1980). As
explained before, maturation is however a long process and can be shifted in time from
spawning considerably. Moreover, no accurate information exist on the duration of gonad
maturation and spawning migration and if gonad maturation mainly takes place inshore or
offshore. For species that spawn offshore and only recruit back into the estuaries as
juveniles, spawning might also be rather timed to maximise food abundance for juveniles
(Poovichiranon, 1992). The rainy season (May - October) includes months, which fall
within (May - July) and outside (August - October) the “high spawning success season” as
observed in this study, and therefore no differences were found.
- Temperature-based season
As could be expected from the positive relationship between the average monthly
temperature and reproductive efficiency, reproductive activities were higher in the warmer
spring-summer season (March - August) compared to the cooler autumn-winter season
(September - February). Actually, the peak spawning season overlaps completely with the
warm season.
A similar pattern for both spawning and maturation was observed for S. serrata in
Australia (Mann et al., 1999a). In this study summer coincided with a peak in spawning and
hence females were on average more mature. A peak of ovarian development recorded in
autumn was attributed to female crabs having undergone the maturity moult and gonadal
development during the warmer summer months (Mann et al., 1999a). Also Heasman et al.
(1985) observed a spawning peak in summer for South-African S. serrata. In autumn
spawning activity rapidly decreased and by mid-autumn spawning totally stopped. Heasman
(1980) determined that female S. serrata can over-winter in advanced states of ovarian
development. These females then apparently contribute to the early rise in spawning activity
in spring (Mann et al., 1999a).
It seems the spawning season of S. paramamosain is to a large extent related to the
average monthly temperature, while the rainy season is probably the environmental cue for
the onset of maturation.
CHAPTER 3 – Broodstock
50
In conclusion of seasonal effects, it should also be noted that rearing conditions in
captivity might differ considerably from natural conditions (e.g. feed abundance and quality,
salinity, light conditions) and therefore reproductive success in captivity should not
necessarily correspond to the natural reproductive season.
Female weight
Larger crabs were observed to have a higher gonado-somatic-index (ovary weight /
total live weight) (Quinn and Kojis, 1987) and higher total egg fecundity (Churchill, 2003).
However, the total number of viable Z1 larvae produced does not always positively
correlate with the total number of eggs produced by a female since other factors (e.g. mating
success, sperm quality) can affect the egg fertilization and hatching rate. In our study,
fertilization rate increased significantly by the increasing female weight in the range of 236
- 415 g. This was however not the case for larger females (425 - 552 g). As no male
breeders were kept in this study, a larger proportion of the smaller females had probably not
yet mated in the wild prior to acquisition. Logically, relative Z1 fecundity positively
correlated with the fertilization rate. Big females (over 500 g) had the highest spawning
success, but relative Z1 fecundities were considerably lower. Due to the large variation
between individual females, most criteria were not significantly different between the 3
weight classes however. Overall, females of the medium weight class (300 - 500 g) tended
to have the highest reproductive efficiency, fertilization rate and total number of Z1
produced, and thus should be preferred as broodstock.
Churchill (2003) also recommended medium-sized S. serrata for optimal reproductive
output. This author moreover found a significant negative correlation between crab size and
the eicosapentaenoic acid (EPA) content of the eggs, which in turn was correlated with
larval quality as measured through stress tests. The author suggested that the optimal size
for S. serrata broodstock females should range between 125 - 145 mm carapace width in
order to assure maximum output in terms of quantity and quality of the produced eggs.
Generally, in the favourable weight class (300 - 500 g), females of about 400 g are
considered to produce the highest quantity of viable Z1 based on the above-mentiond
correlations: (i) the fertilization rate increased significantly by increasing female weight in
the range of 236 - 415 g, (ii) relative Z1 fecundity positively correlated with the fertilization
rate and (iii) larger crabs had higher total egg fecundity.
CHAPTER 3 – Broodstock
51
Time to spawn
The number of females that spawn seemed to decrease with time in captivity. The
majority of the crabs that spawn did so within the first month (65 %). Only 35 % of the
spawning events took place in the second month of captivity. Given the low price of crab
breeders at the moment, it might therefore be uneconomical to maintain breeders for more
then a month under the given conditions. Egg fertilization was not affected by time to
spawn. This confirms that females preserve sperm well regardless long periods (up to 60
days) in captive conditions without decrease in the fertilization rate. It also shows that male
crabs are only required if sub-adult females would be used for broodstock since mating
occurs at the maturity moult and sperm is subsequently stored for long periods by the
females (Du Plessis, 1971).
The overall spawning success in this study (233 out of 786 females = 30 %), was in
between those reported by Hai et al. (2001) (25 %) and (Dat, 1999b) (52 %) for S.
paramamosain. In this study, the highest spawning success (69 %) was obtained in the
earthen pond. This is however considerably lower than the 85 % reported for S. serrata by
Mann et al. (1999a). A universal phenomenon in Scylla populations appears to be offshore
migration of females to spawn (Le Vay, 2001) and hatch their larvae in full strength
seawater. Keenan et al. (1998) argued that S. serrata populations are dominant in oceans
with water salinity above 34 g l-1 and in mangroves that are inundated with high salinity
water for most of the year. For this species, in some areas, conditions suitable for larval
development may occur in inshore coastal areas (Heasman et al., 1985). Conversely, S.
paramamosain populations are more abundant in seas where the salinity is generally below
33 g l-1, and are able to colonize estuarine habitats in which periods of low salinity occur
seasonally (Keenan et al., 1998). It therefore seems possible that under these conditions, S.
serrata already mature inshore, whereas for S. paramamosain females, this only happens
during/after migration. In this respect, it was recognized in this study that female crabs
caught from Central Vietnam (where inshore salinities are normally higher) or netted
offshore by fishermen in the Mekong Delta usually showed higher spawning success and
hatching success compared to the animals that were captured near the coast.
CHAPTER 3 – Broodstock
52
4.2. Artificial incubation of eggs and egg diameter in function of incubation time
Females kept in tanks without substrate often shed their eggs upon spawning. Previous
observations showed that these shedded eggs are generally fertilized and are able to hatch
when incubated artificially in glass cones. Reliable artificial incubation would be a very
useful tool for scientific purposes and could, potentially, also be helpful to further
standardise and control commercial rearing practices. Fungal infection and eggs adhering to
the sides of the incubation containers have so far made this practice impractical however
(Hai et al., 2001; Churchill, 2003). The higher hatching percentage observed in this study
when formalin was added daily, confirms that infestation with parasites and/or microbial
interaction are a key problem in artificial incubation. Bath treatment with 25 µl l-1 formalin
was found to prevent the occurrence of fungal infection on eggs and newly-hatched crab
larvae (Hamasaki and Hatai, 1993a, 1993b).
In contrast to non-viable eggs, the diameter of viable eggs increased steadily during
incubation. Based on this increase, it would be possible to distinguish and reject non-viable
eggs from day 3 onwards. As egg size between batches is subjected to considerable
variation it may however be more practical to postpone the decision until day 5 after hatch
when egg size can be more precisely determined and combined with the appearance of
eyespots in the eggs. The best fitting equation (r2 = 0.96, P < 0.01) to describe the diameter
of viable eggs (Y, in µm) in function of the incubation time (x, in days) was y = 286 + 3 x +
x2. For S. serrata, Churchill (2003) found the same relationship to fit the polynominal
equation y = 299 + 2 x + x2.
5. Conclusions and suggestions
Although more difficult to manage, pond systems resulted in the best reproductive
performance. Tank systems could be a more practical alternative if stocking densities are
kept low and a proper substrate is provided. It was noticed that darkening the broodstock
tanks (as is often done) was not necessary provided shelters for hiding are available.
Broodstock collected from an inshore region where the salinity level is higher and
more stable, tended to perform better than those collected from a region with lower and
variable salinity.
CHAPTER 3 – Broodstock
53
Females of approximately 400 (300 – 500) g produced the highest total number of
viable Z1 and are therefore preferred as breeders. Smaller females were sometimes not
fertilized, while big females (over 500 g) tended to have lower relative Z1 fecundities.
Although detached eggs could be incubated artificially, egg incubation by the females
themselves was the best practice.
The peak spawning season of S. paramamosain in South Vietnam seems to be from
March to July, which would be the easiest period to obtain spawners and hence perform
larval rearing. Gonad maturation in the wild probably takes place from September to
February. With eyestalk ablation, the optimal production period for mud crab could be
extended from February to August.
Although egg quality remained unchanged within 60 days in captivity, given the low
price of crab breeders and the decreasing spawning activity, it is recommended to keep the
broodstock for not more than 30 days.
In conclusion, year-round maturation and spawning of wild breeders of S.
paramamosain for research and pilot production can easily be achieved, especially when
uni-lateral eyestalk ablation is applied. Although individual females achieved high
fecundities and fertilization rates, overall reproductive efficiency (i.e. the percentage of
females that hatched viable larvae) was rather low (maximum 31 % in ponds). A possible
reason might be that the source of the broodstock was restricted to inshore regions.
Broodstock from offshore water might be more mature and therefore more efficient.
In order to avoid further pressure on this valuable resource the use of wild breeders
should however be discouraged and research efforts should be directed towards full
domestication of the species. In this respect, further research is warranted on all aspects of
maturation and fertilization in captive conditions, investigating dietary requirements and
suitable rearing conditions to trigger maturation and spawning in a more natural way.
Acknowledgements
This study was supported by the European Commission (INCO-DC), the Flemish
Inter-University Council (Vl.I.R.-IUC) and the International Foundation for Science (IFS).
CHAPTER 3 – Broodstock
54
Table 1 Average temperature [mean ± standard deviation (number of records)] of rearing water in the broodstock tanks grouped by month and seasons
Group Month/ Season Temperature (°C)
Jan 27.6±1.2f E (188) Feb 28.2±0.9e D (143) Mar 28.7±1.1bcd BCD (186) Apr 29.3±1.0a A (165) May 29.0±1.2ab AB (137) Jun 28.5±1.1cde CD(131) Jul 28.5±1.4cde CD (152) Aug 28.8±1.0bc ABC (135) Sep 28.7±1.0bcd BCD (142) Oct 28.5±1.0cde BCD (152) Nov 28.3±1.0de CD (144)
Month
Dec 27.2±1.5g E (158) Rainy season (May-Oct) 28.7±1.1A (850) Monsoon season Dry season (Nov-Apr) 28.2±1.3B (983) Spring-Summer (Mar-Aug) 28.8±1.2A (906) Temperature-based season Autumn-Winter (Sep-Feb) 28.1±1.2B (927)
Values in the same column for each group followed by the same superscript letter are not statistically different (P ≥ 0.05, regular letters and P ≥ 0.01, capital letters).
Table 2 Number (and percentage ablated over non-ablated) broodstock crabs purchased from East South Vietnam (high-salinity region) and the Mekong Delta (low-salinity region) per year and per month
Females from high-salinity region Female from low-salinity region Stocking
month 1996 1997 1998 1999 2000 2001 1999 2000 2001 2002 Total n
Jan 10 (50) 12 (50) 10 (50) 13 (46) 45 Feb 2 (50) 10 (50) 20 (60) 10 (40) 8 (63) 50 Mar 1 (100) 4 (0) 11 (36) 10 (50) 26 (39) 10 (50) 19 (68) 81 Apr 4 (45) 9 (45) 10 (50) 18 (56) 10 (50) 51 May 3 (33) 2 (100) 15 (33) 10 (50) 10 (60) 20 (45) 10 (50) 70 Jun 5 (20) 5 (100) 13 (62) 14 (43) 10 (50) 3 (0) 18 (61) 10 (50) 18 (100) 96 Jul 15 (47) 12 (50) 20 (50) 12 (50) 59 Aug 3 (67) 13 (46) 7 (43) 4 (50) 20 (50) 10 (50) 57 Sep 3 (100) 4 (50) 12 (58) 10 (50) 6 (0) 20 (50) 10 (50) 65 Oct 2 (100) 10 (80) 10 (60) 15 (100) 10 (50) 20 (50) 10 (50) 77 Nov 2 (100) 10 (50) 9 (67) 20 (50) 10 (50) 51 Dec 3 (100) 12 (42) 9 (100) 7 (29) 20 (45) 10 (50) 61 All groups 8 (100) 11 (36) 34 (64) 120 (48) 50 (72) 116 (48) 32 (44) 222 (50) 125 (45) 45 (77) 763
CHAPTER 3 – Broodstock
55
Table 3 Effects of the eyestalk ablation, tank rearing system and broodstock source on reproductive performance [mean ± standard deviation or percentage (number of observations)]
Factor Eyestalk ablation Tank system
Broodstock source
Parameter Intact Ablated Plastic Cement High salinity
Low salinity
Time to spawn (days)
28±17a (70)
27±14a
(140) 26±13a
(22) 25±13a (42)
27±17a (30)
29±16a
(21)
Ablation-spawn time (days) none 19±13
(140) 18±13a (16)
19±13a (30)
22±15a (18)
24±15a (14)
Survival time in captivity (days)
57±36a (92)§
60±32a (90)§
44±32a
(40) 37±24a
(42) n.d. n.d.
Spawning success (%)
20B (358)
35A (405)
16B (133)
30A
(139) 26a (116)
17a (125)
Hatching success (%)
30a (70)
33a (140)
5a (22)
19a (42)
50a (30)
38a (21)
Reproductive efficiency (%)
6B
(358) 11A
(405) 1b
(133) 6a
(139) 13a
(116) 6a
(125)
Fertilization rate (%)
56±28a
(15) 63±26a (20) n.d. n.d. n.d. n.d.
Egg diameter (µm)
286±8a
(38) 288±7a
(60) n.d. n.d. n.d. n.d.
Relative Z1 fecundity (103 Z1 g-1)
3.0±1.8a
(12) 2.5±1.6a
(33) n.d. n.d. n.d. n.d.
Values in the same row for each factor followed by the same superscript letter are not statistically different (P ≥ 0.05, regular letters or P ≥ 0.01, capital letters). § Including data of 23 females that spawned later than 60 days since stocking. N.d. = not determined. Table 4 Reproductive performance [% (number of observations)] of broodstock reared in an earthen pond or cement tanks
Reproductive performance (%) Pond Tank
Spawning success (number of females spawned / total) 69A (61) 23B (56) Number of females not spawning / total 6B (61) 65A (56) Number of dead or missing females / total 25a (61) 12a (56)
Hatching success (number of females hatching larvae / females spawning) 45a (42) 64a (13) Number of females shedding eggs / females spawning 17a (42) 36a (13) Number of females producing non-viable eggs / females spawning 38a (42) 0b (13) Reproductive efficiency (%) (= Spawning success × Hatching success) 31a (61) 15b (56) Values in the same row followed by the same superscript letter are not statistically different (P ≥ 0.05, regular letters or P ≥ 0.01, capital letters).
CHAPTER 3 – Broodstock
56
Table 5
stocking month on the reproductive performance of the split intact and ablated groups [mean ± Effect ofstandard deviation or percentage (number of observations)]
Time to spawn (days) Spawning success (%) Hatching success (%)
StockiIntac Intact Inta
(4) ) BC (23) 2)
ng month t Ablated Ablated ct Ablated Jan 23±17a 37±16a (7 17abcd A 31ab AB (2 0 (4) 43a (7) Feb 28±21a (2) 31±13a (12)
12)
) )
4) 3)
) (1) ) (1)
9bcd BC (23) 44a AB (27) 0 (2) 50a (12)Mar 34±12a (12) 29±11a (14) 28ab AB (43) 37ab AB (38) 50a A ( 36a (14) Apr 16±12a (6) 26±14a (12) 23abc ABC (26) 48a AB (25) 33ab A (6) 42a (12) May 23±18a (12) 22±11a (11) 32ab AB (37) 33ab AB (33) 42ab A (12) 18a (11) Jun 28±18a (15) 22±14a (23) 41a A (37) 39ab AB (59) 7b A (15) 43a (23) Jul 28±15a (9) 21±11a (15) 30ab AB (30 52a A (29) 56a A (9) 21a (15) Aug 4±0 (2) 29±13a (9) 7cd BC (29) 32ab AB (28 100a A (2) 44a (9) Sep 39±12a ( 36±18a (10) 12bcd ABC (3 31ab AB (32) 0 (4) 30a (10)Oct 33±20a (3) 28±11a (11) 12bcd ABC (26) 22b B (51) 0 (3) 27a (11) Nov / 19±13a (10) 0 (23) 36ab AB (28 / 20a (10) Dec 60 37±12a (6) 4d C (28 18b B (33) 0 0 (6) Values in the lumn foll me letter are y di (P ≥ 0.05, tters
Table stocking month on the reproductive performance of breeders (all females) [mean ± standard deviation
Stocki month Ablation-spawn Spawning )
Hatching )
Reproductive
same co owed by the sa superscript not statisticall fferent regular leand P ≥ 0.01, capital letters).
6 Effect ofor percentage (number of observations)]
Time to spawn ng (days) time (days) success (% success (% efficiency (%)
Jan 32±17a (11) 27±16a (7) 24ab AB (45) 27a (11) 7a (45) Feb 31±14a (14) 21±13a (12)
lation with (r2)
28ab AB (50) 43a (14) 12a (50) Mar 31±12a (26) 20±10a (14) 32ab AB (81) 42a (26) 14a (81) Apr 22±14a (18) 15±11a (12) 35ab AB (51) 39a (18) 14a (51) May 23±15a (23) 14±8a (11)
33ab AB (70) 30a (23) 10a (70)
Jun 25±16a (38) 14±10a (23) 40ab AB (96) 29a (38) 11a (96) Jul 23±13a (24) 15±12a (15) 41a A (59)
) 33a (24) 14a (59)
Aug 24±16a (11) 19±16a (9)
19bc AB (57 55a (11) 11a (57) Sep 36±16a (14) 20±16a (10) 22bc AB (65) 21a (14) 5a (65) Oct 29±12a (14) 23±10a (11) 18bc B (77) 21a (14) 4a (77) Nov 19±13a (10) 15±11a (10) 20bc AB (51) 20a (10) 4a (51) Dec 40±14a (7) 34±11a (6) 11c B (61) 0 (7) 0 (61) Corremonth temperature (-)0.35* (-)0.64** 0.25 0.37* 0.37*
Values in the same column followed by the sa pt letter are not statisticall nt (P ≥ 0.05, regular letters me superscri y differeand P ≥ 0.01, capital letters). * and ** = significant correlation (P < 0.05 and P < 0.01, respectively).
CHAPTER 3 – Broodstock
57
Table 7
f seasons on the reproductive performance of breeders [mean ± standard deviation or percentage
Monsoon season
Effects o(number of observations)]
Temperature-based season
Reproductive characteristic Rainy ct) Apr)
Sprin r
Time to spawn (days) ) ) (May-O
Dry (Nov-
g-Summer Autumn-Winte(Mar-Aug) (Sep-Feb)
26±15a (124 29±14a (86) 25±14b (140 31±15a (70)Ablation-spawn time (days)
(%) 2) ) )
17±12a (79) 20±13a (61) 16±11B (84) 22±14A (56) Spawning success (%) 29a (424) 25a (339) 34A (414) 20B (349) Hatching success (%)
y31a (124) 34a (86) 36a (140) 24a (70)
Reproductive efficienc 9a (424) 3)
9a (339) 12A (414) 5B (349) Fertilization rate (%)
61±29a (2 56±25a (1 62±28a (26 56±25a (9
Relative Z1 fecundity (103 Z1 g-1) 2.4±1.4a (24) 3.0±.19a (21) 2.9±1.6a (32) 2.0±1.7a (13)
Values in the same row of each seas ed b rscri stati (P ≥
Table
female weight on reproductive performance of breeders [mean ± standard deviation or percentage
Hatching Reproductive Fertilization Total number Relative Z1
on group follow y the same supe pt letter are not stically different0.05, regular letters or P ≥ 0.01, capital letters).
8 Effect of(number of observations)]
Weight Spawning class (g)
success (%)
success (%)
efficiency (%)
rate (%)
of Z1 (103 Z1)
fecundity ) (103 Z1 g-1
[100-300[ ) 2a (6) (6) 23a (167 31a (39) 7a (167) 34±2 770±717a 2.9±2.7a (6) [300-500[ 30a (538) 31a (163) 9a 163) 65±24a (24) 975±605a (36) 2.7±1.5a (36)[500-700[ 41a (56) 17a (23) 7a (23) 59±36a (4) 968±727a (3) 1.8±1.3a (3) All groups 30 (761) 30 (225) 9 (761) 59±27 (34) 947±616 (45) 2.6±1.7 (45) Values in the sam ollow ame pt lette tica
Table on between a number of reproductive characteristics and female weight (y = β + αx) [mean ± standard
Details of y Details of x r2 n
e column f ed by the s superscri r are not statis lly different (P ≥ 0.05).
9 Correlatideviation (min-max)]. n = number of observations
Correlation y x
A Egg diameter male weight ) .01 (µm)
Fe(g)
288±10 (258-312)
390±97 (228-700 (-)0 78
B ) 0.52** 24
ization rate ale weight
ndity
** 21
Fertilization rate (%)
Female weight (g)
62±24 (12-93)
334±54 (236-415
C Fertil(%)
ization rate
Fem(g)
e to spawn
54±33 (10-93)
475± 42 (425-552) 0.01 10
D Fertil(%)
ization rate
Tim(days)
e Z1 fecu
60±27 (10-95)
30±19 (2-74) 0.00 35
E Fertil(%)
Relativ(103 Z1 g-1)
69±23 (12-93)
2.0±1.2(1.8-4.1)
0.47
** = significant correlation (P < 0.01).
CHAPTER 3 – Broodstock
58
Table 10
eproductive performance of breeders in function of time in captivity before spawning (days) [mean ± eviation or percentage (number of observations)]
(days) event success (%) tion
rate (%) Egg diameter (µm)
Relative Z1 fecundity (103 Z1 g-1)
Rstandard d
Time to spawn % of spawning Hatching Fertiliza
0-15 30a (210) 32a (62) 58±32a (12) 286±7a (33) 2.4±1.2a (13) 16-30 35a (210)
3532a (74) 63±21a (11)
60±28286±7a
(32)
288±73.2±1.8a (18) 2.1±1.331-60 a (210) 31a (74) a (12) a (33) a (14)
Values in the same owed e superscript letter are not stat nt (
Table 11 of eggs during incubation (µm) (mean ± standard deviation). n = 42 and 65 batches of viable eggs iable eggs, respectively
Non-viable eggs
column foll by the sam istically differe P ≥ 0.05).
Diamet r and non-v
e
Day after hatch Viable eggs
0 287±10 a a288±9 1 291±11a 292±9a
2 295±11a 294±10296±10B
a 3 301±14A
297±10b4 309±15A 5 318±16A 298±11B 6 333±19A 302±18B
299±16B 7 347±19A 8 353±17A 308±9B 9 364±15A 312±1 B 4
/ 10 387±16 Values i e same y the ript letter are not statistically different (P ≥ 0.05, regular letters or P ≥ 0.01, ca letters)
n th row followed b same superscpital .
CHAPTER 3 – Broodstock
59
Figure 1. Broodstock collection sites in South Vietnam: in high-salinity region (Vung Tau) and low-salinity region (Vinh Chau). Experimental hatchery is located in Can Tho.
CHAPTER 4
Optimal feeding schedule for mud crab (Scylla paramamosain) larvae
Nghia, T.T.*1, Wille, M.2 and Sorgeloos, P.2
1 College of Aquaculture and Fisheries, Can Tho University, Vietnam. Email: [email protected] 2 Laboratory of Aquaculture & Artemia Reference Center, Ghent University, Belgium. Email: [email protected]
Abstract The ability of the different zoeal stages to catch and consume Artemia nauplii was first
investigated in 20-ml vials containing individual larvae. Results showed that crab larvae could only start catching noticeable quantities of newly-hatched Artemia from the zoea 2 stage onwards. The ability of zoea 2 to catch newly-hatched Artemia was however variable between batches and individuals. From the zoea 2 stage onwards, the number of Artemia consumed increased with each larval stage. In a next step, two experiments were conducted to determine a suitable first feed for zoea 1 stage larvae of the mud crab Scylla paramamosain. Micro-algae, rotifers and artificial diets were compared as first feed. Of the 3 feeds tested, rotifers gave the best results. Micro-algae and artificial diets resulted in very low survival and growth. Although micro-algae were not the proper initial feed for early crab stages, they proved beneficial in improving the nutritional quality of rotifers, resulting in higher survival in later zoeal stages (zoea 4 and 5) and a more successful metamorphosis to the megalopa stage. In an attempt to simplify the feeding schedule, a series of experiments were carried out where rotifers were replaced by different forms of Artemia (live and heat-killed umbrella-stage Artemia and frozen or heat-killed Artemia nauplii). Live umbrella-stage Artemia were the best replacement for rotifers for feeding zoea 1 - zoea 2 larvae compared to other Artemia forms. The unselective feeding behaviour (especially at the early stages) seems promising to develop artificial diets in order to substitute live feed and to reduce the dependency on rotifers. In a last step, the optimal time to shift from rotifers to Artemia was investigated. Results showed that rotifers should be replaced by Artemia already in zoea 2 stage. A transition period to shift from one diet to another seemed not necessary. Prolonged feeding of rotifers beyond the zoea 2 stage tended to reduce survival and delay larval development. Although Artemia are more difficult to capture for zoea 2 larvae compared to rotifers, they probably enhanced crab larval performance due to their higher nutritional value compared to that of rotifers. The nutritional value of rotifers and Artemia is however not consistent and therefore optimal feeding schedules might depend on local facilities and culture zootechnics. From the zoea 3 stage onwards, crab larvae can ingest enriched Artemia meta-nauplii.
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1. Introduction
Mud crab (Scylla spp.) fishing represents a valuable component of small-scaled
coastal fisheries in many countries in tropical and subtropical Asia. Since wild stocks are
reported to decrease in many countries (Angell, 1992; Keenan, 1999a), aquaculture of mud
crabs, is becoming increasingly popular. To date aquaculture already contributes a
considerable proportion to the world production of the genus (FA0, 1999). In Vietnam, the
mud crab Scylla paramamosain is the second most important cultured species (next to
shrimp) in the coastal zone.
Aquaculture operations however currently rely almost entirely on wild seed stock, for
which there has been a similar trend of increased exploitation in recent years (Angell, 1992;
Keenan, 1999a). It is therefore generally accepted that the main obstacle for the further
development of mud crab farming is the establishment of hatchery-techniques for controlled
production of seed (Keenan, 1999a; Liong, 1992; Mann et al., 2001; Rattanachote and
Dangwatanakul, 1992; Shelley and Field, 1999; Xuan, 2001).
In contrast to fish, there has been little study of the feeding processes of decapod
crustacean larvae (Harvey and Epifanio, 1997). First feeding (more specifically optimal prey
size, prey density, and its nutritional value) is however of utmost importance and forms the
basis for the development of successful hatchery techniques (Suprayudi et al., 2002a).
Mud crab zoeae are considered to be essentially zooplanktivorous and are able to feed
just minutes after hatch (Mann and Parlato, 1995). Most research to date has agreed that
rotifers (Brachionus spp.) and Artemia (Artemia spp.) are the most suitable feed for Scylla
larvae (Brick, 1974; Heasman and Fielder, 1983; Marichamy and Rajapackiam, 1992;
Zainoddin, 1992; Zeng and Li, 1992). These preys are however often used in combination,
and therefore the optimal live feed species or size for each larval stage has not been clearly
determined yet.
Differences in the quality of the live feed and the rearing systems and zootechnics
applied, also often lead to contradictory results. Quinitio et al. (2001) reported that for S.
serrata the best feeding sequence is to feed rotifers throughout the zoeal stages, to introduce
artificial diets for shrimp at the late zoea 1 stage and finally to introduce Artemia from zoea
3. Ruscoe et al. (2004) on the other hand found that rotifers are best replaced by Artemia
already in the zoea 2 stage. In this study, co-feeding of rotifers and Artemia resulted in
poorer performance. The recent re-classification of the genus Scylla into four separate
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species (Keenan et al., 1998; Keenan, 1999b) moreover makes it necessary to investigate
differences among the different species. Suprayudi et al. (2002a) in this respect remarked
that the feeding preference of S. serrata seems to be slightly different from that of S.
paramamosain.
From the start of our study, it was obvious that rotifers could sustain a certain survival
rate of crab larvae to the megalopa stage. However, newly-hatched Artemia are preferred to
rotifers since the former live feed are commercially available and readily produced, while
rotifers require a laborious and sometimes unpredictable culture. Therefore, in commercial
applications, rotifers are replaced by newly-hatched Artemia as soon as possible. In this
respect, the earliest stage then crab larvae could capture newly-hatched Artemia was
investigated. From the experience observed in larviculture of Penaeid shrimp, the possibility
to replace rotifers as first feed for crab larvae with micro-bound diets and/or micro-algae
was tried since these feeds are commercially available and cultured more easily than
rotifers. In an other attempt not to rely entirely on rotifers in the early stages, different
alternative inert forms of Artemia were tested. Finally, feeding schedules were investigated
in order to find out the best time for shifting from rotifer to Artemia feeding.
2. Materials and methods
2.1. Broodstock rearing
Fully gravid crabs were bought from local markets and transported to the hatchery.
Prior to stocking in the hatchery, the crabs were bathed in a 100 µl l-1 formalin solution for 1
hour. The crabs were housed individually in 100-l compartments of a roofed 2 × 2 × 0.5 m
cement tank, equipped with a biofilter. Rearing water (30 ± 1 g l-1) was diluted from brine
(90 - 110 g l-1) with tap water and chlorinated before use. Water temperature was not
controlled, but fluctuated slightly around 28 °C. Each crab was fed a daily ration of 10 - 15
g of fresh marine squid, bivalve or shrimp meat alternately.
After 3 - 5 days of acclimation, unilateral eyestalk ablation was applied to induce
spawning. After spawning, berried crabs were again bathed in a 100 µl l-1 formalin solution
for 1 hour and transferred to a 70-l plastic tank connected to a biofilter for incubation. Daily
management consisted of siphoning out waste material and shedded eggs from the tank
bottom and controlling temperature (30 °C), salinity (30 g l-1) and ammonia and nitrite
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levels. Every other day, the crab was bathed in a 50 µl l-1 formalin solution for 1 hour to
reduce or prevent infestation of the eggs with fungi and bacteria. During egg incubation, the
crabs were not fed.
One to two days prior to hatching, the female was moved to a 500-l fibreglass tank.
When the hatching process was completed, larvae were selected based on their photo-tactic
behavior: aeration in the hatching tank was turned off for several minutes and the larvae that
were actively swimming up to the surface were collected by gently scooping them from the
surface.
The larvae were then transferred to the rearing containers. In order to slowly acclimate
the larvae to the new rearing conditions, they were placed in a 50-l plastic mesh bucket and
slowly rinsed with water from the larval rearing containers for 20 to 30 minutes, before
releasing them.
2.2. Live feed culture and enrichment
Start cultures of the micro-algae Chaetoceros calcitrans and Chlorella vulgaris were
cultured indoor with Walne solution in seawater of 30 g l-1 at 25 °C. Large-scale production
was performed indoor in 500-l tanks under a transparent roof.
The same rotifer strain, Brachionus plicatilis L-strain with lorica length and width of
164 ± 22 and 120 ± 22 µm, respectively, was used in all experiments. Rotifers were cultured
indoor in 100-l fiberglass tanks operated in batch mode, following the procedure described
in Sorgeloos and Lavens (1996). Rotifers were initially grown on baker yeast, but one week
before use as feed for the larvae, the yeast was replaced by Culture Selco® (INVE
Aquaculture, Belgium). Temperature and salinity were controlled at 25 °C and 25 g l-1,
respectively. Rotifers were harvested on a 60 µm screen and rinsed. In some experiments
the rotifers were enriched with micro-algae or artificial enrichment media before being fed
to the crab larvae. Enrichment with Chlorella or Chaetoceros was performed at a density of
5 106 cells ml-1 for 3 hours (Dhert, 1996). A hemocytometer was used to count micro-algal
densities. In some experiments rotifers were also enriched with Dry Immune Selco® (DIS,
INVE Aquaculture, Belgium), using two separate doses of 0.05 g l-1 at a 3-hour interval.
Rotifer enrichment was performed at a density of 500 rotifers ml-1. The water in the
enrichment vessel was slowly heated to 29 - 30 °C to avoid exposing the rotifers to thermal
shock when they were added to the larval rearing tanks. Before being fed to the larvae,
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enriched rotifers were rinsed and re-suspended in clean seawater at the same temperature of
the crab rearing tanks. Whenever the crab larvae were fed enriched rotifers, algae- or DIS-
enriched rotifers were used on alternate days.
Artemia nauplii (Vinh Chau strain) were hatched as described by Van Stappen (1996).
Both newly-hatched and enriched Artemia nauplii were used in the experiments of this
study. The nauplii were either enriched with Chaetoceros (maintained at 5 106 cells ml-1) for
12 hours or with DIS® (using two separate doses of 0.3 g l-1 at a 6-hour interval). In
experiment 6 only, Artemia were enriched with A1 Selco® (using 1 dose of 0.3 g added to 2
g cysts l-1 for 30 hours upon starting of cyst incubation). The temperature and salinity were
maintained at 30 °C and 30 g l-1, respectively during Artemia enrichment. The density of
Artemia during enrichment was 200 ml-1. Before feeding to the crab larvae, the Artemia
were rinsed with disinfected seawater and suspended at a known density in seawater.
2.3. Larval rearing: objectives, experimental design and techniques
The experimental conditions of the different experiments are summarized in Table 1.
An overview of the different feeding schedules used in experiment 2 to 8 is presented in
Table 2.
Experiment 1
From the very first experiments, rotifers proved to be able to sustain a certain survival
rate of crab larvae to the megalopa stage. However, rotifer culture is labor consuming and
often not consistent. Therefore, most hatchery owners prefer to replace rotifers by Artemia
that are commercially available as soon as possible. In this respect, experiment 1 was
planned to investigate from what stage crab larvae could capture newly-hatched Artemia. At
the same time, the number of prey consumed per larva was quantified. This test was
conducted in small rearing vials where individual crab larvae and a determined number of
Artemia were cultured together for a certain period of time as described by Zeng (1998).
Prior to the actual experiment, the experimental animals were reared together in 30-l
cylindro-conical fibreglass tanks in a recirculating system under standard rearing conditions.
Rotifers were fed to the crab larvae from DAH 0 - 6. Artemia meta-nauplii were supplied
from DAH 6 onwards. Rotifers and Artemia were alternately enriched with Chaetoceros or
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DIS®. Rotifers and Artemia were added daily at 45 ml-1 and 5 - 10 ml-1 to the rearing tank,
respectively.
At each larval stage, 10 larvae were sampled from the stock tank and individually
housed in 20-ml glass vials stocked with 200 newly-hatched Artemia nauplii. Each time, 10
control vials, containing the same number of Artemia nauplii but without crab larva were
incubated as well. Before the start of the test, the selected larvae were kept in a beaker with
slight aeration and starved for 3 hours. The glass vials were incubated in a waterbath
controlled at 29 °C.
After 24 hours, the water in the vials was poured into a petri dish and examined by
means of a dissecting microscope. Those vials that were found to contain weak or dead crab
larvae or crab exuvia were eliminated. The animals in the remaining vials were killed with
a few drops of lugol and the remaining number of Artemia in each vial were counted and the
crab larvae staged. The difference between the number of Artemia in the experimental and
control vials was considered to be the number of prey consumed by the crab larvae.
This test was repeated in three times, each with a different batch of larvae, making
sure that in total at least 20 measurements for each larval stage were determined.
Experiment 2
From experience in larviculture of Penaeid shrimp, the possibility to replace rotifers as
first feed for crab larvae by micro-bound diets and/or micro-algae was tried since these
feeds are commercially available and cultured more easily than rotifers. For this purpose, in
the second experiment four different feeds, i.e. (i) a commercial micro-bound diet for
shrimp, (ii) micro-algae, (iii) rotifers and (iv) a combination of micro-algae and rotifers,
were compared as first feed. A starvation treatment was included as a control.
The crab larvae were reared at a density of 40 l-1 in 2-l glass bottles operated in batch
mode. Rather strong aeration was provided to reduce the settlement of the micro-bound diet
and micro-algae. Daily, larvae were siphoned out using a large-tip pipette and gently
transferred to new bottles containing fresh seawater of the same temperature and salinity.
Lanzy PZ® (a commercial diet for shrimp zoeal stages; INVE Aquaculture, Belgium)
was fed in 4 rations to give a total ration of 1 mg l-1 day-1 according to the manufacturer’s
recommendations. Micro-algae Chaetoceros were counted with a hemocytometer and added
to give a final concentration of approximately 150,000 ± 50,000 cells ml-1 in the treatments
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“micro-algae” and “rotifers and micro-algae”. The density of rotifers was 30 and 15 ml-1 in
the “rotifers only” and “rotifers and micro-algae” treatments, respectively. Rotifers were not
enriched.
Experiment 3
Experiment 3 was a replication of the previous experiment. This time formalin was
used as a prophylactic chemical to increase overall larval survival. In this experiment, four
treatments from experiment 2, i.e. (i) starvation, (ii) micro-algae, (iii) rotifers and (iv)
micro-algae and rotifers were repeated. On DAH 9 and again on DAH 12, the surviving
larvae were pooled per treatment and the water volume adjusted to obtain similar larval
densities (20 l-1) for all treatments. On DAH 12, the treatment “rotifers and micro-algae”
was split up into 2 sub-treatments, i.e. one part continued to receive rotifers; the other half
was fed Artemia from then onwards. Micro-algae were supplemented in both sub-
treatments.
Rearing conditions were similar to those in experiment 2. In this experiment rotifers
were however supplied at a higher density (45 ml-1 compared to 15 - 30 ml-1 in experiment
2). Also 20 µl l-1 formalin was applied every other day to reduce or prevent fungi and
bacteria to develop.
Experiment 4
Through previous experiments, rotifers proved the most suitable live feed for early
crab larvae. However in practice, availability of rotifer stocks is sometimes problematic for
large-scale production as. Therefore, in an attempt not to rely entirely on rotifers in the early
stages, different alternative inert forms of Artemia were tested. Five treatments were
compared: (i) starvation, as negative control, (ii) rotifers, as positive control, (iii) heat-killed
umbrella-stage Artemia, (iv) heat-killed instar-1 Artemia, (v) live instar-1 Artemia and (vi)
frozen instar-1 Artemia.
Static 1-l PVC cones were used in this experiment. Larvae were stocked at a density
of 50 l-1. As in experiments 2 and 3, the crab larvae were manually transferred to new cones
daily. Rearing conditions were also similar. Aeration was however increased to maintain the
non-moving feed items in suspension. Newly-hatched Artemia and umbrella-stage Artemia
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were heat-killed by dipping in water of 80 °C for 15 minutes. Instar-1 Artemia were put in a
-20 °C freezer for 2 hours to make “frozen Artemia”. Umbrella-stage Artemia were
produced by harvesting already after 12 hours incubation. The feeding density was identical
(10 ml-1) for all Artemia forms. In the positive control, 45 rotifers ml-1 were fed. Rotifers
and Artemia were not enriched in this experiment.
Experiment 5
Experiment 5 was the replication of experiment 4 but in larger cylindro-conical tanks
operated in recirculating mode in order to better keep the inert feed in suspension and to
increase overall survival. The same treatments as in the previous experiment were designed,
i.e. (i) starvation, as negative control, (ii) rotifers, as positive control, (iii) heat-killed
umbrella-stage Artemia, (iv) heat-killed instar-1 Artemia, (v) live instar-1 Artemia and (vi)
frozen instar-1 Artemia). Here the larvae were however reared in 30-l cylindro-conical
fibreglass tanks that were operated in recirculation mode. The stocking density of Z1 was
also higher than that used in experiment 4 (150 instead of 50 l-1 in the previous experiment).
General rearing techniques were similar to those described in experiment 1. In the
positive control treatment (feeding rotifers only), rotifers were enriched with Chlorella and
DIS® alternately and supplied to the larvae at a density of 45 ml-1. The different forms of
Artemia were prepared and fed (10 ml-1) to the crab larvae identically as in experiment 4.
Experiment 6
From experiments 4 and 5, heat-killed umbrella-stage Artemia shows some promise as
a (partial) replacement for rotifers. Live umbrella-stage Artemia, which stay in suspension
better (due to the alveolar layer of the cyst shell) and have a superior nurtritional quality,
might even be better. For that reason, in this experiment, live umbrella-stage Artemia were
tried as an alternative for rotifers as first feed. Live umbrella-stage Artemia were the only
feed used for the early crab larvae (Z1 - Z2) from DAH 0 - 6. From DAH 7, enriched (A1
Selco® and Chaetoceros alternately) Artemia nauplii were fed. The trial was conducted in
eight 500-l tanks and was operated in batch mode from DAH 0 - 6 and in recirculating mode
from DAH 7 onwards. It differed from the previous experiments in this way that the total
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feed amount was fed in 2 - 3 rations (of approximately 2 umbrella-stage Artemia ml-1) per
day instead of the routine regimen using a higher density (5 - 10 Artemia ml-1) once per day.
Experiment 7
In experiment 7 the optimal time to shift from feeding rotifers to Artemia was more
precisely determined. Six feeding schedules, 1/1, 2/2, 3/3, 4/4, 5/5 and 6/4, differing in the
time when rotifers were replaced by Artemia were compared. Feeding schedule 1/1 implies
that rotifers were supplied on DAH 0 and flushed out on DAH 1 and Artemia was supplied
from DAH 1 onwards; feeding schedule 2/2: rotifers were supplied on DAH 0 and DAH 1
and flushed out on DAH 2 and Artemia was supplied from DAH 2 onwards, etc.
The larvae were reared in 30-l cylindro-conical fibreglass tanks that were operated in
recirculation mode. General rearing techniques were similar to those described in
experiment 1. Rotifers were not enriched and instar-1 Artemia were used.
Experiment 8
In this experiment, feeding schedules 6/4 (Artemia introduced in the Z2 stage), 9/7
(Artemia introduced in the Z3 stage) and 12/9 (Artemia introduced in the Z4 stage) were
compared. All three treatments had a 2-day overlap in feeding rotifers and Artemia, i.e.
feeding schedule 6/4 implies that rotifers were supplied from DAH 0 - 6 and Artemia were
supplied from DAH 4 onwards; feeding schedule 9/7: rotifers were supplied from DAH 0 -
9 and Artemia were supplied from DAH 7 onwards, etc.
The rearing containers, rearing techniques and feeding practices were similar to those
of experiment 7.
2.4. Evaluation criteria
In experiments 2, 3 and 4 (using small containers) the average survival rate was
calculated by individually counting all surviving larvae in each replicate. The survival rates
in the other experiments (using 30 to 500-l containers) were estimated by volumetric
sampling. Depending on the tank volume and the density of the surviving larvae, triplicate
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300- to1000-ml samples were taken from each tank. Megalopae (M) were counted
individually.
Larval development was monitored every three days by identifying the average zoeal
instar stage of a sample of larvae and assigning it a value: first zoea (Z1) = 1; second zoea
(Z2) = 2, etc. To compare the larval development between treatments, an average larval
stage index (LSI) was calculated from the average LSI value of all replicate containers in
the same treatment. In experiments (1, 3 and 4), using small containers, all larvae were
staged visually upon counting daily survival. For larger containers (experiments 5, 6, 7, and
8), 5 or 10 larvae (in 30-l and 500-l tanks respectively) were sampled from each tank to
calculate the average LSI. For the latter method, the sampled larvae were staged under a
dissecting microscope.
2.5. Statistical analysis
One-way analyis of variance (ANOVA) was used to compare data. Homogeneity of
variance was tested with the Levene statistic (P or α value was set at 0.05). If no significant
differences were detected between the variances, the data were submitted a one-way
ANOVA. The Tukey HSD post-hoc analysis was used to detect differences between means
and to indicate areas of significant difference. If significant differences were detected
between variances, data were transformed using the arcsine-square root (for percentage, i.e.
survival rate) or logarithmic transformations (for other parameters) (Sokal and Rohlf, 1995).
The two-tailed Fisher exact test (modified from the contingency table method) was used to
compare ratios (expressed in percent) for the survival of pooled data. All data are presented
as means ± standard deviation when using the Tukey test or as ratio/percentage without
standard deviation when the Fisher exact test was used. P was set at both 0.05 and 0.01.
Whenever differences are significant at P < 0.01, this is also indicated. All analyses were
performed using the statistical program STATISTICA 6.0.
3. Results
Table 3 shows the Artemia consumption in the successive larval stages in experiment
1. In all three data sets, the number of remaining Artemia in the experimental vials is
significantly different from the controls from Z3 onwards (P < 0.01). This shows that crab
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larvae can consume live Artemia nauplii from that stage. For the Z2 stage, results were less
clear. The number of Artemia remaining in tanks with Z2 larvae was always lower than the
number in controls but this was significant (P < 0.05) in only one of the experiments. These
results indicate that the ability of Z2 larvae to catch and ingest Artemia nauplii is still rather
limited and might be variable between different batches and individuals. Z1 seemed not
capable of consuming large numbers of live Artemia nauplii, indicating that they probably
require a different first feed. The average Artemia consumption in the different larval stages
is presented in Figure 1. From this it can be seen that consumption increased from
approximately 6 Artemia nauplii larva-1 24 h-1 in the Z2 stage to more then 100 in the
megalopa stage.
The average survival rates of the crab larvae fed artificial diets, algae, rotifers or a
combination of rotifers and algae in experiments 2 and 3 are presented in Table 4. In both
experiments, starved larvae could not survive beyond DAH 3. Larvae fed an artificial
shrimp (experiment 2) diet died gradually from DAH 3 and complete mortality occurred on
day DAH 5. In this treatment only a few larvae could moult to Z2. Also treatment “micro-
algae” resulted in very low survival on DAH 3 and 6 and the larvae could not moult to the
next stage, which shows that micro-algae are not a suitable first feed for S. paramamosain
larvae. The “rotifers only” treatment always result in the best survival. The survival rate of
the “rotifers only” treatment was generally also higher than the “algae and rotifers”
treatment (although only significantly different at P < 0.01 on DAH 3 in experiment 2). The
better performance of treatment “rotifers only” compared to treatment “algae and rotifers”
in experiment 2 could be explained by the lower rotifer density fed to the larvae in the latter
(15 compared to 30 ml-1 in treatment “rotifers only”). However, in experiment 3, both
treatments received the same amount of rotifers, and still there was quite lower survival
(although not significant) in treatment “rotifers and algae”. Therefore, supplementation of
micro-algae to a diet of rotifers does not seem to enhance the survival of early-stage crab
larvae. Table 5 shows the average survival rates at the later larval stages in experiment 3.
On DAH 9, treatment “rotifers only” performed still better than treatment “rotifers and
algae” (P < 0.05). After pooling and restocking the larvae on DAH 9 and again on DAH 12,
significantly better survival was however obtained in treatment “rotifers and algae” as
compared to “rotifers only” (P < 0.01). On DAH 15, feeding only rotifers resulted in a
significantltly lower (P < 0.01) survival compared to those of treatments with micro-algae
supplementation. Consequently, feeding only rotifers could not furthermore support
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metamorphosis to the megalopa stage. The combination of rotifers and micro-algae on the
other hand yielded megalopae. A slightly higher metamorphosis rate was obtained at DAH
22 however when rotifers were replaced by Artemia on DAH 12.
In experiments 4 and 5, some alternative forms of Artemia were tested as a
replacement for rotifers to feed crab larvae in the Z1 stage. Table 6 summarizes survival and
larval development rate (expressed as LSI) of the larvae in the different treatments as well
as average water quality parameters. The results on DAH 3 and 6 confirm that rotifers are
the best live feed in terms of survival and larval development for Z1 and Z2 larvae (P <
0.01). Among the remaining treatments, treatment “heat-killed umbrella-stage Artemia”
tended to have the highest survival rates on DAH 3 and 6. Compared to the positive control
(rotifers), LSI was however compromised in all treatments. Live and frozen instar-1 Artemia
nauplii resulted in slightly better (not significant) growth compared to heat-killed Artemia
forms (instar-1 and umbrella-stage) on DAH 6 in experiment 5.
In experiment 6 a pilot-scale production trial (data not shown in the tables) was
performed using live umbrella-stage Artemia to feed Z1 and Z2 stages (DAH 0 - 6). These
umbrella-stage Artemia were fed in 2 or 3 rations of 2 - 3 individuals ml-1 day-1. The
survival rates on DAH 3, 6 and 9 were 79 ± 9, 36 ± 2 and 30 ± 5 %, respectively. The LSI
values on the same sampling days were 1.7 ± 0.1, 2.9 ± 0.2 and 3.9 ± 0.2, respectively,
representing a normal development rate.
In Tables 7 and 8, the survival and development rates are shown of larvae subjected to
different rotifer/Artemia feeding schedules (experiment 7 and 8, respectively). In
experiment 7, a higher survival rate on DAH 3 was observed for treatment 4/4 compared to
the other treatments (although only significantly different with treatment 4 at P < 0.05).
Where it is possible that treatments 1/1 and 2/2 gave low survival because of the early
introduction of Artemia; theoretically, the survival rates on DAH 3 of the last four
treatments (3/3, 4/4, 5/5 and 6/4) should be similar as the same live feed (i.e. rotifers) was
offered from DAH 0 - 3. On DAH 6 no difference were observed between any of the
treatments. On DAH 9 a significantly (P < 0.05) higher survival was found for treatment 4/4
compared to treatments 1/1 and 3/3. Treatments 2/2, 5/5 and 6/4 showed intermediate
results. This could indicate that rotifers should not be replaced by Artemia before DAH 3
(Z1 stage). It furthermore suggests that rotifers are best replaced by Artemia already early in
the Z2 stage and that an overlap in feeding rotifers and Artemia is not really necessary.
Although no statistical differences were found, LSI values of the larvae in treatments
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receiving Artemia in the Z1 stage (treatments 1/1, 2/2 and 3/3) tended to be lower than those
in the other treatments where Artemia were offered only in the Z2 stage (treatments 3/3, 4/4,
5/5 and 6/4), which confirms the survival results.
In experiment 8 (Table 8), delaying the introduction of Artemia until the Z3 and Z4
stage (treatment 9/7 and 12/9, respectively) resulted in a significantly lower LSI value on
DAH 9 compared to early supplementation of Artemia from the Z2 stage (treatment 6/4) at
P < 0.01. The late Artemia feeding schedules also tended to reduce the survival and
development rate of crab larvae from DAH 9 - 15 although the data of all treatments shows
no significant difference.
4. Discussion
All publications to date have agreed that rotifers and Artemia are the most suitable live
food for Scylla larvae (Brick, 1974; Heasman and Fielder, 1983; Marichamy and
Rajapackiam, 1992; Zainoddin, 1992; Zeng, 1998). These preys are however often used in
combination, and there remains uncertainty about the optimal live feed species or size for
each larval stage.
From the results of experiment 1, it became clear that from the Z3 stage onwards, S.
paramamosain larvae can easily catch and consume Artemia nauplii. Although rotifers as a
sole feed could support crab larvae to reach the last zoeal stage, it might be beneficial to
supplement micro-algae of shift to a larger prey at a certain point in the rearing process. In
this respect it was shown in this study that micro-algae were necessary in combination with
rotifers in order for the larvae to successfully complete the first metamorphosis.
Live prey size seems however to be most crucial for early Z1 and Z2 larvae. Using
Artemia nauplii at start-feeding for S. paramamosain larvae usually results in low survival
(Li et al., 1999). S. serrata larvae can however be grown successfully on an exclusive diet
of Artemia, and the insignificant differences (compared to feeding rotifers) in larval survival
and growth when fed newly-hatched nauplii or even enriched meta-nauplii from the Z2
stage onwards indicate that live prey size within the range of 463 to 658 µm does not affect
larval performance of S. serrata (Mann et al., 2001).
As there are indications that feeding abilities might differ among the four Scylla
species, the best time to shift to Artemia still needs to be determined more precisely for
CHAPTER 4 – Feeding schedule
74
every species within the genus Scylla. The following discussion concentrates on the suitable
prey size for Z1-Z2 stages for S. paramamosain.
4.1. Ability of S. paramamosain zoeae to catch instar-1 Artemia
The ability of S. paramamosain zoeae to catch Artemia nauplii and the number of prey
consumed in each stage was checked by individually rearing larvae in small containers. The
results of experiment 1 showed that crab Z1 could not catch large numbers of newly-
hatched Artemia. This is probably due to the fast movement and relative big size of the
latter. Harvey and Epifanio (1997) estimated the swimming speed of rotifers, instar-1
Artemia and Z1 larvae of the mud crab Penopeus herbstii at 3, 6 and 2 mm s-1, respectively.
From this it is clear that rotifers are a much easier prey than Artemia for crab larvae. Zeng
and Li (1999) concluded that due to their relatively larger size and faster swimming
behaviour, Artemia as a sole diet for early S. paramamosain larvae generally yields low
survival rates compared to rotifers and it was observed that early larvae seem unable to
capture and ingest Artemia as effectively as rotifers.
When comparing the different species within the genus Scylla, there is evidence that
S. serrata Z1 can cope better with Artemia as first feed, resulting in higher survival
compared to S. paramamosain. For example, a survival rate of 48 % at the Z5 stage was
obtained by Baylon and Failaman (1999) when S. serrata zoeae were fed only Artemia. In
our trials with S. paramamosain, feeding only Artemia to Z1 larvae, resulted in extremely
low or zero survival. Table 9 compares the size of some popular live feeds, the carapace
width of the different larval stages of S. paramamosain and egg size of the four Scylla
species. The data of the diameter of newly-spawned eggs demonstrates a smaller egg size in
S. paramamosain (288 ± 10 µm) compared to S. serrata (329 ± 8 µm). When comparing all
four Scylla species, S. paramamosain seems to have the smallest newly-spawned eggs
among all. It is likely that the smaller egg diameter in S. paramamosain coincides with a
smaller Z1 size, which results in a lower capability to catch larger preys like Artemia,
making prey size even more crucial.
At the Z2 stage, few Artemia (7 larva-1 24 hours-1 on average) were consumed and the
capture ability seemed to vary from one batch to another and even between individual
larvae. In Table 9, it can be seen that the carapace width of Z2 was similar to the size of
newly-hatched Artemia resulting in difficulty for crab larvae to ingest the whole prey. That
CHAPTER 4 – Feeding schedule
75
S. paramamosain Z2 can survive under these conditions, can probably be explained by the
fact that Z2 are observed to consume only part (e.g. appendages) of its prey (Zeng and Li,
1999). Similarly for S. serrata, Baylon et al. (2004) observed that crab larvae hold the
Artemia nauplius for a while but eventually released them. The released prey had missing
body parts such as the head and appendages when they were examined under a microscope.
The authors suggested that although the early stage larvae could not consume the entire
Artemia nauplius, they managed to ingest bits and pieces of the prey.
From Z2 stage onwards, the number of consumed prey increased by each larval stage.
The average number of Artemia nauplii consumed were 7, 15, 25, 37 and 114 individuals
for Z2, Z3, Z4, Z5 and M, respectively. For comparison, Baylon et al. (2004) found for S.
serrata larvae ingestion rates increasing from approximately 2 Artemia nauplii larva-1 day-1
in the Z1 stage to almost 50 in the Z5 stage. The consumption of Artemia nauplii by the crab
larvae also seemed to be affected by their physiological status (e.g. moulting period) and by
the quality of the larvae. In this respect, it was observed that weak or
moulting/metamorphosing crab larvae consumed significantly less prey. In a comparable
experiment, Zeng (1998) observed that independent from prey density (ranging from 2 to 20
Artemia nauplii ml-1), larval feeding rate showed a similar disturbed pattern related to the
moulting cycle, which was characterized by a sharp decline in feeding close to moult or
metamorphosis to the megalopa stage and as soon as larvae completed metamorphosis, the
feeding rate immediately shot back to its maximum. Baylon et al. (2004) found that when S.
serrata larvae were cofed with Artemia and rotifers, there was an increased consumption of
Artemia nauplii on the day before moulting or metamorphosing and increased ingestion of
Brachionus on the day after larvae had developed to the next stage.
4.2. Suitable first feed
In experiments 2 and 3, Z1 larvae, starved at 29.5 - 30.6 °C (Table 1) could not
survive more than 3 days and could not moult to Z2. Similarly, Z1 larvae of S. serrata
starved for 48 to 72 hours (at temperatures from 27 to 29 °C) incurred high mortalities
(Lumasag and Quinitio, 1998). Also at lower temperature, newly-hatched zoeae which were
continuously starved were not able to moult to the second zoeal stage and the time to reach
100 % mortality was 5.5 and 5.9 days at 28 and 24 °C, respectively (Mann and Parlato,
1995). It therefore seems likely that the maximal starvation time for Scylla larvae, even at
CHAPTER 4 – Feeding schedule
76
low temperatures, is 6 days. This also means that, under our conditions, larvae that survived
beyond DAH 3 in the “feeding treatments” were capable to catch and ingest at least some of
the feed offered.
All larvae fed an artificial shrimp diet in experiment 2 died before DAH 6. Similarly,
Quinitio et al. (1999) found that artificial shrimp diets were not a suitable first feed for mud
crab larvae. In that study, the larvae could not survive to the Z3 stage and as a consequence
of water pollution and bacterial contamination, mortality was positively correlated with the
amount of artificial feed given.
In experiments 2 and 3, feeding only algae resulted in almost complete mortality of
the crab larvae before DAH 6. Davis (2003) reported that larvae probably merely ingest
micro-algae by chance when swallowing water. Brick (1974) also found that S. serrata
zoeae fed Chlorella did not actively ingest noticeable quantities of the algae. Although the
presence of micro-algae in the water prolonged the survival of Z1, they could not moult to
Z2 unless the diet was supplemented with zooplankton (Brick, 1974). Micro-algae therefore
are not a proper feed for early S. paramamosain larvae.
From experiments 2 and 3 it is clear that rotifers were the most suitable live feed for
Z1 compared to the other feeds tested here. Ruscoe et al. (2004) found that also for S.
serrata larvae, rotifers were necessary in the feeding regime for optimal growth and
survival. The size of Z1 larvae is much bigger compared to the size of rotifers (Table 9),
which facilitates capture and ingestion. Similarly, Baylon et al. (2004) reported that the
smaller size of Brachionus (220 - 240 µm) and their slow swimming movement makes them
easy prey for the early-stage zoeae of S. serrata to capture and to consume, compared with
the larger Artemia (460 - 500 µm).
The lower survival rate of treatment “rotifers and algae” compared to “rotifers only”
in experiment 2 could be explained by the lower density of rotifers offered in the former. In
this respect, Zeng and Li (1999) concluded that rotifers (Brachionus plicatilis) are a suitable
feed for early larvae (Z1 and Z2) of the mud crab S. serrata, but their density significantly
affected larval survival and development. Crustacean larvae are generally passive feeders
and do not pursue prey actively (Baylon et al., 2004). They only feed if there is a chance
encounter with the food organism (Heasman and Fielder, 1983). Harvey and Epifanio
(1997) reported an increased ingestion by common mud crab Panopeus herbstii larvae of
Brachionus and Artemia with increasing density. Minagawa and Murano (1993) found a
CHAPTER 4 – Feeding schedule
77
decrease in the survival of the red frog crab Ranina ranina with decreasing prey density
(0.05 -5.0 organisms ml-1).
In experiment 3, where the same number of rotifers were fed in both treatments,
treatment “rotifers only” still outperformed treatment “rotifers and algae” however. Also the
quality of the algae may have affected the results. The algae were often observed to die due
to the low light intensity (about 1,600 lux) in the rearing containers and in this way polluted
the water and created a greenish mucous biofilm on the sides and bottom of the rearing
containers that was observed to trap early crab zoeae. Chythanya and Savan (1999)
indicated that in some cases biofilm bacteria can also cause large scale infection and
mortality of fish and crustaceans. Also Mann (2001) noted that one pattern of early rapid
mortality, which occasionally occurs in crab hatcheries, seems associated with development
of a characteristic mucilaginous matrix on the bottom of the tank. However attempts to
isolate the causative bacteria and re-inoculate cultures have been unsuccessful. Removing
this layer daily however significantly improved the survival rates of crab larvae reared in 5-l
bowls (Williams et al., 1998, 1999a).
Towards the end of experiment 3 however, a significantly higher survival was
observed in the treatment “rotifers and algae” (79 % compared to 25 % in the treatment
“rotifers only” on DAH 12). During this period, the layer of dead algae was removed every
day instead of every few days, earlier in the experiment. On DAH 22, 48 % of megalopae
were obtained in treatment “rotifers and algae”, while the surviving larvae in treatment
“rotifers only” could not pass the first metamorphosis and started dying on DAH 21. A
positive effect of micro-algae on the survival and development of Scylla larvae in later
stages has been found in other studies. For S. serrata, the presence of Chlorella left zoeal
survival unaffected while stimulating production of megalopae (Brick, 1974). Several recent
studies pointed out the potential beneficial roles of micro-algae in aquaculture rearing
systems, such as maintaining the quality of live feed (Makridis and Olsen, 1999), stabilizing
water quality via either ammonia uptake or oxygen production and producing natural
antibiotics (Tseng et al., 1991). The exact mode of action (e.g. source of micronutrients,
source of immunostimulants, water quality conditioner, and microbial conditioner) of
“green-water” (i.e. high concentrations of selected species of micro-algae) in the
commercial larviculture of several species of marine fish remains however unclear and
therefore requires further study (Sorgeloos, 1995). Most likely, the addition of micro-algae
to the rearing system supported rotifer growth and maintained their nutritional quality, in
CHAPTER 4 – Feeding schedule
78
this way indirectly benefiting the larvae. It can therefore be concluded that although micro-
algae are not a proper initial feed for early crab stages and might cause certain negative
effects during early larval rearing, they seem important to improve the rotifer quality and in
this way improve growth and metamorphosis of the crab larvae.
4.3. Alternative Artemia forms as first feed
In experiments 4 and 5, the positive control (rotifers) always resulted in the best
survival and highest LSI throughout the whole rearing period, confirming that rotifers are
the best first feed.
Among the other treatments, heat-killed umbrella Artemia gave the best survival in
both experiments. Live Artemia nauplii seemed the best in terms of LSI in experiment 5.
The relative high survival and good growth of the treatment “live instar-1 Artemia” was
somehow contradictory to the results of experiment 1. In that experiment Z1 seemed unable
to catch newly-hatched Artemia. Whereas in experiment 1, no aeration was applied, in
experiments 4 and 5, the rearing medium was thoroughly aerated and mixed however.
Possibly, the water turbulence maximised the probability of Z1 to encounter prey, resulting
in a higher incidence of contact and more chance to capture the nauplii. In this respect, the
suitability of live feed and prey selection by the larvae can be explained by the relative
vulnerability of prey items (Dutil et al., 1977; Greene and Landry, 1985; Yen, 1982).
Vulnerability can be defined as the product of two terms, the probability of encounter and
the probability of capture (Pastorok, 1981). The rate of encounter varies directly with the
size and swimming speed of prey, but also with physical properties of the system, while the
efficiency of capture varies inversely with these factors (Swift and Fedorenko, 1975). This
also stresses the impact zootechnical factors such as system design can have on feeding.
Zeng and Li (1999) often found Z1 larvae holding Artemia for a long time but finally
abandoning them usually only having the head or appendages removed. This way, although
not an ideal prey, Artemia can sustain growth and survival of zoeal larvae to some extent.
However, Li et al. (1999) noticed that Z1 larvae (S. paramamosain) fed with Artemia
nauplii usually resulted in lower survival (compared to rotifers), which confirms our results.
Both heat-killed and frozen instar-1 Artemia tended to sink to the bottom of the
rearing containers rapidly and so reduced feed densities in the water column considerably.
Frozen Artemia moreover decomposed rapidly in the rearing water resulting in increased
CHAPTER 4 – Feeding schedule
79
NH4+ and NO2
- concentrations. This probably explains why in both experiments mortality
was highest in this treatment.
In terms of nutritional quality, fresh (live or frozen) Artemia seemed to be better than
heat-killed Artemia, resulting in higher LSI values. Heat treatment of Artemia can cause
protein denaturation, resulting in decreased specific energy, protein solubility and enzyme
activities (García-Ortega et al., 1995). According to these authors, 11-day-old larvae of the
African catfish Clarias gariepinus showed slower growth when fed treated cysts compared
to live Artemia nauplii or untreated cysts that may reflect the effect of the destruction of
enzymes required for feed digestion and absorption.
Despite the poor performance of the larvae receiving non-moving food, the
observation that not all the larvae died in these treatments and were found catching and
consuming the food, shows that zoeae are not restricted to live prey. This non-selective
feeding at early stages also shows a direction to develop artificial feed particles as suitable
alternatives for live feed for mud crab larvae especially at early stages. Levine and Sulkin
(1984b) concluded that calcium alginate microcapsules, or parts thereof, clearly can be
ingested by newly-hatched crab larvae of Eurypanopeus depressus indicating that
brachyuran larvae are capable of capturing and ingesting non-living, non-motile prey. This
suggests that they are not obligate carnivores, but are omnivores whose nutritional needs
could be satisfied by a variety of feed types. Recently, Genodepa et al. (2004) have shown
that microbound diets incorporated with 14C-labelled rotifers are readily ingested by S.
serrata zoeae and megalopae.
In experiment 6, a pilot-scale larval rearing trial was performed using live umbrella-
stage Artemia as first feed. Due to the size of the experiment, it was not possible to run and
compare different treatments. Acceptable survival and growth were obtained however,
comparable to previous trials where rotifers were used as a first feed. Live umbrella-stage
Artemia combine a number of advantageous characteristics in that they are smaller than
Artemia nauplii, are non-moving and thus easy to catch, cause little water deterioration, stay
in suspension quite well (due to the attached chorion) and are nutritionally similar or even
better than Artemia nauplii, making them an acceptable replacement for rotifers. Although
survival and growth of the crab larvae fed live umbrella-stage Artemia was not higher as
previous trials using rotifers, this feeding regimen could be used as a rotifer supplement or
replacement when rotifer production fails.
CHAPTER 4 – Feeding schedule
80
4.4. Feeding schedule
Rotifers and Artemia have been commonly used as the main live feeds for Scylla
larvae in most studies. In many cases, uncertainty of the right time to offer the most suitable
prey that are easily captured, digested and assimilated by the crab larvae at a certain stage
has led to the cofeeding of both prey organisms for a prolonged rearing period. Ruscoe et al.
(2004) recently noted that while the provision of various species of with differing physical
and nutritional characteristics throughout the rearing period may ensure the larvae have a
suitable prey at all times, the difficulties in terms of culture and hatchery management may
make the overall hatchery unprofitable. Moreover, feeding more than one prey type in the
same larval stage, created a competition on dissolved oxygen between the prey and the
cultured larvae and resulted in low water quality due to higher levels of toxic metabolites
(including ammonia) being produced (study on Penaeus semisulcatus, Samocha et al.,
1989). The poor quality of the culture medium in its turn may have an impact on larval
health, feeding ability, and ultimately growth and survival (Ruscoe et al., 2004). For these
reasons, determination of the feeding schedule, i.e. the timing of introduction and cessation
of a prey organism into a culture system needs to be addressed as precisely as possible.
In experiment 7, replacing rotifers by Artemia before DAH 4 (the second day of Z2
stage) clearly compromised the survival and development of the crab larvae. Therefore,
rotifers should be fed to Z1 in order to achieve higher survival.
Results furthermore indicated that rotifers should be replaced by Artemia already in
the Z2 stage. An abrupt shift from rotifers to Artemia on DAH 4 (the second day of Z2)
tended to give similar survival than when rotifers and Artemia were cofed for an extra 2
days. A transition period to shift from one diet to another therefore seemed not necessary.
When rotifer feeding was maintained beyond the zoea 2 stage, lower survival and a delay in
larval development was experienced. This is in accordance with the findings of Ruscoe et al.
(2004) in an experiment where rotifer feeding was discontinued at every larval stage. From
this experiment, the authors concluded that while rotifers are a valuable inclusion in the
feeding regime for larval S. serrata, their use should be limited to the first zoeal stage only
to maximize growth and survival.
When comparing S. paramamosain larvae fed rotifers and Artemia, Zeng (1987; cited
in Li et al., 1999) found no difference in the dry weight; and carbon, nitrogen and hydrogen
content in larvae of both treatments. This suggested that the nutritional value of rotifers can
CHAPTER 4 – Feeding schedule
81
meet the requirements for development of Z2 stage larvae. However, as larvae entered Z3,
those fed with Artemia had considerably higher values and the difference compared to
larvae fed rotifers grew wider as larvae developed. The author suggested that diet
replacement should take place at Z3.
For S. paramamosain, it is apparent that Z2 larvae are capable to ingest rotifers more
easily than Artemia nauplii. The beneficial impact of Artemia feeding on the performance of
Z2 probably is due to their nutritional value rather than their size compared to that of
rotifers. However, from a nutritional perspective, both rotifers and Artemia are far from
ideal and show nutritional inconsistency (Southgate and Partridge, 1998). Whether rotifers
or Artemia are used for Z2 stage larvae might therefore depend on their nutritional value and
general culture conditions and zootechnics. For example, in the above-mentioned analysis
study of Zeng (1987; cited in Li et al., 1999), rotifers did still meet the nutritional
requirements of crab larvae until the Z2 stage and shifting to Artemia feeding was suggested
to take place only in the Z3 stage. While our study and the study by Ruscoe et al. (2004)
indicated this should already be done at Z2 stage.
Based on the feeding schedule applied in experiment 1 (in the stock rearing tanks) and
6, crab larvae were found to be capable of catching and ingesting Artemia meta-nauplii
enriched with emulsions containing highly unsaturated fatty acids from the Z3 stage
onwards.
5. Conclusions and suggestions
Rotifers were the most suitable live feed for Z1 of S. paramamosain. Although micro-
algae were not a suitable first feed for early crab stages, they seemed to improve the quality
of the rotifers resulting in a more successful metamorphosis to the megalopa stage.
Crab larvae could start catching instar-1 Artemia nauplii from the Z2 stage onwards
and the number of consumed prey increased for each consecutive larval stage. The ability of
Z2 to catch Artemia nauplii was slightly variable between batches and individuals.
Artemia are best introduced at the Z2 stage already in order to maximise survival and
growth. Optimal feeding schedules should however also take into account the nutritional
composition of both live foods. An overlap in feeding rotifers and Artemia seems not really
necessary.
CHAPTER 4 – Feeding schedule
82
Live umbrella-stage Artemia were the best replacement for rotifers for feeding Z1 - Z2
compared to other Artemia forms.
The non-selective feeding at early crab larval stages indicates there might be
possibilities to develop artificial diets as alternatives for live feed, in order to reduce the
dependency on rotifer cultures.
From the Z3 stage onwards, crab larvae can catch and ingest enriched Artemia meta-
nauplii.
Acknowledgements
This study was supported by the European Commission (INCO-DC), the Flemish
Inter-University Council (Vl.I.R.-IUC) and the International Foundation for Science (IFS).
CHAPTER 4 – Feeding schedule
83
Table 1 Overview of experimental conditions in experiment 1 to 8
Experiment Container volume (l)
Stocking density (Z1 l-1)
Number of replicates
Temperature (°C)
Salinity (g l-1 )
1 0.02 50 See text 29.0±0.7 30.0±0.5 2 2 40 6 29.5±0.7 29.9±1.0 3 2 40 3/12§ 30.6±1.4 30.0±0.5 4 1 50 3 29.1±1.2 30.0±0.5 5 30 150 5 29.2±0.4 30.5±1.4 6 500 100 8 29.4±0.7 30.0±0.6 7 30 100 4 29.4±1.1 30.1±0.5 8 30 50 5 29.2±0.5 30.1±0.4 § 12 replicates for treatment “rotifers and micro-algae”.
CHAPTER 4 – Feeding schedule
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Table 2 Overview of feeding schedules used in experiment 2 to 8. DAH = day after hatch, Z = zoea, M = megalopa, C1 = first crab, Rot. = rotifers, (Micro-)algae = Chaetoceros spp., HK = heat-killed
DAH 0 1 2 3 4 5 6 7 8 9 10 11 12 … 15 … 22-30
Z1 Z3 Z5 C1 LARVAL STAGE Z2 Z4 M
EXPERIMENT 2 Starvation Artificial diet Micro-algae Rotifers only Rot. + algae
<------ Starvation ------------------------><------ Shrimp diet ----------------------><------ Micro-algae ----------------------><------ Rotifers ---------------------------><------ Rotifers+Micro-algae ---------->
EXPERIMENT 3
<------ Starvation ------------------------><------ Algae ----------------------------->
<------ Rotifers ----------------------------------------------------------------------------------------------><-----Rotifers+algae------->
Starvation Micro-algae Rotifers only Rot. + algae <------ Rotifers+Micro-algae --------------------------------------------> <-----Artemia+algae-------> EXPERIMENT 4 and 5 Starvation Rotifers HK umbrella HK nauplii Live nauplii Frozen nauplii
<------ Starvation ------------------------><------ Rotifers ---------------------------><------ HK umbrella Artemia ----------><------ HK Artemia nauplii -----------><------ Live Artemia nauplii -----------><------ Frozen Artemia nauplii -------->
EXPERIMENT 6 Live umbrella <--- Live umbrella Artemia ------------><---- Enriched Artemia ---> EXPERIMENT 7 1/1 2/2 3/3 4/4 5/5
<-Rot.-> <--- Artemia ---------------------------------------><---- Rot.----> <--- Artemia --------------------------------><---- Rot.----------> <--- Artemia --------------------------><---- Rot.-----------------> <--- Artemia -------------------><---- Rot.------------------------> <-Artemia --------------->
<--- Rot.-----------------------------> 6/4 <------ Artemia -----------------> EXPERIMENT 8
<------ Rotifers --------------------------> 6/4 <------- Artemia -------------------------------------------------->
<--- Rotifers -------------------------------------------> 9/7 <------- Artemia ------------------------------>
<--- Rotifers ----------------------------------------------------------------> 12/9 <------ Artemia ----------------->
CHAPTER 4 – Feeding schedule
85
Table 3 Number of Artemia nauplii remaining after incubating different larval stages of S. paramamosain for 24 hours together with 200 newly-hatched Artemia nauplii in 20-ml vials (experimental vials). Control vials were incubated with the same number of Artemia nauplii, but without crab larvae. Z = zoea, M = megalopa. Experiment 1 Larval stage Number of replicates Experimental vial Control vial TEST 1 Z1 / n.d. n.d. Z2 21 185±9a 196±5a
Z3 26 180±8B 197±3A
Z4 30 173±14B 199±3A
Z5 29 161±27B 199±2A
M 13 43±18B 194±5A
TEST 2 Z1 20 198±2a 199±2a
Z2 20 195±2b 199±2a
Z3 27 187±7B 199±2A
Z4 13 180±11B 200±1A
Z5 / n.d. n.d. M / n.d. n.d. TEST 3 Z1 21 200±1a 200±1a
Z2 34 196±3a 200±1a
Z3 42 187±6B 199±2A
Z4 19 174±11B 198±2A
Z5 38 164±20B 199±2A
M 9 149±23B 199±2A
Values in the same row followed the same superscript letter are not significantly different (P ≥ 0.05, regular letters or P ≥ 0.01, capital letters). N.d. = not determined. Table 4 Survival (%) and larval stage index (LSI) up to DAH 6 of S. paramamosain larvae fed different feeds. Experiments 2 and 3
Survival rate (%) LSI
Experiment 2 Experiment 3 Experiment 2
Experiment 3 Treatment
DAH 3 DAH 6 DAH 3 DAH 6 DAH 3 DAH 6 DAH 3 DAH 6 Starvation 0 / 2±2c C 0 / / 1.0±0.0b B / Artificial diet 1±2c C 0 / / 1.2±0.0b B / / / Micro-algae 7±6bc BC 0 29±26bc BC 1±2b A 1.0±0.0c C / 1.0±0.0b B 1.0±0.0b B
Rotifers only 39±12a A 17±18a 79±7a A 41±37a A 1.4±0.1a A 2.8±0.0a 1.5±0.0a A 2.9±0.0a A
Rotifers+algae 15±4b B 8±2a 54±14ab AB 19±8ab A 1.4±0.1a A 2.8±0.1a 1.5±0.1a A 2.8±0.1a A
Values in the same column followed the same superscript letter are not significantly different (P ≥ 0.05, regular letters and P ≥ 0.01, capital letters).
CHAPTER 4 – Feeding schedule
86
Table 5 Survival (%) from DAH 9 to DAH 22 of S. paramamosain larvae fed different diets. Algae = micro-algae (Chaetoceros spp.). Experiment 3
Experimental design Treatment No of replicates
DAH 9 (Z3)
DAH 12 (Z3-Z4)
DAH 15 (Z4-Z5)
DAH 22 (Megalopa)
Rotifers only 3 22±6a Initial Rotifers + algae 12 11±5b Rotifers only 2 100 25B After pooling on
DAH 9 Rotifers + algae 3 100 79A Rotifers only 1 100 39b B 0 Rotifers + algae 1 100 85a A 48aAfter pooling on
DAH 12 Artemia + algae 1 100 88a A 60a
Values in the same column followed the same superscript letter are not significantly different (P ≥ 0.05, regular letters and P ≥ 0.01, capital letters). Data of initial design are treated by Tukey test (with standard deviation). Pooled data are treated by Fisher exact test (without standard deviation). Table 6 Survival (%), larval stage index (LSI) and water quality parameters of S. paramamosain larvae fed different feeds. Experiments 4 and 5
Survival rate (%) LSI Treatment
DAH 3 DAH 6 DAH 3 DAH 6 NH4
+
(mg l-1) NO2-
(mg l-1)
EXPERIMENT 4 Starvation 2±3b B / n.d. / 0.23±0.15b B 0.03±0.03a
Rotifers 43±9a A / n.d. / 0.50±0.12b AB 0.04±0.03a
Heat-killed umbrella-stage Artemia 20±10ab A / n.d. / 0.45±0.13b AB 0.10±0.06a
Heat-killed instar-1 Artemia 12±16b A / n.d. / 0.40±0.08b B 0.09±0.05a
Live instar-1 Artemia 12±3ab A / n.d. / 0.40±0.08b B 0.06±0.03a
Frozen instar-1 Artemia 0 / n.d. / 0.86±0.23a A 0.12±0.08a
EXPERIMENT 5 Starvation 0 / / / / / Rotifers 75±24a A 37±17a A 1.5±0.1a A 2.8±0.1a A n.d. n.d. Heat-killed umbrella-stage Artemia 47±6b AB 10±1b B 1.0±0.0b B 2.0±0.2b B n.d. n.d. Heat-killed instar-1 Artemia 34±10bc B 6±3b B 1.0±0.0b B 2.0±0.2b B n.d. n.d. Live instar-1 Artemia 26±4bc B 9±3b B 1.0±0.0b B 2.2±0.3b B n.d. n.d. Frozen instar-1 Artemia 23±7c B 9±3b B 1.0±0.0bB 2.1±0.2b B n.d. n.d. Values in the same column for the same experiment followed the same superscript letter are not significantly different (P ≥ 0.05, regular letters and P ≥ 0.01, capital letters). N.d. = not determined.
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Table 7 Survival (%) and larval stage index (LSI) of S. paramamosain larvae receiving different rotifer/Artemia feeding schedules. For treatment descriptions refer to Table 2. Experiment 7
Survival rate (%) LSI Feeding schedule
DAH 3 DAH 6 DAH 9 DAH 3 DAH 6 DAH 9 1/1 61±18b A 47±26a 34±11b A 2.0±0.1a 2.9±0.2a 4.0±0.1a
2/2 86±13ab A 57±14a 49±10ab A 2.0±0.1a 2.8±0.2a 4.0±0.1a
3/3 74±21ab A 44±17a 33±12b A 2.0±0.0a 2.9±0.1a 4.0±0.1a
4/4 97±4a A 75±25a 69±17a A 2.0±0.0a 3.0±0.0a 4.0±0.0a
5/5 88±12ab A 56±18a 41±9ab A 2.0±0.0a 3.0±0.0a 4.0±0.0a
6/4 90±14ab A 73±27a 59±23ab A 2.0±0.0a 3.0±0.0a 4.0±0.0a
Values in the same column followed the same superscript letter are not significantly different (P ≥ 0.05, regular letters and P ≥ 0.01, capital letter). Table 8 Survival (%) and larval stage index (LSI) of S. paramamosain larvae receiving different rotifer/Artemia feeding schedules. For treatment descriptions refer to Table 2. Experiment 8
Survival rate (%) LSI Feeding schedule
DAH 9 DAH 12 DAH 15 DAH 9 DAH 12 DAH 15 6/4 36±08a 21±10a 17±10a 3.9±0.3a A 4.3±0.4a 4.5±0.4a
9/7 35±10a 14±8a 11±7a 3.6±0.3b A 4.0±0.2a 4.2±0.5a
12/9 32±15a 11±6a 6±3a 3.6±0.3b A 3.8±0.3a 3.9±0.4a
Values in the same column showing the same superscript letter are not significantly different (P ≥ 0.05, regular letters and P ≥ 0.01, capital letters). Table 9 Sizes (µm) of used live feeds and those of Scylla eggs and larvae. n = number of observations Length Width Diameter n LIVE FEED Adult rotifers 164±22 120±22 50 Artemia cysts (with shells) 235±15 100 Newly-hatched Artemia 601±101 378±55(1) 15 24h-enriched Artemia 763±102 469±51 15 NEWLY-SPAWNED EGGS S. paramamosain(2) 287.6±9.9c C 2910 S. tranquebarica(3) 299.5±9.2b B 27 S. olivacea (3) 300.6±4.4b B 22 S. serrata(3) 329.1±8.3a A 27 CARAPACE OF S. paramomasain LARVAE(4) Z1 452 Z2 571 Z3 714 Z4 1058 Z5 1577 M 1280 840 Values in the same column followed the same superscript letter are not significantly different (P ≥ 0.05 and P ≥ 0.01). (1) Width of Artemia including appendages (around 150 µm excluding appendages); (2) Our data, sampled from 97 batches of eggs (30 eggs batch-1); (3) Data from Emilia T. Quinitio, Southeast Asian Fisheries Development Center, pers. com.; (4) Measured from drawings in Jones et al. (in press) and measuring methods based on Bigford (1978).
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Mean; Box: Mean-SD, Mean+SD; Whisker: Min, Max
No
of re
mai
ning
Arte
mia
Experiment vialControl vial
Z1 Z2* Z3** Z4** Z5** M**
Stage of crab larvae
20
40
60
80
100
120
140
160
180
200
Figure 1. Number of remaining Artemia after incubating different S. paramamosain larval stages for 24 hours together with 200 Artemia nauplii in 20-ml vials (experimental vials). Control vials were incubated with the same number of Artemia nauplii, but without crab larvae. Z = zoea, M = megalopa. Mean = average of 3 tests. * and ** for P < 0.05 and P < 0.01, respectively indicate that the number of remaining Artemia in the experimental vial are significantly different with those in the control vial. Experiment 1.
CHAPTER 5
Influence of the content of highly unsaturated fatty acids in the live feed on larviculture success of mud crab
(Scylla paramamosain)
Nghia, T.T.*1, Wille, M.2, Vandendriessche, S.2 and Sorgeloos, P.2
1 College of Aquaculture and Fisheries, Can Tho University, Vietnam. Email: [email protected] 2 Laboratory of Aquaculture & Artemia Reference Center, Ghent University, Belgium. Email: [email protected]
Abstract Four experiments were carried out to investigate the effects of the level of
docosahexaenoic acid (DHA), eicosapentaenoinc acid (EPA) and arachidonic acid (ARA) in the live feed on survival, growth and metamorphosis success of mud crab Scylla paramamosain larvae. Six different lipid emulsions, varying in the level of total n-3 and n-6 highly unsaturated fatty acids (HUFA), DHA, EPA and ARA were used to manipulate the fatty acid profile of the live feed (rotifers and Artemia). Fatty acid profiles of the live feed and crab larvae at zoea 1, 3 and 5 stages were analyzed to study uptake of HUFA by the crab larvae. Larviculture success was measured through survival, larval development rate (expressed as larval stage index) and success of metamorphosis (survival and time and duration of first and second metamorphosis).
The fatty acid content of the live feed affected the fatty acid profiles of the crab larvae. In most experiments, survival rate in the zoeal stages was not statistically different among treatments. Larval development rate and metamorphosis success were however more strongly affected by the dietary treatments. In this respect, the DHA/EPA ratio in the live feed seems to be a key factor. Enrichment emulsions with very high (50 %) total HUFA content but low DHA/EPA ratio (0.6) or zero total HUFA content caused growth retardation and/or metamorphosis failure. An emulsion with moderate total HUFA (30 %) and high DHA/EPA ratio (4) was the best in terms of larval development rate during the zoeal stages and resulted in good metamorphosis. The optimal DHA/EPA ratio of live feed enrichment emulsions for early stages (Z1 - Z2) could however be lower than 4. Dietary arachidonic acid seemed to improve first metamorphosis, but its exact role needs further clarification. For the larval rearing of Scylla paramamosain, it is recommended to use enrichment media with a total n-3 HUFA content of approximately 30 %, with a DHA/EPA ratio of minimum 1. Further research needs to be performed on the total HUFA and DHA/EPA ratio requirements for each larval crab stage. The role of ARA in metamorphosis also needs to be further elucidated.
CHAPTER 5 – Feed quality
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1. Introduction
Mud crabs, Scylla spp., are large, primarily carnivorous portunid crabs, which are
strongly associated with the mangrove areas throughout the Pacific and Indian oceans
(Keenan, 1999a). They form the basis of substantial, but mainly artisanal fishery operations
throughout their distribution. South East Asia has a long history in traditional forms of mud
crab farming (e.g. fattening), but a number of factors have triggered a renewed interest in
mud crab farming in recent years. Johnston and Keenan (1999) listed a number of benefits
of mud crab over shrimp farming as providing more reliable income and higher profit
margins and return of initial investment resulting from higher survival because of superior
adaptation to the mangrove environment, higher price per kg with little capital or food input,
high growth rate and lower disease risk. Mud crab farming however currently relies entirely
on wild seed stock and the main obstacle for the development of mud crab culture is the
availability of hatchery-reared seed (Liong, 1992; Keenan, 1999a; Mann et al., 2001;
Shelley and Field 1999; Xuan, 2001). Of the four Scylla species, Scylla paramamosain is
dominant in Vietnam (Keenan et al., 1998; Keenan, 1999b).
One of the major factors influencing the survival and growth of larvae of marine
species is the dietary HUFA composition. Sorgeloos et al. (2001) reviewed the history of
research on dietary HUFAs. In the 1980’s, most attention was dedicated to the presence of
eicosapentaenoic acid (20:5n-3, EPA) in Artemia as a guarantee for successful production of
marine fish larvae. In the late 1980’s and early 1990’s, more attention was paid to the level
of docosahexaenoic acid (22:6n-3, DHA) because good survival appeared to be correlated
with EPA, but DHA improved larval quality and growth. The importance of DHA, more
particularly the requirement for high DHA/EPA ratios in promoting growth, stress
resistance, and pigmentation was also revealed (Sorgeloos et al., 2001). Recent work
showed that besides DHA not only highly unsaturated fatty acids of the n-3 series are
important but that also arachidonic acid (20:4n-6, ARA) may play a significant role (Castell
et al., 1994; Estévez et al., 1999, Koven et al., 2000). ARA may improve larval growth and
pigmentation in several marine fish species since it provides precursors for eicosanoid
production.
Many studies on supplementation of essential fatty acids through the live feed, mainly
rotifers (Dhert et al., 2001; Olsen et al., 1993) and Artemia (Sorgeloos et al., 2001;
Watanabe, 1982) have been performed in marine and freshwater organisms, including fish
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(e.g. Ashraf, 1993; Sargent et al., 1995; Watanabe, 1993), shrimp (e.g. Lavens and
Sorgeloos, 2000; Rees et al., 1994) and bivalves (e.g. Caers et al., 1998; Coutteau et al.
1996). Although data on the nutritional requirements of larval stages of brachyuran crabs
are rather limited, the requirement for dietary fatty acids has been demonstrated for several
species, including Scylla spp. (Hamasaki et al., 1998; Kobayashi et al., 2000; Levine and
Sulkin, 1984a; Suprayudi et al., 2002b; Takeuchi et al., 1999; Takeuchi et al., 2000).
Despite these findings, problems to develop reliable zootechnics and the often low and
inconsistent larval survival for Scylla larvae have hampered nutritional research
considerably and contradictory results have been reported. Takeuchi et al. (2000) for
example showed that mud crab larvae fed rotifers and Artemia enriched with HUFAs
presented increased survival and performance. Hamasaki et al. (2002b) on the other hand
reported that elevated levels of EPA in the live feed resulted in abnormal development and
mortality of the larvae at metamorphosis. In a study comparing different Artemia strains and
Artemia enrichment products, Mann et al. (2001) found no influence of the n-3 HUFA level
on the ability of the larvae to complete development. From this it is clear that exact dietary
requirements for n-3 HUFA of mud crab larvae have not been established yet.
In order to define more clearly the importance of n-3 HUFA in the diet of Scylla
larvae, the present study evaluates the effect of the level and ratio of specific n-3 HUFAs
(DHA and EPA) and ARA in the live feed on the fatty acid composition and culture
performance of mud crab S. paramamosain larvae.
2. Materials and methods
2.1. Source of larvae
Fully gravid crabs were bought from local markets in the coastal area and transported
to the hatchery. Prior to stocking in the hatchery, the crabs were bathed in a 100 µl l-1
formalin solution for 1 hour. The crabs were housed individually in 100-l compartments of a
roofed 2 × 2 × 0.5 m cement tank, equipped with a biofilter. Rearing water (30 ± 1 g l-1) was
diluted from brine (90 - 110 g l-1) with tap water and chlorinated before use. Water
temperature was not controlled, but varied around 28 °C. Each crab was daily fed 10 - 15 g
of fresh marine squid, bivalve or shrimp meat alternately.
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After 3 - 5 days of acclimation, unilateral eyestalk ablation was applied to induce
spawning. After spawning, berried crabs were again bathed in a 100 µl l-1 formalin solution
for 1 hour and transferred to a 70-l plastic tank connected to a biofilter for incubation. Daily
management consisted of siphoning out waste material and shedded eggs from the tank
bottom and controlling temperature (30 °C), salinity (30 g l-1) and ammonia and nitrite
levels. Every other day, the crabs were bathed in a 50 µl l-1 formalin solution for 1 hour to
reduce or prevent infestation of the eggs with fungi and bacteria. During egg incubation, the
crabs were not fed.
One to two days prior to hatching, the female was moved to a 500-l fibreglass tank in
order to provide a clean and spacious environment for the hatching larvae. At 30 °C,
hatching normally occurred after 10 days of incubation.
When the hatching process was completed, larvae were selected based on their photo-
tactic behaviour. Aeration in the hatching tank was therefore turned off for several minutes
and the larvae that were actively swimming up to the surface were collected by gently
scooping them from the surface. In order to slowly acclimate the larvae to the new rearing
conditions, the larvae were then placed in a 50-l plastic mesh bucket and slowly rinsed with
water from the larval rearing containers for 20 to 30 minutes.
2.2. Larval rearing
Larval rearing systems and procedures
Two different rearing systems were applied in this study. In experiments 1 and 2,
larvae were reared in the recirculating system consisting of 30-l (experiment 1) or 100-l
(experiment 2) cylindro-conical fibreglass tanks connected to a submerged biological filter.
An upwelling system with a water renewal rate of 100 % every 3 - 4 hours was used. Water
was evacuated at the water surface and passed through a 70 or 300 µm filter screen during
the rotifer and Artemia feeding stage respectively, and thus retained larvae and live feed in
the culture tank. Gentle aeration was applied to all rearing tanks. Formalin at a
concentration of 20 µl l-1 was applied to the whole system every 2 days to prevent or reduce
fungi and bacteria development.
In experiments 3 and 4 a small-scale static rearing mode consisting of 1-l acrylic
bowls was used. The bowls were randomly distributed over a heated water bath to provide
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93
identical rearing conditions. Like for the recirculating system, gentle aeration was applied to
all bowls. Each day the remaining larvae were pipetted into new bowls containing fresh
seawater. These “new” bowls were incubated beforehand in the same water bath to
equilibrate temperature. In order to exclude bacterial interference, 10 mg l-1 Oxytetracycline
was applied to the bowls daily. In experiment 3, each treatment was also repeated in two
100-l fibreglass tanks (operated in recirculation mode as in experiment 1 and 2).
Only in experiments 3 and 4 the larvae were reared through to the megalopa and first
crab stage. In experiment 3, megalopae were separated daily from the rearing containers and
redistributed by treatment in 1-l bowls at a density of 6 - 8 megalopae bowl-1. Substrate (1-
cm3 pieces of PVC sponge) as for hiding space was provided in each bowl to prevent
cannibalism. In experiment 4, megalopae were separated from the culture several times per
day and reared individually to crab stage in 100-ml cups without aeration.
Source and treatment of the rearing water for the larvae was the same as for
broodstock rearing.
Larval rearing conditions for all experiments are summarized in Table 1.
Live feed culture and enrichment
- Culture practices
The same rotifer strain, Brachionus plicatilis L-strain with lorica length and width of
164 ± 22 and 120 ± 22 µm, respectively, was used in all experiments. In experiments 1, 2
and 4, rotifers were cultured outdoor in an integrated recirculating system. The system
consisted of two 10-m3 tanks inoculated with Chlorella spp. and stocked with Tilapia (1 kg
m-3), connected to a 4-m3 fibreglass rotifer culture tank. When the algal concentration
reached 10 million cells ml-1, rotifers were stocked at 100 ml-1 and the system was
recirculated (1 complete exchange every 4 hours). Starting from the third day, part of the
rotifer population was then harvested daily as feed for the crab larvae. In experiment 3,
rotifers were cultured on baker yeast in an indoor recirculating system in 100-l fiberglass
tanks (Suantika, 2001). Water recirculation rate in this system was 100 % day-1.
Artemia (Vinh Chau strain) cysts were disinfected with chlorine, incubated and
hatched following standard methods (Sorgeloos et al., 1986).
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- Enrichment practices
In order to manipulate the fatty acid content of the live feed, rotifers and Artemia were
enriched with different ICES (International Council for Exploration of the Sea) standard
reference emulsions (ICES, 1997).
Rotifer enrichment was performed at a density of 500 ml-1, using two separate doses
of 0.125 g l-1 each at a 3-hour interval. The temperature and salinity of the water was
maintained at 25 to 30 °C and 25 to 30 g l-1, respectively.
For Artemia enrichment, newly-hatched Artemia were concentrated to 200 ml-1 and
the oil emulsions were used at two separate doses of 0.3 g l-1 each at a 12-hour interval.
Temperature and salinity were kept at 30 °C and 30 g l-1, respectively.
In experiment 2, control Artemia were kept in an algal suspension (5 ± 1 million cells
of Chaetoceros ml-1) for the duration of the enrichment period of the other treatments, to
maintain a uniform size with the enriched Artemia meta-nauplii in the other treatments.
In experiment 3, control Artemia were starved at 30 °C for 24 hours.
In experiment 4, the control rotifers and Artemia were enriched with the commercial
product Culture Selco® (28 % n-3 HUFA with a DHA/EPA ratio of 1; INVE Aquaculture
NV, Belgium), under similar conditions (live feed density, salinity and temperature,
enrichment dose and time) as for the ICES emulsions.
Feeding
In experiment 1, rotifers were the sole live feed for all crab larval stages. Rotifers
were added daily (45 ml-1) to the rearing tanks after the old ones were flushed out almost
completely (3 - 5 ml-1 left).
In experiments 2, 3 and 4, crab larvae were fed rotifers from DAH 0 - 6 (Z1 - Z2) at
the same density as experiment 1 and then Artemia afterwards (7 - 10 ml-1 daily).
2.3. Experimental design
The design of the 4 experiments is presented in Table 2. Six different ICES emulsions
or combinations thereof were tested for rotifer and Artemia enrichment: emulsion 0/- (a
coconut emulsion, free of HUFA, mainly consisting of saturated fatty acids); emulsion
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95
30/0.6 (containing approximately 30 % n-3 HUFA on total fatty acids with a DHA/EPA
ratio of 0.6); emulsion 30/4 (30 % n-3 HUFA with a DHA/EPA ratio of 4; emulsion 50/0.6
(50 % n-3 HUFA with a DHA/EPA ratio of 0.6); emulsion 30/1/ARA (having
approximately 30 % n-3 HUFA and a ratio of ARA/EPA/DHA of 1/1/1) and emulsion
30/4/ARA (a mixture of 75 % of the 30/4 emulsion and 25 % of an emulsion containing 40
% arachidonic acid).
In experiments 2 and 3, the same enrichment emulsions were used for rotifers and
Artemia enrichment. In experiment 4, one treatment was included (0.6-4) where rotifers
were enriched with the 30/0.6 emulsion and Artemia with the 30/4 emulsion. Hence,
treatments in experiment 4 were designated as treatment 0.6-0.6 (corresponding to treatment
30/0.6 in the other experiments), treatment 4-4 (i.e. treatment 30/4) and treatment 0.6-4. For
each experiment, a control treatment was included (see section “Live feed culture and
enrichment” and Table 2). Where appropriate, the control treatments are clarified as:
“Chlorella” control, “Chaetoceros” control, “starvation” control and “1-1” control for the
control treatments of experiments 1, 2, 3 and 4 respectively.
2.4. Evaluation criteria
Fatty acid composition
In experiment 3, the ICES emulsions, the different enriched live feeds (rotifers and
Artemia) and the crab larvae (Z1, Z2 and Z5) were sampled for fatty acid analysis. Samples
were washed with freshwater and stored under –80°C until fatty acid methyl ester (FAME)
analysis. Fatty acid composition was determined by gas chromatography. FAMEs were
prepared via a procedure modified from Lepage and Roy (1984). The method consists of a
direct acid-catalized transesterification of dry samples (ranging from 10 - 150 mg) without
prior lipid extraction. An internal standard 20:2(n-6) or 22:2(n-6) was added prior to the
reaction. FAMEs were extracted with hexane. After evaporation of the solvents the FAMEs
were prepared for injection by re-dissolving them in iso-octane (2 mg ml-1). Quantitative
determination was done by a Chrompack CP9001 gaschromatograph equipped with an auto-
sampler and a TPOCI (Temperature programmable on-column injector). Injections (0.5 µl)
were performed on column into a polar 50 m capillary column, BPX70 (SGE Australia),
with a diameter of 0.32 mm and a layer thickness of 0.25 µm, connected to a 2.5 m methyl
CHAPTER 5 – Feed quality
96
deactivated precolumn. The carrier gas was H2, at a pressure of 100 kPa and the detection
mode was FID (flame ionization detection). The oven was programmed to rise from the
initial temperature of 85 to 150 °C at a rate of 30 °C min-1, from 150 to 152 °C at 0.1 °C
min-1, from 152 to 172 °C at 0.65 °C min-1, from 172 to 187 °C at 25 °C min-1 and to stay at
187 °C for 7 min. The injector was heated from 85 to 190 °C at 5 °C sec-1 and stayed at 190
°C for 30 min. Identification was based on standard reference mixtures (Nu-Chek-Prep, Inc.,
U.S.A.). Integration and calculations were done on computer with a software program
Maestro (Chrompack). Each sample was analyzed twice. The results are expressed as mg
FAME per gram of dry weight (mg g-1 DW or mg g-1).
Only the most important essential fatty acids and groups (ARA, EPA, DHA, total n-3,
total n-6 and total HUFA) and ratio’s thereof (DHA/EPA, ARA/EPA and Σn-3/Σn-6) are
reported in the results section. Total HUFA was defined as all fatty acids with more then 20
carbon atoms and 3 or more unsaturated carboxyl bonds, total n-3 HUFA as all n-3 fatty
acids ≥ 20:3n-3.
Larval performance
In experiments 1 and 2 (in 30 to 100-l containers, respectively), the average survival
rate in the zoeal stages was estimated by volumetric sampling. Depending on the tank
volume and the density of the surviving larvae, triplicate 300- to 1000-ml samples were
taken from each tank. In experiments 3 and 4 (in 1-l bowls) the average survival rates were
calculated by individually counting all surviving larvae in each replicate.
Zoeal development was monitored every three days by identifying the zoeal instar
stage of a sample of larvae and assigning it a value: first zoea (Z1) = 1; second zoea (Z2) =
2, etc. To compare the larval development in each treatment, an average larval stage index
(LSI) was calculated from the average LSI value of replicate tanks in the same treatment.
Five or ten larvae (in 30-l and 100-l tanks respectively) were sampled from every tank in
experiments 1, 2 and 3 to calculate the average LSI of each tank. The sampled larvae were
staged under a dissecting microscope. Only in experiment 4, larvae were staged visually
upon daily counting the surviving larvae.
Two parameters were used to evaluate the success of metamorphosis in experiments 3
and 4:
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- Firstly the metamorphosis rate (MR, %) or the percentage survival through metamorphosis
was determined. Since there are 2 metamorphoses, the 2 metamorphosis rates were
calculated: (i) the percentage of Z5 that survives through metamorphosis to megalopa
(MR1) and (ii) the percentage of megalopa that survives through metamorphosis to crab
stage (MR2).
- Secondly also the average time needed for the larvae to go through metamorphosis and the
duration (minimum and maximum time needed) of both metamorphoses were recorded.
- Lastly the survival rates of Z1 to megalopa and crab stage (only for experiment 4) were
graphically presented based on the pooled data of all replicates in each treatment.
2.5. Statistical analysis
One-way analysis of variance (ANOVA) was used to compare data. Homogeneity of
variance was tested with the Levene test (P or α value was set at 0.05). If no significant
differences were detected between the variances, the data were submitted to a one-way
ANOVA. The Tukey HSD post-hoc analysis was used to detect differences between means
and to indicate areas of significant difference. If significant differences were detected
between variances, data were transformed using the arcsine-square root (for percentage, i.e.
survival rate) or logarithmic transformations (for other parameters) (Sokal and Rohlf, 1995).
The two-tailed Fisher exact test (modified from the contingency table method) was used to
compare ratio (expressed in percent) data of pooled treatments. The Pearson’s coefficient
was used to examine the correlation between the fatty acid composition of the live feed and
the crab larvae; and the correlation between the fatty acid composition of the live feed and
larval development (i.e. LSI values and metamorphosis rates). All data are presented as
mean ± standard deviation when using the Tukey test or as a ratio/percentage (without
standard deviation) when the Fisher exact test was used. P was set at both 0.05 and 0.01.
Whenever differences are significant at P < 0.01, this is also indicated. All analyses were
performed using the statistical program STATISTICA 6.0.
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3. Results
3.1. Fatty acid composition of live feed and crab larvae (experiment 3)
Only in experiment 3, the fatty acid profile of the live feed (Table 4) and the larvae
(Table 5) was determined. The composition of the emulsions (the same batch was used for
all experiments) is presented in Table 3. We assume that, except for the control treatments
(which differed between experiments), similar live food enrichment levels were obtained in
the other experiments. Rotifer composition was clearly influenced by the enrichment
treatments. Total n-3 content of the rotifers increased from 4 mg g-1 in the control to 26 to
35 mg g-1 in treatments 30/0.6, 30/4 and 30/4/ARA. The highest n-3 level (49 mg g-1) was
obtained in treatment 50/0.6. Treatment 0/- resulted in a level similar to the control. As
expected, treatment 50/0.6 resulted in the highest EPA level in the rotifers (29 mg g-1).
DHA/EPA ratio of the rotifers was highest for treatments 30/4 and 30/4/ARA (2.57 and 2.1,
respectively), compared to 0.71 to 0.84 for the other treatments. Treatment 30/4/ARA
resulted in elevated ARA and total n-6 levels and hence a high ARA/EPA ratio. The control
and 0/- rotifers were especially low in ARA, EPA and DHA. Similar patterns were observed
for Artemia. Using the same enrichment medium, DHA levels and the DHA/EPA ratio
were however lower in Artemia compared to rotifers (except for treatment 30/4, where the
DHA level of Artemia was slightly higher than that of rotifers). Especially the DHA level in
the control Artemia was extremely low, giving a very low DHA/EPA ratio. Absolute levels
of most other fatty acids were however higher in Artemia. Compared to un-enriched rotifers,
the control Artemia also contained relatively high amounts of EPA, total n-3 and ARA.
Overall, the rotifer and Artemia composition reflected very well the total n-3, EPA, DHA
and ARA levels of the emulsions and thus the theoretic design of the experiments.
The fatty acid composition of the different crab larval stages in experiment 3 is given
in Table 5. The dietary fatty acid level in its turn affected the composition of the larvae.
Treatment 50/0.6 led to the highest total n-3 and EPA level in Z3 and Z5, while intermediate
results were obtained for treatments 30/0.6, 30/4 and 30/4/ARA. The DHA level and
DHA/EPA ratio was increased in all treatments except 0/-, but was highest for 30/4 and
30/4/ARA. A very low DHA/EPA ratio was observed for Z5 in the control and 0/-
treatments (0.11 and 0.16, respectively). The ARA content of Z3 and Z5 in the 30/4/ARA
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99
treatment (5 and 7 mg g-1, respectively) was higher then those in the other treatments.
Treatment 30/4/ARA also resulted in the highest ARA/EPA ratio in the crab larvae.
In Table 6, the correlation coefficients between specific fatty acid levels in the crab
larvae and those of the live feed are summarized. For Z3, the EPA level, and the DHA/EPA,
ARA/EPA and n-3/n-6 ratio were significantly correlated to those of the rotifers (all at P <
0.01). The level of ARA, DHA and total n-3 were weakly correlated however (r2 = 0.36,
0.62 and 0.65, respectively). For Z5, all investigated fatty acid levels and ratios were
significantly correlated to those of the Artemia (P < 0.01, except ARA/EPA ratio at P <
0.05).
3.2. Zoeal survival
Survival during the zoeal stages in experiments 1 to 4 is presented in Table 7 to 10.
Survival was generally low in later larval stages and high variability was observed between
replicates. This makes comparison difficult and hence not many significant differences in
survival were observed between any of the treatments. In experiment 3 (Table 9) a quite low
survival (although not significantly different) was observed on DAH 15 for treatment 30/4,
resulting from a high mortality from DAH 12 to 15. In experiment 4 (Table 10), on DAH 9,
survival was lower in treatment 0.6-0.6 compared to the control (P < 0.05).
3.3. Larval development rate during the zoeal stages
Experiment 1
Larval development rates (expressed as LSI) in the different treatments are presented
in Table 7. From DAH 6 to 12, the LSI values in treatment 0/- were significantly lower than
in the “Chlorella” control and treatments 30/0.6 and 30/4 (P < 0.01, except on DAH 9 at P
< 0.05). Treatment 50/0.6 had intermediate values. By DAH 15 however, no significant
differences were observed anymore. Overall, a better growth rate was observed for
treatments 30/0.6, 30/4 and the control compared to the other treatments. Whereas the
control and treatment 30/0.6 performed better in early larval stages (from DAH 3 - 9),
treatment 30/4 became better in late larval stages (from DAH 12 - 15). Treatment 30/0.6
tended to have higher LSI values than treatment 50/0.6 on all sampling days.
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Experiment 2
Table 8 presents the LSI values in experiment 2. On DAH 6, a significantly higher
development rate was observed for treatment 30/4 compared to the control and treatments
0/-, 30/0.6 and 50/0.6 (P < 0.01). LSI values of treatment 30/1/ARA were higher than the
control and treatments 30/0.6 and 50/0.6 (P < 0.05). On DAH 9 and 12, the LSI in
treatments 30/4 and 30/1/ARA were higher than in 0/-, 30/0.6 and 50/0.6 (P < 0.01). The
“Chaetoceros” control had intermediate results. LSI values of 30/1/ARA were slightly
lower than those of treatment 30/4. LSI values of the control treatment tended to be higher
than those of treatments 0/-, 50/0.6 and 30/0.6. In contrast with the previous experiment, the
LSI values of treatment 50/0.6 tended to be better than those of treatment 30/0.6 on most
sampling days.
Experiment 3
Larval development rates of experiment 3 are presented in Table 9. On DAH 3, the
LSI values of treatments 30/4 and 30/4/ARA were significantly higher than for all other
treatments (P < 0.01). Although not always statistically significant, this difference persisted
throughout the larval rearing period. The “starvation” control and treatment 0/- always gave
the lowest LSI values. For treatments 50/0.6 and 30/0.6, intermediate larval development
rates were found.
Experiment 4
LSI values in experiment 4 are shown in Table 10. On DAH 3, LSI values of the “1-1”
control (Culture Selco enriched rotifers and Artemia) and treatment 4-4 (or 30/4) were
significantly higher (P < 0.01, except treatment 4-4 at P < 0.05) than those in treatments
0.6-0.6 (or 30/0.6) and 0.6-4. LSI values in these treatments remained high throughout
larval development. No differences were detected between the control treatment and
treatment 4-4 on any of the sampling days.
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3.4. Metamorphosis
In this study, larvae were only reared to the megalopa and first crab stage in
experiments 3 and 4.
Experiment 3
The metamorphosis rates of Z5 to megalopa and megalopa to first crab (MR1 and
MR2, respectively) are presented in Table 11. MR1 of treatment 30/4/ARA was
significantly higher (50 %) in the other treatments (ranging from 30 to 34 %) at P < 0.05.
For MR2, the “starvation” control and treatment 0/- had a significantly lower percentage of
megalopa reaching crab stage compared to the other treatments (P < 0.01). The treatments
with a high DHA/EPA ratio (30/4 and 30/4/ARA) gave a MR2 of over 90 %; the treatments
with a low DHA/EPA ratio (50/0.6 and 30/0.6) resulted in a metamorphosis rate of 73 %,
while in the treatments with a low total n-3 HUFA content (control and 0/-) only about 10 %
of the megalopae could metamorphose to crabs.
Also the average time needed to reach metamorphosis and the time period (minimum-
maximum) needed for all larvae within a treatment to go through the first and second
metamorphosis (Table 12) were affected by the dietary treatments. The first megalopae
appeared on DAH 15 in treatments 30/0.6 and 30/4/ARA. In the ‘starvation” control, the
first megalopae only appeared by DAH 18. For the first metamorphosis, a significant
difference (P < 0.01) in the mean metamorphosis time was found between the control and
treatments 30/0.6 and 30/4/ARA. On average, larvae in the control needed 1 to 2 days more
to metamorphose to megalopae compared to the other treatments. For the second
metamorphosis period, the average time to metamorphose to the first crab stage was
significantly shortest (P < 0.01) in treatments 30/4 and 30/4/ARA compared to treatment
50/0.6. In general, metamorphosis time, as well as its total duration was shorter for
treatments 30/4 and 30/4/ARA (24 days); intermediate values were obtained for treatment
30/0.6 (26 days) and the control and treatments 0/- and 50/0.6 had the longest
metamorphosis time (± 27 days).
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Experiment 4
The first (MR1) and second (MR2) metamorphosis rates and its duration in
experiment 4 are shown in Tables 13 and 14 respectively. No statistical differences were
found between the treatments for both metamorphosis rates. The highest MR1 value was
found for treatment 4-4 while the lowest was in the “1-1” control treatment. Also the second
metamorphosis did not differ significantly between treatments. The MR2 value was
however highest in treatment 4-4, followed by treatments 0.6-0.6, 0.6-4 and the control. The
average first metamorphosis period of treatment 4-4 was significantly shorter (± 18 days)
compared to treatment 0.6-4 (± 20 days) (P < 0.01); while those of the “1-1” control and
treatment 0.6-0.6 were in between (± 19 days) (P < 0.01). The average time needed to
complete second metamorphosis was significantly longest for treatment 0.6-4 (± 27 days)
and shortest in the control treatment (± 25 days) (P < 0.01). Those of treatments 0.6-0.6 and
4-4 were intermediate (± 26 days).
First and second metamorphosis success are also illustrated in Figures 1 and 2. It can
be seen that first metamorphosis starts around DAH 16 in all treatments. From the normal
distribution curves it is obvious that first metamorphosis is centred around DAH 18 and 19
in treatments 4-4 and the “1-1” control, while it was protracted over more than one week in
treatments 0.6-0.6 and 0.6-4. Figure 2 shows that there were 2 peaks in the metamorphosis
to crab stage (around DAH 25 and 29) in treatment 0.6-4, while only 1 peak was found in
the other treatments.
In Figure 3, the cumulative metamorphosis rates from Z1 to megalopa and crab of the
different treatments is presented. From this it is obvious that treatment 4-4 outperforms all
other treatments (P < 0.01). The “1-1” control, was not different from treatment 0.6-4, but
significantly better then 0.6-0.6 (P < 0.05 and 0.01 for the first and second metamorphosis,
respectively).
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3.5. Correlation between the fatty acid composition of the live feed and the crab larvae, and
larval development rate and metamorphosis success
LSI in relation to the fatty acid composition of the live feed
The significant correlation (P < 0.01) between the rotifer DHA/EPA ratios and the LSI
values on DAH 3 and 6 (r2 = 0.86 and 0.87, respectively) indicates that the DHA/EPA ratio
was the most important criterion in the dietary fatty acid composition for early-stage crab
larvae (Table 15).
The significant correlations between the DHA content (P < 0.01), total n-3 content (P
< 0.05) and DHA/EPA ratio (P < 0.01) of Artemia with the LSI values of crab larvae from
DAH 9 - 15 (r2 ranging from 0.77 to 0.98) proves that besides the DHA/EPA ratio, also the
absolute DHA and total n-3 content become more imperative for the crab larvae in the
Artemia-feeding stage.
The content of the other fatty acids (ARA and EPA) or ratios (ARA/EPA and Σn-
3/Σn-6) of the live feeds (rotifers and Artemia) were not correlated with the LSI values of
the crab larvae.
LSI in relation to the fatty acid composition of the crab larvae
Similar to the correlation between the fatty acid composition of the live feed with the
LSI, only the DHA/EPA ratio of the Z3 larvae was found significantly correlated with the
LSI values of crab larvae sampled on DAH 6 (r2 = 0.91, P < 0.01).
Besides the highly significant correlation between the DHA/EPA ratio of Z5 and the
LSI value on DAH 15 (r2 = 0.96, P < 0.01), also the larval DHA and total n-3 contents were
significantly correlated with the LSI value of the larvae (r2 = 0.94, P < 0.01 and 0.78, P <
0.05 respectively).
Metamorphosis success in relation to the fatty acid composition of the live feed and the crab
larvae
Following the same pattern as for the LSI, the DHA and total n-3 contents, and the
DHA/EPA ratio of Artemia (r2 ranging 0.8 to 0.92) and Z5 (r2 ranging from 0.69 to 0.93)
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correlated significantly with the second metamorphosis rate (MR2). All correlations were
significant at P < 0.01, except P < 0.05 for total n-3.
For the first metamorphosis however, only the ARA content (of both Artemia and Z5)
and the ARA/EPA ratio (of Artemia) correlated significantly with the MR1 value (P < 0.05).
4. Discussion
4.1. Fatty acid composition of live feed and crab larvae
As was expected, the ICES emulsions influenced the ARA, EPA, DHA and total n-3
contents, and thus the relating DHA/EPA, ARA/EPA and Σn-3/Σn-6 ratios, of the live feed
significantly at P < 0.01 (treated by Pearson correlation from Tables 3 and 4, not shown in
the tables).
In general, the elevated HUFA content in the enriched live feed (rotifers and Artemia)
resulted in a significant increase of these fatty acids in the crab larvae. This confirms the
finding of previous studies that dietary HUFAs are readily assimilated by Scylla larvae
(Davis, 2003; Hamasaki et al., 1998; Kobayashi et al., 2000; Levine and Sulkin, 1984a;
Mann et al., 2001; Suprayudi et al., 2002b; Takeuchi et al., 1999; Takeuchi et al., 2000) and
on other aquaculture species including prawn, shrimp, fish and molluscs. For most fatty
acids however (except for the ARA/EPA ratio), a better correlation was found between the
composition of Artemia and Z5 compared to rotifers and Z3 (i.e. the ARA, DHA and total n-
3 contents were not significantly correlated for the latter). This probably indirectly results
from the different digestion and assimilation of HUFA in rotifers and Artemia. For example,
it has been well known that Artemia catabolize DHA much more than rotifers and thus the
former are considered to be much more difficult to “enrich” with this fatty acid (Bell et al.,
2001; Dhert et al., 1993; Navarro et al., 1999; Wouters et al., 1997). Furthermore, ARA,
EPA and DHA were the main HUFA’s present in both live feed and larvae; therefore any
changes in the level of each HUFA would certainly affect those of the others.
4.2. Survival in the zoeal stages
In experiments using large rearing tanks (30 and 100 l, in experiments 1 and 2,
respectively) without antibiotics, survival to Z5 was very low (1 to 19 %) and subjected to
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large variations. In the small rearing containers (1-l bowls, in experiments 3 and 4) where
the water was exchanged daily and antibiotics were applied daily, the survival rates were
much higher (20 to 55 %) and only there differences in survival were detected. In
experiment 3, on DAH 15, a slightly lower (through not significant) survival was observed
for treatment 30/4 compared to the other treatments. In experiment 4 however, this
treatment (30/4) gave good survival on all sampling days. Similarly, in experiment 4 on
DAH 9, treatment 0.6-0.6 (or 30/0.6) had a significantly lower survival compared to the “1-
1” control. This was not however confirmed in any of the other experiments. We therefore
conclude that survival during the zoeal stages was not affected by the dietary treatments.
In experiment 3, live feed with the lowest total n-3 contents (4 - 5 and 9 - 11 mg g-1 in
rotifers and Artemia, in the “starvation” control and treatment 0/- respectively) overall
resulted in comparable survival rates in the zoeal stages compared to those in treatments
with higher total n-3 contents (26 - 49 and 45 - 65 mg g-1 in rotifers and Artemia,
respectively). Similarly on S. serrata, survival through the first metamorphosis was not
significantly different among treatments using live feed that contained a wide range of total
n-3 HUFA (9 - 18 and 2 - 54 mg g-1 in rotifers and Artemia, respectively) (Davis, 2003).
Mann et al. (2001) also found that feeding S. serrata larvae with Artemia nauplii enriched
with a commercial lipid booster did not significantly affect larval survival when the total n-3
level of the live feed in the different treatments varied from 3 to 84 mg g-1.
In this respect, it was observed in our study, that although the total n-3 level of rotifers
in the “starvation” control and treatment 0/- were very low, Z3 in both treatments could still
accumulate these n-3 HUFAs from their feed to obtain a level of 11 - 12 mg g-1, which is
only slightly lower than those in most other treatments (15 - 16 mg g-1, except for the high
level of 32 mg g-1 in treatment 50/0.6). Furthermore, the high initial total n-3 content in Z1
(22 mg g-1) together with trace amounts derived from the diet might be sufficient to
overcome deficiencies and maintain high survival up to the last zoea stage. For the red frog
crab Ranina ranina, it was found that dietary energy is utilized for survival first, molting
second and morphogenesis last (Minagawa, 1992). Similarly in fish, the effects of feeding
HUFA-deficient and HUFA-enriched Artemia nauplii are not always apparent in survival
and growth (Ashraf, 1993).
In the Artemia feeding stage however, Z5 larvae of the “starvation” control and
treatment 0/-, could not maintain their total n-3 content (9 and 13 mg g-1, respectively)
above that of the Artemia they were fed with (9 and 11 mg g-1, respectively). The same
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observation could be made for other HUFAs. This means that, in contrast to the rotifer
feeding stages, the larvae fed with HUFA-deficient Artemia could not accumulate these
fatty acids. However, the survival rates in these later stages were, on most sampling days,
still not significantly different between treatments. This once again shows that larval
response to a nutritional factor does not come to expression immediately. Therefore,
although effects are maybe not immediately evident, all larval stages should be fed with
properly enriched live feed in order to build up sufficient HUFA reserves that are essential
for high survival beyond the zoeal stages. Takeuchi et al. (2000) similarly noticed that S.
paramamosain require both n-3 HUFA enriched rotifers and Artemia nauplii and should be
given the enriched Artemia nauplii from Z3 stage in order to attain a high survival rate of
the first crab stage.
4.3. Larval development rate during the zoeal stages
Larval development rate expressed as LSI was more affected by the dietary treatments
than survival rate. Anger et al. (1981) noticed that in brachyuran crabs, nutrition influences
development more directly than survival that is affected by a variety of other factors.
In most experiments, treatment 30/4 resulted in the highest larval development rate
among treatments. Enrichment media with a similar or higher total n-3 content, but lower
DHA/EPA ratio (treatments 30/0.6 and 50/0.6) usually performed significantly less.
Treatment 30/0.6 tended to have slightly higher LSI values than those of treatment
50/0.6 on most sampling days in experiment 1, but contradictory results were obtained in
experiments 2 and 3. This shows that high levels of HUFAs (particularly EPA as in
treatment 50/0.6) as such are neither needed nor beneficial for the larval performance. High
HUFA levels in crustacean larvae do not necessarily result in improved performance
(González-Félix et al., 2002) and the performance of S. serrata can even be compromised
when HUFA is supplied at excessive levels (Suprayudi et al., 2002b). In this study, an n-3
HUFA level of 30 % in the emulsions proved to be sufficient for live feed enrichment.
One of the suggested causes for the discrepancy in mud crab larval nutrition studies
has been the variability in quality between different batches of larvae as in S. paramamosain
(Djunaidah et al., 2003; Zeng and Li, 1999) and in S. serrata (Davis, 2003; Mann et al.,
1999a; Millamena and Bancaya, 2001). The larval quality was linked to the HUFA content
as was illustrated by Churchill (2003) in S. serrata larvae (particular EPA and total n-3).
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Furthermore (Davis, 2003) showed that larvae containing different fatty acid profiles at
hatch may require different levels or ratios of certain essential fatty acids in the diet. Larval
quality is in turn affected by the changing egg quality relating to different HUFA contents
from different batches that has been found commonly in the wild-caught spawners
(Churchill, 2003; Djunaidah et al., 2003). Although the selected broodstock crabs were fed
the same diet, they spent different time in captivity. Hence, the nutritional status of female
mud crabs varied and consequently affected the quality of eggs and Z1 larvae (Davis, 2003).
Treatment 0/-, using a HUFA-deficient emulsion, produced low LSI values in all
experiments throughout the rearing period. This shows that live feed enrichment does not
improve larval development through merely supplying extra energy.
As the control treatment differed from one experiment to another, also the LSI values
of the control treatments in the four experiments were different. In experiment 1, where
control rotifers were grown on Chlorella, overall LSI values in the control treatment seemed
to be better than those of treatments 0/- and 50/0.6. The positive effects of micro-algae were
also evident in experiment 2. In this experiment, Artemia in the control treatment were
enriched with Chaetoceros prior to feeding to the crab larvae. Where initially, LSI values of
the “Chaetoceros” control treatment were similar to those of treatments 0/-, 50/0.6 and
30/0.6, by DAH 9 to 12 they became significantly higher than the latter and similar to those
in treatments 30/4 and 30/1/ARA. Enriching the Artemia with Chaetoceros probably
boosted the n-3 HUFA content of the Artemia and therefore probably increased larval
development rate in the later zoeal stages to a level similar to treatment 30/4. Chaetoceros
gracilis is after all known to contain high n-3 HUFA, and more specifically EPA levels
(Napolitano, 1990; Volkman et al., 1989). However, Chen and Jeng (1980) found that the
addition of Chlorella to the culture medium of S. serrata did not influence growth and
survival of the larvae. Although Brick (1974) reported a similar finding for the same mud
crab species up to the last zoeal stages, he observed an improved rate of larval
metamorphosis to the megalopa stage. This inconsistency in the micro-algal effect might be
caused by the variable content of n-3 HUFAs (mainly EPA and DHA) between algal species
and even from culture to culture within the same species (Olsen, 1989). As there was no
positive response of the S. serrata larvae to the addition of Nannochloropsis oculata and
Tahitian Isochrysis galpana, Mann et al. (2001) suspected that they did not improve the
larval nutrition. Johns et al. (1981) also found that enriching Artemia nauplii with Isochrysis
galbana did not positively influence the survival of Rhithropanopeus harrisii mud crab
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larvae. Mann et al. (2001) suggested that the improvement may have been due to the water
conditioning effect of the micro-algae rather than nutritional influences. The algae may also
have delivered other essential nutrients (such as vitamins, polysaccharides, nucleotides,
etc.), which makes it difficult to define the exact factor involved.
In experiment 3, LSI values in the “starvation” control treatment were always lower
than those of the “HUFA-rich” treatments. In this trial rotifers were grown on baker’s yeast
and Artemia starved and thus especially deficient in HUFA.
In experiment 4, control live feed was “enriched” using the commercial product
Culture Selco, which contains considerable amounts of n-3 HUFA. The LSI values of the
“1-1” control treatment were always higher than those of treatment 0.6-0.6 (30/0.6). In early
larval stages, the “1-1” control also outperformed treatment 0.6-4, however LSI values
became similar towards the end of the rearing period. As the “1-1” control, which only has a
DHA/EPA ratio of 1, resulted in similar LSI values (and survival rates) compared to
treatment 4/4, it seems an emulsion with a total HUFA content of approximately 30 % with
a DHA/EPA value of 1 is sufficient to satisfy the requirements for larval development in the
zoeal early stages as the effects of dietary HUFAs would manifest in the metamorphosis
stages.
The importance of DHA and its ratio to EPA for larval development was also obvious
from the correlation coefficients between the LSI and the fatty acid profile of the live feed.
The LSI values on DAH 3 and 6 were only significantly correlated with the DHA/EPA level
of the rotifers, and not with the DHA or the total n-3 level. In contrast, during the Artemia
feeding stage, also the absolute DHA level and total n-3 level significantly influenced larval
development. This difference can probably be explained by the difference in enrichment
kinetics between rotifers and Artemia. Artemia is known to selectively catabolize DHA
during enrichment, resulting in relatively lower total DHA levels (Bell et al., 2001; Dhert et
al., 1993; Navarro et al., 1999; Wouters et al., 1997). This could explain why also the
absolute DHA level (and hence total n-3 level) becomes more important during Artemia
feeding.
Similarly, growth was also significantly correlated with the fatty acid composition of
the Z3 and Z5. Again growth during the rotifer feeding stage was only correlated with the
DHA/EPA ratio of the larvae, while in the Artemia-stage Z5 also the DHA and total n-3
content became important.
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4.4. Metamorphosis
Metamorphosis rate
In experiment 3, the first metamorphosis rate (from Z5 to megalopa) of treatment
30/4/ARA was significantly higher (50 %) then in the “n-3 HUFA treatments” (ranging
from 30 to 34 %). No differences were detected between any of the other treatments. This
was confirmed by the fact that the ARA content of both Artemia and Z5 and the ARA/EPA
ratio of Artemia correlated significantly with the MR1 values.
The role of arachidonic acid in first metamorphosis remains unclear and requires
further investigation. Koven et al. (2000) suggested that besides DHA, not only highly
unsaturated fatty acids of the n-3 series are important but that also ARA may play a
significant role for the larvae of gilthead seabream (Sparus aurata). ARA may improve
larval growth and pigmentation in several marine fish species since it provides precursors
for eicosanoid production (Castell et al., 1994; Estévez et al., 1999). Turbot fed ARA as the
only HUFA yielded higher growth and survival compared to any of the DHA/ARA mixtures
or DHA alone (Castell et al., 1994). It is likely that, at specific stages in the life cycle of
fish, higher levels of ARA may be required to cope with periods of environmental stress
(Bell and Sargent, 2002). The requirement of ARA in fish, however, seems to depend on the
fish species and larval development, and needs to be dosed with extreme care since it may
act in a different way depending on the DHA concentration (Castell et al., 1994; Koven et
al., 2000). Studies on arachidonic acid in crustaceans are rather scarce. Glencross and Smith
(2001) investigated arachidonic acid requirements in Penaeus monodon. They concluded
that ARA is not really essential if linoleic acid (18:2 n-6), the natural precursor to ARA, is
present in sufficient amounts. These authors suggested that possible effects of ARA may lie
in the importance of the balance of n-3 to n-6 fatty acids in the diet.
Also in experiment 4, the MR1 values of the different n-3 HUFA enrichment
treatments were not significantly different. It therefore seems that the various total n-3
HUFA levels (28 - 50 %) and DHA/EPA ratios (0.6 - 4) tested in this experiment could
satisfy the requirements for first metamorphosis. There might however also be an interaction
between metamorphosis success, survival in the zoeal stages and the quality of the surviving
larvae. As in experiments 3 and 4, survival in the zoeal stages was affected by the dietary
treatments, it could be hypothesized that in the “bad” treatments the weaker larvae had died
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before reaching Z5 stage and the survivors were capable to metamorphose successfully,
whereas in the assumed “better” treatments, also weaker larvae could reach the Z5 stage but
then failed to metamorphose to megalopae. This might have leveled out metamorphosis
results.
The effects of dietary HUFA on the second metamorphosis rate were more
pronounced. The effects of dietary HUFA often become obvious in later stages only when
the larvae have used up all reserves built up in earlier stages. In this respect, it has been
reported that n-3 HUFA are among the last components to be utilized (Galois, 1987; cited in
Wouters et al., 1997), probably because they play an important role as fatty acid groups of
polar lipids in cell membranes (Sargent et al., 1991; Watanabe, 1993).
In experiment 3, three groups could be distinguished on the basis of their MR2 rates:
high survival through second metamorphosis for the treatments with a high DHA/EPA ratio
(30/4 and 30/4/ARA, over 90 %), intermediate survival for treatments with a low DHA/EPA
ratio (50/0.6 and 30/0.6, approximately 70 %) and low survival for treatments with low total
n-3 (or total HUFA) content (“starvation” control and 0/-, approximately 10 %),
respectively. Following the same pattern as for larval development rate in the zoeal stages,
the DHA and total n-3 content, and the DHA/EPA ratio of the Artemia and Z5 were hence
significantly correlated with the second metamorphosis rate. Takeuchi et al. (2000) found
that S. paramamosain larvae require both n-3 HUFA enriched rotifers and Artemia and that
it is necessary to feed enriched Artemia nauplii from the Z3 stage onwards in order to attain
a high survival rate to first crab. Similarly, for S. tranquebarica, a high survival rate and
maximum carapace width of the first crab stage was obtained when the larvae were fed
DHA-enriched Artemia (Takeuchi, in press).
In experiment 4, no real negative control (low n-3 HUFA) was included. MR2 values
were hence rather similar in all treatments. Again the MR2 values of the four treatments
might have been leveled out by the survival in the preceding stages and the quality of the
surviving larvae as explained in the discussion of MR1 values.
Timing and duration of metamorphosis
Onset of metamorphosis was largely dictated by the larval development rate during
the zoeal stages. In experiment 3, the treatments using the emulsions containing a medium
total n-3 content (30/0.6, 30/4 and 30/4/ARA) reached first metamorphosis earlier and had a
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shorter metamorphosis period than the treatments with high (50/0.6) or low (control and 0/-)
total n-3 HUFA content. No difference was observed however between high and low
DHA/EPA treatments. A similar trend was observed for the second metamorphosis.
In experiment 4, the average first metamorphosis time was shortest for treatment 4-4;
intermediate for treatments control and 0.6-0.6; and significantly longer for treatment 0.6-4.
The histograms and normal distribution curves of the first metamorphosis clearly
demonstrate that the duration of metamorphosis was prolonged in treatments 0.6-0.6 and
0.6-4. The average time to reach the second metamorphosis was longest for treatment 0.6-4,
shortest in the “1-1” control treatment and intermediate values for treatments 0.6-0.6 and 4-
4. Again, the survival rate could have affected the completion of metamorphosis processes
as the assumed “best” treatment (i.e. treatment 4-4) had many more surviving larvae, what
probably resulted in a wider variation in metamorphosis time in contrast to the assumed
“worst” treatment (i.e. treatment 0.6-0.6) that had few but “strong” survivors.
Figure 1 shows that in treatment 0.6-4 a number of megalopae appeared very late on
DAH 26 and 28. This could point out a kind of recovery after switching to an emulsion with
higher DHA/EPA ratio from Z3 onwards in this treatment. Zeng (1998) observed that crab
larvae that were put on a too low ration, prolonged the zoeal stage by developing a sixth
zoeal stage before metamorphosing into megalopa. The author suggested that these larvae
adopted a strategy of extending zoeal development to accumulate enough nutritional and
energy reserves necessary for this metamorphosis. Figure 2 also shows 2 peaks of
metamorphosis to crab stage in the treatment 0.6-4 while only 1 peak was found in the other
treatments. This could be linked to the late first metamorphosis of some megalopae as
presented in Figure 1.
4.5. Survival of Z1 to megalopa and the first crab (M/Z1 and C1/Z1 survival rates)
In our study, dietary HUFAs did not always bring an instant response on larval
performance. However, due to the accumulation of responses there usually is an inflection
point where larval survival or growth diverges between different treatments (Mann et al.,
2001). Using Penaeus stylirostris as a test organism, it appeared that the EPA and DHA
content in the zoea diet only showed a major impact on survival and growth in later stages,
when animals had already been switched to another diet (Léger et al., 1985). The effects of
early nutrition are often only manifested during the later stages of development and
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particularly at metamorphosis to the post larval stages (megalopa and crabs) (Davis, 2003;
Harvey, 1996; Jeff et al., 1996; Ribeiro and Jones, 2000; Sulkin, 1978). Therefore, the
overall survival of Z1 to megalopa or C1 that are the product of zoeal survival and
metamorphosis rate are a good means to evaluate the final effect of treatments. These
overall survival rates are also the primary concern for hatchey managers. In this respect, the
M/Z1 and C1/Z1 survival rates are presented in Figure 3. This figure clearly shows that both
overall survival rates were significantly higher in treatment 4-4. The higher M/Z1 and
C1/Z1 survival rates in treatment 0.6-4 compared to those of treatment 0.6-0.6 proves that
the crab larvae can recover their growth at later stages when DHA-rich Artemia was offered
from the Z3 stage onwards. However, survival rates to M and C1 in treatment 0.6-4 were
still lower than those of treatment (4-4) and the “1-1” control. This proves that an emulsion
with DHA/EPA level lower than 1 should not be used for live feed enrichment for early
stages.
Several other studies used overall survival rates for evaluating the effect of live food
enrichment. Mann et al. (2001) found that feeding the S. serrata larvae with only Artemia
enriched with the commercial lipid booster (Super Selco®, INVE Aquaculture) did not affect
larval survival or growth to DAH 18 (megalopa stage). This commercial lipid booster
increased the DHA/EPA ratio of Artemia from 0 - 0.04 (in un-enriched Artemia) to 0.3 - 0.4,
which is similar to the DHA/EPA ratio of Artemia enriched with the emulsion 30/0.6 in
experiment 3 of our study (= 0.4). These authors concluded that the requirement for EPA,
DHA and other fatty acid are met in un-enriched newly-hatched Artemia nauplii. Our study
however indicates that the low DHA/EPA levels (0.3 - 0.4) in enriched Artemia in their
experiment were not sufficient to improve the larval performance when Artemia is the sole
feed for all crab larval stages. This is confirmed by a study on S. serrata by Davis et al.
(2003). In that study, the zoeal survival and development, first metamorphosis rate and
overall survival of Z1 to megalopa in treatments using enriched rotifers and enriched/un-
enriched Artemia (with Super Selco®) were significantly improved compared to the
treatments without rotifer enrichment or to treatments where only enriched/un-enriched
Artemia were fed (Davis, 2003). Boosting FAME levels (including HUFAs) in the rotifers
fed at hatch may have given the larvae a significant advantage which was carried through
the rearing process and culminated in improved survival through metamorphosis,
particularly when both rotifers and Artemia were enriched and the HUFA supply was thus
not uninterrupted (Davis, 2003).
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4.6. Use of antibiotics for studying nutritional requirements Although we don’t want to encourage the use of antibiotics for commercial farming
practices, the application of antibiotics (experiments 3 and 4) as a prophylactic improved
overall survival considerably and made results more reproducible. After all, it should not be
overlooked that there are various interactions possible between pathogenic bateria and
nutritional treatments. For example, although the enriched live food was thouroughly rinsed
before being fed to the larvae, it can not be excluded that residual oil enters the system this
way. Moreover, the decomposition of uneaten enriched live feed might be a source of extra
nutrients and this way supporting the development of bacteria. In this respect, it was noticed
that treatments using emulsions with a high n-3 HUFA percentage (50/0.6) or high
DHA/EPA ratio (30/4) tended to give lower survival compared to treatments 0/- and 30/0.6
and the control towards the end of experiment 1. It might be that the use of emulsions with a
high n-3 HUFA level resulted in more waste material being produced higher accumulation
of fatty acids sourced from decomposed waste products and therefore deteriorated the
culture medium more rapidly. Possibly, HUFA’s with their higher number of carbon atoms
and many more double bonds might also be a more readily available substrate for bacterial
growth (Mead et al., 1986). Conversely, it is also known that short chain saturated fatty
acids (which are relatively abundant in the coconut oil-based ICES 0/-) have bacteriostatic
properties (Rickle, 2003). In experiments 1 and 2, the nutritional benefits arising from
feeding enriched live feed might thus have been leveled out by a negative microbial
interaction, where in experiment 3 and 4 the daily application of antibiotics should have
separated out the purely nutritional effects.
5. Conclusions and suggestions
The ICES emulsions influenced the fatty acid profiles of the live feed and, in turn, the
fatty acid profiles of the crab larvae.
No real differences in survival in the zoeal stages were found between the different
enrichment treatments tested here. The “nutritional impact” of HUFAs on the zoeal survival
was probably obscured by other more decisive factors such as the batch quality, micro-biota,
zootechnics. The significantly lower metamorphosis success in the low HUFA treatments
proved however that HUFA-rich live food enrichment emulsions are needed to attain high
CHAPTER 5 – Feed quality
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survival to the crab stage. Not that much the total n-3 level, but more particularly the DHA
level and the DHA/EPA ratio seem of crucial importance.
Also larval development rate was very much affected by the dietary n-3 HUFA level
and its DHA/EPA ratio. In all experiments, the LSI values of treatments 30/4 tended to be
higher than those of treatments 50/0.6 and 30/0.6 throughout the zoeal stages. This means
that a DHA/EPA ratio of 0.6 in enrichment emulsions is not sufficient to support larval
development. Based on the results of experiment 4, a DHA/EPA ratio of approximately 1
might be sufficient for early stages (Z1 - Z2).
There was also evidence that DHA/EPA requirements might change during
development. Therefore it might be better if this ratio is increased gradually in time (e.g.
emulsions with DHA/EPA ratio of 1 for Z1-Z2 stages, of 2 - 3 for Z3 - Z4 and of 4 for Z5
onwards). Exact requirements for each larval stage should therefore be investigated in
function of the diet type (rotifers, Artemia, micro-bound diets). This should however be
verified using more precisely formulated emulsions.
For the total n-3 HUFA content, no differences were found between live feed
enrichment products containing 30 or 50 % n-3 HUFA. An inclusion level of 30 % therefore
seems to be sufficient.
Supplementation of arachidonic acid had no effect on survival nor growth during the
zoeal stages. First metamorphosis rate was however improved by the addition of dietary
ARA. Further research on the suitable levels of ARA in the enrichment diet for crab larvae
is therefore worth pursuing.
Acknowledgements
This study was supported by the European Commission (INCO-DC), the Flemish
Inter-University Council (Vl.I.R.-IUC) and the International Foundation for Science (IFS).
CHAPTER 5 – Feed quality
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Table 1 Overview of larval rearing conditions and water quality parameters in the 4 experiments
Container Water quality parameters Experi-ment
Culture period (days)
Stocking density (Z1 l-1)
Volume (l) Type
Water exchange Temp.
(°C) Salinity (g l-1)
NH4+
(mg l-1) NO2
-
(mg l-1) NO3
+
(mg l-1)
1 15 50 30 Tank Recirculation 29.6 ±0.4
29.6 ±1.1
0.08 ±0.1
0.05 ±0.02
3.0 ±2.0
2 12 100 100 Tank Recirculation 31.1 ±0.7
30.1 ±0.5
0.13 ±0.1
0.26 ±0.28 -
29 50 1 Bowl Batch 3 15 50 100 Tank Recirculation 4 31 100 1 Bowl Batch
30.0 ±1.0
30.0 ±1.0 < 1.0 < 0.3 -
Table 2 Overview of the treatments used in the 4 experiments. In experiment 1, rotifers were the only live feed for all larval stages. In the remaining experiments, rotifers were fed to the crab larvae from DAH 0 - 6 (Z1 - Z2) and Artemia were replaced rotifers from the evening of DAH 6 (onset of Z3 stage)
Treatment Experi-ment Control 0/- 50/0.6 30/0.6
(0.6-0.6)§ 30/4 (4-4)§ 0.6-4 30/1/
ARA 30/4/ ARA
Number of repli-cates
1
freshly harvested un-enriched rotifers, cultured on Chlorella
rotifers enriched with emulsion 0/-
rotifers enriched with emulsion 50/0.6
rotifers enriched with emulsion 30/0.6
rotifers enriched with emulsion 30/4
4
2
un-enriched rotifers and Artemia enriched with Chaetoceros
rotifers and Artemia enriched with emulsion 0/-
rotifers and Artemia enriched with emulsion 50/0.6
rotifers and Artemia enriched with emulsion 30/0.6
rotifers and Artemia enriched with emulsion 30/4
rotifers and Artemia enriched with emulsion 30/1/ARA
3
3
un-enriched rotifers and starved Artemia
rotifers and Artemia enriched with emulsion 0/-
rotifers and Artemia enriched with emulsion 50/0.6
rotifers and Artemia enriched with emulsion 30/0.6
rotifers and Artemia enriched with emulsion 30/4
rotifers and Artemia enriched with emulsion 30/4/ARA
4 in bowls 2 in tanks
4
rotifers and Artemia enriched with Culture Selco
rotifers and Artemia enriched with emulsion 30/0.6
rotifers and Artemia enriched with emulsion 30/4
rotifers enriched with emulsion 30/0.6 and Artemia enriched with emulsion 30/4
3
§ = Treatment names used in experiment 4.
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Table 3 Fatty acid composition of the ICES emulsions used to enrich the live feed in the 4 experiments. ARA = Arachidonic acid, EPA = Eicosapentaenoic acid, DHA = Docosahexaenoic acid, Σn-3 = Total n-3 HUFA, Σn-6 = Total n-6 HUFA, ΣHUFA = Total HUFA, FAME = Fatty acid methyl esters = Total fatty acid content ICES emulsion Fatty acid content
0/- 50/0.6 30/0.6 30/4 30/1/ARA 30/4/ARA
ARA (mg g-1) 0.00 10.40 5.25 4.71 124.70 94.51 EPA (mg g-1) 0.00 261.40 103.70 42.13 125.96 31.58 DHA (mg g-1) 0.00 163.60 63.73 161.87 122.60 121.46 Σn-3 (mg g-1) 0.00 499.80 167.43 204.00 248.56 156.19 Σn-6 (mg g-1) 2.88 59.50 36.57 50.91 207.79 159.93 ΣHUFA (mg g-1) 0.00 524.40 172.68 208.72 373.26 251.17 DHA/EPA - 0.63 0.61 3.84 0.97 3.85 ARA/EPA - 0.04 0.05 0.11 0.99 2.99 Σn-3/Σn-6 0.00 8.40 4.58 4.01 1.20 0.98 Σn-3/FAME (%) 0.00 51.41 29.29 30.48 28.40 22.34 Table 4 Fatty acid composition of rotifers and Artemia used in experiment 3. ARA = Arachidonic acid, EPA = Eicosapentaenoic acid, DHA = Docosahexaenoic acid, Σn-3 = Total n-3 HUFA, Σn-6 = Total n-6 HUFA, ΣHUFA = Total HUFA. Con. = Control
Rotifers Artemia
Fatty acid content Con. 0/- 50/0.6 30/0.6 30/4 30/4/ ARA Con. 0/- 50/0.6 30/0.6 30/4 30/4/
ARA ARA (mg g-1) 1.08 0.75 2.15 1.65 1.03 7.13 3.86 4.16 6.50 2.50 8.34 25.77 EPA (mg g-1) 2.33 2.65 28.61 18.91 7.19 11.26 8.94 10.49 40.09 29.60 23.29 27.49 DHA (mg g-1) 1.94 2.10 19.99 13.34 18.45 23.61 0.35 0.21 19.53 11.20 20.61 19.07 Σn-3 (mg g-1) 4.27 4.76 48.60 32.25 25.64 34.88 9.49 11.05 64.99 45.10 46.64 48.79 Σn-6 (mg g-1) 7.05 14.96 17.17 12.38 11.36 23.15 7.67 14.56 16.49 14.10 22.82 47.72 ΣHUFA (mg g-1) 5.35 5.51 50.75 33.90 26.67 42.01 14.00 16.02 73.51 50.60 58.00 77.96 DHA/EPA 0.84 0.79 0.70 0.71 2.57 2.10 0.04 0.02 0.49 0.38 0.88 0.69 ARA/EPA 0.46 0.28 0.08 0.09 0.14 0.63 0.43 0.40 0.16 0.08 0.36 0.94 Σn-3/Σn-6 0.61 0.32 2.83 2.61 2.26 1.51 1.24 0.76 3.94 3.20 2.04 1.02
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Table 5 Fatty acid composition of S. paramamosain zoea 1 (= newly-hatched zoea), zoea 3 and zoea 5 fed different enriched live feed in experiment 3. ARA = Arachidonic acid, EPA = Eicosapentaenoic acid, DHA = Docosahexaenoic acid, Σn-3 = Total n-3 HUFA, Σn-6 = Total n-6 HUFA, ΣHUFA = Total HUFA, Con. = Control
Zoea 3 Zoea 5
Fatty acid content
Zoea 1
Con. 0/- 50/0.6 30/0.6 30/4 30/4/ARA Con. 0/- 50/0.6 30/0.6 30/4 30/4/
ARA ARA (mg g-1) 6.59 4.56 2.73 4.03 1.84 2.27 4.93 3.77 4.74 4.41 3.70 4.73 6.76 EPA (mg g-1) 12.13 7.43 7.29 18.78 8.76 6.33 6.84 8.17 10.92 16.79 12.12 11.44 12.66 DHA (mg g-1) 9.70 4.03 4.32 13.07 6.77 8.20 8.00 0.87 1.78 6.21 4.34 6.78 6.84 Σn-3 (mg g-1) 21.83 11.46 11.61 31.85 15.53 14.53 14.84 9.04 13.25 23.00 16.46 19.07 19.50 Σn-6 (mg g-1) 11.47 15.50 12.59 14.22 6.20 5.54 11.00 6.10 15.54 9.86 7.89 11.55 12.80 ΣHUFA (mg g-1)
28.42 16.02 14.33 35.88 17.36 16.79 19.77 12.81 19.18 27.41 20.15 24.88 26.25
DHA/EPA 0.80 0.54 0.59 0.70 0.77 1.29 1.17 0.11 0.16 0.37 0.36 0.59 0.54 ARA/EPA 0.54 0.61 0.37 0.21 0.21 0.36 0.72 0.46 0.43 0.26 0.30 0.41 0.53 Σn-3/Σn-6 1.90 0.74 0.92 2.24 2.50 2.62 1.35 1.48 0.85 2.33 2.09 1.65 1.52
Table 6. Pearson correlation coefficients (r2) between the fatty acid composition of the live feeds and those of the crab larvae. ARA = Arachidonic acid. EPA = Eicosapentaenoic acid. DHA = Docosahexaenoic acid, Σn-3 = Total n-3 HUFA, Σn-6 = Total n-6 HUFA
Fatty acid Correlation
ARA EPA DHA Σn-3 DHA/EPA ARA/EPA Σn-3/Σn-6
Rotifers - Zoea 3 0.36 0.72* 0.62 0.65 0.90** 0.96** 0.86** Artemia - Zoea 5 0.91** 0.83* 0.98** 0.91** 0.97** 0.81* 0.86** * and ** = significant correlations (P < 0.05 and P < 0.01, respectively). Table 7 Survival rates and larval stage index (LSI) values (mean ± standard deviation) of S. paramamosain larvae fed different enriched rotifers in experiment 1. For treatment descriptions refer to Tables 2 and 3. DAH = days after hatch Treatment DAH 3 DAH 6 DAH 9 DAH 12 DAH 15 Survival rate (%) Control 82±22a 67±38a 48±34a 42±30a 18±13a
0/- 99±0a 82±8a 53±40a 25±17a 19±17a
50/0.6 92±10a 59±26a 44±14a 21±6a 10±9a
30/0.6 96±8a 79±18a 71±19a 23±2a 15±6a
30/4 85±31a 74±24a 63±20a 29±17a 9±7a
LSI value Control 2.0±0.1a 3.0±0.0a A 3.8±0.3a A 4.1±0.1ab A 4.3±0.1a
0/- 2.0±0.1a 2.2±0.4b B 2.8±0.3b A 3.3±0.3c B 3.8±0.3a
50/0.6 1.8±0.3a 2.7±0.1ab AB 3.1±0.5ab A 3.8±0.3b AB 4.0±0.0a
30/0.6 2.0±0.0a 2.9±0.1a A 3.7±0.4a A 4.1±0.1ab A 4.2±0.3a
30/4 2.0±0.1a 2.9±0.2a A 3.6±0.3a A 4.3±0.1a A 4.8±0.0a
Survival rates or LSI values in the same column followed by the same superscript letter are not statistically different (P ≥ 0.05, regular letters and P ≥ 0.01, capital letters.
CHAPTER 5 – Feed quality
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Table 8 Survival rates and larval stage index (LSI) values (mean ± standard deviation) of S. paramamosain larvae fed different enriched rotifers and Artemia in experiment 2. For treatment descriptions refer to Tables 2 and 3. DAH = days after hatch Treatment DAH 3 DAH 6 DAH 9 DAH 12 Survival rates (%) Control 66±17a 28±17a 5±4a 1±2a
0/- 64±26a 39±18a 22±10a 11±7a
50/0.6 74±28a 21±8a 15±11a 5±5a
30/0.6 62±18a 35±10a 17±1a 10±3a
30/4 70±12a 26±13a 11±10a 7±5a
30/1/ARA 79±9a 39±8a 11±9a 7±6a
LSI Control 1.7±0.2a 2.4±0.2cd BC 3.5±0.2bc BC 4.7±0.1a A
0/- 1.7±0.1a 2.5±0.1bc BC 3.2±0.1cd C 4.3±0.1b B
50/0.6 1.7±0.2a 2.4±0.2cd BC 3.1±0.1cd C 4.3±0.1b B
30/0.6 1.6±0.4a 2.2±0.2d C 3.1±0.1d C 4.1±0.1b B
30/4 1.8±0.2a 2.9±0.1a A 4.0±0.1a A 4.9±0.1a A
30/1/ARA 1.8±0.2a 2.8±0.2ab AB 3.7±0.2ab AB 4.8±0.1a A
Survival rates or LSI values in the same column followed by the same superscript letter are not statistically different (P ≥ 0.05, regular letters and P ≥ 0.01, capital letters. Table 9 Survival rates and larval stage index (LSI) values (mean ± standard deviation) of S. paramamosain larvae fed different enriched rotifers and Artemia in experiment 3. For treatment descriptions refer to Tables 2 and 3. DAH = days after hatch Treatment DAH 3 DAH 6 DAH 9 DAH 12 DAH 15 Survival rates (%) Control 96±2a 92±5a 88±4a 64±5a 28±11a
0/- 94±5a 89±8a 83±10a 59±7a 34±7a
50/0.6 95±4a 92±4a 85±8a 63±15a 35±14a
30/0.6 93±3a 86±5a 80±4a 67±7a 43±6a
30/4 96±4a 87±5a 85±6a 56±17a 20±10a
30/4/ARA 96±3a 89±6a 81±3a 65±13a 41±11a
LSI Control 1.7±0.1b B 2.2±0.1b C 3.0±0.1c C 3.5±0.1c B 4.4±0.1c C
0/- 1.6±0.0c B 2.0±0.1b C 2.9±0.1c C 3.4±0.1c B 4.4±0.1c BC
50/0.6 1.7±0.0b B 2.2±0.1b BC 3.3±0.1ab AB 3.8±0.1ab A 4.7±0.1ab A
30/0.6 1.6±0.1bc B 2.2±0.1b BC 3.2±0.1b B 3.7±0.1b A 4.6±0.1b AB
30/4 1.9±0.0a A 2.6±0.2a A 3.4±0.1ab AB 3.9±0.1a A 4.8±0.1a A
30/4/ARA 1.9±0.0a A 2.5±0.1a AB 3.3±0.1a A 3.8±0.1ab A 4.8±0.3ab A
Survival rates or LSI values in the same column followed by the same superscript letter are not statistically different (P ≥ 0.05, regular letters and P ≥ 0.01, capital letters).
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Table 10 Survival rates and larval stage index (LSI) values (mean ± standard deviation) of S. paramamosain larvae fed different enriched rotifers and Artemia in experiment 4. For treatment descriptions refer to Tables 2 and 3. DAH = days after hatch Treatment DAH 3 DAH 6 DAH 9 DAH 12 DAH 15 Survival rate (%) Control 87±6a 80±6a 76±5a A 66±7a 55±12a
0.6-0.6 81±7a 64±17a 28±24b A 26±25a 22±24a
0.6-4 83±3a 63±13a 49±16ab A 45±15a 39±12a
4-4 83±12a 76±14a 71±16ab A 63±17a 50±18a
LSI Control 1.9±0.1a A 2.7±0.4a 3.4±0.1a 4.0±0.1a 4.4±0.2a
0.6-0.6 1.0±0.0b B 2.1±0.1a 2.3±0.5a 3.8±0.4a 4.5±0.2a
0.6-4 1.0±0.0b B 2.1±0.1a 3.2±0.2a 4.0±0.2a 4.2±0.3a
4-4 1.7±0.5a AB 2.6±0.4a 3.6±0.3a 4.1±0.2a 4.5±0.4a
Survival rates or LSI values in the same column followed by the same superscript letter are not statistically different (P ≥ 0.05, regular letters and P ≥ 0.01, capital letters). Table 11 Survival through first (M/Z5, MR1) and second (C1/M, MR2) metamorphosis rates of S. paramamosain larvae fed different enriched rotifers and Artemia in experiment 3. For treatment names refer to Tables 2 and 3. Z5 = zoea 5, M = megalopa, C1 = crab 1 Treatment Survival through first
metamorphosis (M/Z5) (%)
Survival through second metamorphosis (C1/M) (%)
Control 31b A 13b B
0/- 32b A 11b B
50/0.6 30b A 73a A
30/0.6 34b A 73a A
30/4 30b A 92a A
30/4/ARA 50a A 93a A
Values in the same column followed by the same superscript letter are not statistically different (P ≥ 0.05, regular letters and P ≥ 0.01, capital letters). Table 12 Average and minimum and maximum time (days after hatch) to reach first and second metamorphosis of S. paramamosain larvae fed different enriched live feed in experiment 3. For treatment descriptions refer to Tables 2 and 3
First metamorphosis Second metamorphosis Treatment mean ±
standard deviation minimum - maximum
mean ± standard deviation
minimum - maximum
Control 19.3±1.6a A 18-22 27.0±1.4ab AB 26-28 0/- 18.2±1.5ab AB 17-22 27.5±2.1ab AB 26-29 50/0.6 18.3±1.5ab AB 16-23 27.0±1.6a A 24-29 30/0.6 17.1±1.0b B 15-19 25.9±1.2ab AB 24-28 30/4 17.8±1.6ab AB 16-21 24.3±1.4b B 22-27 30/4/ARA 17.3±1.4b B 15-21 24.8±1.4b B 22-27 Values in the same column followed by the same superscript letter are not statistically different (P ≥ 0.05, regular letters and P ≥ 0.01, capital letters).
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Table 13 Survival through first (M/Z5, MR1) and second (C1/M, MR2) metamorphosis of S. paramamosain larvae fed different enriched rotifers and Artemia in experiment 4. For treatment names refer to Tables 2 and 3. Z5 = zoea 5, M = megalopa, C1 = crab 1
Treatment Survival through first metamorphosis (M/Z5) (%)
Survival through second metamorphosis (C1/M) (%)
Control 48±9a 67±20a
0.6-0.6 65±14a 78±24a
0.6-4 50±16a 67±20a
4-4 66±20a 81±3a
Values in the same column followed by the same superscript letter are not statistically different (P ≥ 0.05). Table 14 Average time (days after hatch) to reach first and second metamorphosis of S. paramamosain larvae fed different enriched live feed in experiment 4 (mean ± standard deviation). For treatment descriptions refer to Tables 2 and 3 Treatment First metamorphosis Second metamorphosis Control 19.3±1.7ab AB 25.7±2.3b B
0.6-0.6 19.4±2.1ab AB 26.6±2.1ab AB
0.6-4 20.1±2.5a A 27.1±2.0a A
4-4 18.6±1.5b B 26.5±2.0ab AB
Values in the same column followed by the same superscript letter are not statistically different (P ≥ 0.05, regular letters and P ≥ 0.01, capital letters). Table 15 Pearson correlation coefficients (r2) of the fatty acid composition of the live feed (rotifers and Artemia) and crab larvae (zoea 3 and zoea 5) with larval stage index (LSI) values and metamorphosis rates (MR, %) in experiment 3. DAH = days after hatch, MR1 = survival through first metamorphosis (M/Z5), MR2 = survival through second metamorphosis (C1/M), ARA = Arachidonic acid, EPA = Eicosapentaenoic acid, DHA = Docosahexaenoic acid, ΣHUFA = Total highly unsaturated fatty acid, Σn-3 = Total n-3 HUFA, Σn-6 = Total n-6 HUFA, DAH = days after hatch Fatty acid Correlation
ARA EPA DHA Σn-3 DHA/EPA ARA/EPA Σn-3/Σn-6
Rotifers – LSI DAH 3 0.30 0.00 0.52 0.13 0.86** 0.10 0.08 Rotifers – LSI DAH 6 0.18 0.00 0.56 0.17 0.87** 0.01 0.19 Artemia – LSI DAH 9 0.20 0.58 0.97** 0.79* 0.94** 0.00 0.24 Artemia – LSI DAH 12 0.28 0.60 0.98** 0.81* 0.94** 0.02 0.21 Artemia – LSI DAH 15 0.32 0.57 0.95** 0.77* 0.93** 0.03 0.15 Zoea 3 – LSI DAH 6 0.00 0.05 0.14 0.00 0.91** 0.07 0.25 Zoea 5 – LSI DAH 15 0.28 0.39 0.98** 0.78* 0.96** 0.02 0.24 Artemia - MR1 0.83* 0.02 0.09 0.04 0.09 0.69* 0.15 Artemia - MR2 0.30 0.61 0.92** 0.80* 0.90** 0.02 0.20 Zoea 5 - MR1 0.73* 0.00 0.14 0.04 0.17 0.36 0.02 Zoea 5 - MR2 0.20 0.33 0.93** 0.69* 0.92** 0.02 0.35 * and ** = significant correlations (P < 0.05 and P < 0.01, respectively).
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121
16 17 18 19 20 21 22 23 24 25 26 27 28
Day after hatch
0
5
10
15
20
25
30
35
40
45N
o of
obs
erva
tions 4-4
"1-1" control
0.6-4
0.6-0.6
"1-1" control 0.6-0.6 0.6-4 4-4
Figure 1. Histogram and normal distribution curves of the metamorphosis to megalopa of S. paramamosain larvae fed different enriched live feed in experiment 4. For treatment descriptions refer to Tables 2 and 3.
CHAPTER 5 – Feed quality
122
22 23 24 25 26 27 28 29 30 31
Day after hatch
0
2
4
6
8
10
12
14
16
18
20
No
of o
bser
vatio
ns
4-4
0.6-4
0.6-0.6
"1-1" control
"1-1" control 0.6-0.6 0.6-4 4-4
Figure 2. Histogram and normal distribution curves of metamorphosis to crab 1 of S. paramamosain larvae fed different enriched live feed in experiment 4. For treatment descriptions refer to Tables 2 and 3.
CHAPTER 5 – Feed quality
123
0
5
10
15
20
25
30
351
16 17 18 19 20 21 22 23 22 23 24 25 26 27 28 29 30 31
Day after hatch
Cum
ulat
ive
% o
f M/Z
1 an
d C
1/Z
"1-1" control0.6-0.60.6-44-4
b AB
c B
bc B
a A
b B
b BC
c C
a A
Megalopa Crab 1
Figure 3. Cumulative metamorphosis rates from zoea 1 to megalopa and first crabs of S. paramamosain larvae fed different enriched live feed in experiment 4. For treatment descriptions refer to Tables 2 and 3. Curves for each survival followed by the same letter are not statistically different (P ≥ 0.05, regular letters and P ≥ 0.01, capital letters).
CHAPTER 6 Improved larval rearing techniques for mud crab
(Scylla paramamosain)
Nghia, T.T.*1, Wille, M.2 and Sorgeloos, P.2
1 College of Aquaculture and Fisheries, Can Tho University, Vietnam. Email: [email protected] 2 Laboratory of Aquaculture & Artemia Reference Center, Ghent University, Belgium. Email: [email protected]
Abstract Eight larval rearing trials were carried out with the purpose to develop optimal rearing
techniques for the mud crab (Scylla paramamosain). Based on the method of water exchange (discontinuous partial water renewal or
continuous treatment through biofiltration) and micro-algae (Chlorella or Chaetoceros) supplementation (daily supplementation with low levels of 0.1 - 0.2 million cells ml-1 or maintenance at high levels of 1 - 2 millions cells ml-1), six different types of rearing systems were tried.
The combination of a green-water batch system for early stages and a recirculating system with micro-algae supplementation for later stages resulted in the best overall performance of the crab larvae.
A stocking density of 100 Z1 l-1 combined with a rotifer density of 45 ml-1 for early stages and Artemia feeding density of 20 nauplii ml-1 appeared to produce the best performance of S. paramamosain larvae. Optimal rations for crab larvae should however be adjusted depending on various factors such as species, larval stage, larval status, prey size, rearing system and zootechnics. A practical feeding ration could be 30 - 45 rotifers ml-1 for Z1 - Z2 and 5 - 10 Artemia (meta) nauplii ml-1 from Z3 onwards.
Although not encouraged for commercial practices, antibiotics improved survival considerably, which shows bacterial disease is one of the key factors underlying the high mortality. Ozonation and probiotics as alternatives to prophylactic chemicals are worth investigating.
Cannibalism is also an important cause of high larval mortality at later larval stages and could be overcome by provision of suitable substrates/shelters and feeding larger Artemia meta-nauplii.
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1. Introduction
Aquaculture of mud crabs, Scylla spp., contributes a large proportion to the world
production of the genus (FA0, 1999). Mud crab moreover represent a valuable component
of small-scaled coastal fisheries in many countries in tropical and subtropical Asia, for
which there has been a general trend of increased exploitation in recent years (Angell, 1992;
Keenan, 1999a). In Vietnam, the mud crab Scylla paramamosain is the second most
important marine species next to shrimp, being cultured widely in the coastal area. Mud
crab farming however currently relies entirely on the wild for seed stock and the main
obstacle for expansion is the unavailability of hatchery-reared seed (Liong, 1992; Keenan,
1999a; Mann et al., 2001; Rattanachote and Dangwatanakul, 1992; Shelley and Field, 1999;
Xuan, 2001).
Zootechnics, disease and nutrition are the three main areas of research, which have
supported commercial controlled production of marine fish and crustacean larvae
(Sorgeloos and Léger, 1992). These three aspects are to a large extent interconnected and
developing hatchery techniques for a “new” species is not possible unless all three are
addressed. Strictly speaking, the design of rearing systems covers purely zootechnical
aspects. Sub-optimal rearing conditions (e.g. physical stress, lack of oxygen or sub-optimal
water quality) however affect larval health and can cause mass mortality by the outbreak of
diseases. Similarly, system design influences (live) feed quality and its availability for the
predator larvae.
There has been a great deal of progress in marine larval rearing technology since its
beginning in the 1960’s (Howell et al. 1998; Shelbourne 1964). Many of the modern
technical improvements developed over the past decades could, with some modifications, be
applied for mud crab. An overview of the rearing systems currently applied for larviculture
of mud crabs is presented in Chapter 2. Although much experience and knowledge has been
obtained from these systems, there is a need to further optimize rearing techniques in order
to maximize larval survival and quality. Furthermore, techniques should be adapted for each
Scylla species in function of local conditions (seawater source, status of hatchery
management, local resources).
This paper describes and discusses the main larval rearing techniques for S.
paramamosain that have evolved in the present study in order to further improve seed
production of this species.
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2. Materials and methods
2.1. Source of larvae
Fully gravid crabs were bought from local markets and transported to the hatchery.
Prior to stocking in the hatchery, the crabs were bathed in a 100 µl l-1 formalin solution for 1
hour. The crabs were housed individually in 100-l compartments of a roofed 2 × 2 × 0.5 m
cement tank, equipped with a biofilter. Rearing water (30 ± 1 g l-1 salinity) was diluted from
brine (90 - 110 g l-1) with tap water and chlorinated before use. Water temperature was not
controlled, but fluctuated slightly around 28 °C. Every crab was fed a daily ration of 10 - 15
g of fresh marine squid, bivalve or shrimp meat alternately.
After 3 - 5 days of acclimation, unilateral eyestalk ablation was applied to induce
spawning. After spawning, berried crabs were again bathed in a 100 µl l-1 formalin solution
for 1 hour and transferred to a 70-l plastic tank connected to a biofilter for egg incubation.
Daily management consisted of siphoning out waste material and shedded eggs from the
tank bottom and controlling temperature (30 °C), salinity (30 g l-1) and ammonia and nitrite
levels. Every other day, the crab was bathed in a 50 µl l-1 formalin solution for 1 hour to
reduce or prevent infestation of the eggs with fungi and bacteria. During egg incubation, the
crabs were not fed.
One to two days prior to hatching, the female was moved to a 500-l fibreglass tank.
When the hatching process was completed, larvae were selected based on their photo-tactic
behaviour. Aeration in the hatching tank was therefore turned off for several minutes and
the larvae that were actively swimming up to the surface were collected by gently scooping
them from the surface.
The larvae were then transferred to the rearing containers. In order to slowly acclimate
the larvae to the new rearing conditions, they were placed in a 50-l plastic mesh bucket and
slowly rinsed with water from the larval rearing containers for 20 to 30 minutes, before
releasing them.
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2.2. Food and feeding
Start cultures of the micro-algae Chaetoceros calcitrans and Chlorella vulgaris were
cultured indoor with Walne solution in seawater of 30 g l-1 at 25 °C. Large-scale production
was performed indoor in 500-l tanks under a transparent roof. A hemocytometer was used to
count micro-algal densities.
Rotifer culture and enrichment
The same rotifer strain, Brachionus plicatilis L-strain with lorica length and width of
164 ± 22 and 120 ± 22 µm, respectively, was used in all experiments. Rotifers were cultured
indoor in 100-l fiberglass tanks operated in batch mode, following the procedure described
in Sorgeloos and Lavens (1996). Rotifers were initially grown on baker yeast, but one week
before use as feed for the larvae, the yeast was replaced by Culture Selco® (INVE
Aquaculture, Belgium). Temperature and salinity were controlled at 25 °C and 25 g l-1,
respectively. They were harvested through a 60 µm screen and rinsed thoroughly.
Rotifers were enriched with micro-algae or artificial enrichment media before being
fed to the crab larvae. Enrichment with Chlorella was performed at a density of 5 106 cells
ml-1 for 3 hours (Dhert, 1996). Rotifers were also enriched with Dry Immune Selco® (DIS,
INVE Aquaculture, Belgium), using two separate doses of 0.05 g l-1 at a 3-hour interval.
Enrichment was performed at a density of 500 rotifers ml-1. The water in the enrichment
vessel was slowly heated to 29 - 30 °C to avoid exposing the rotifers to thermal shock when
they were added to the larval rearing tanks. Before being fed to the larvae, enriched rotifers
were rinsed and re-suspended in clean seawater at the same temperature of the crab rearing
tanks.
Artemia culture and enrichment
Artemia nauplii (Vinh Chau strain) were hatched as described by Van Stappen (1996).
Both newly-hatched or enriched Artemia nauplii were used in the experiments of this study.
Artemia were enriched with Chaetoceros in the same micro-algal density as for rotifer
Micro-algae culture
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enrichment. The nauplii were also enriched with DIS® (using two separate doses of 0.3 g ml-
1 at a 6-hour interval). The temperature and salinity were maintained at 30 °C and 30 g l-1,
respectively during Artemia enrichment. The density of Artemia during enrichment was 200
ml-1. Before feeding to the crab larvae, the Artemia were rinsed with disinfected seawater
and suspended at a known density in seawater.
Feeding
Rotifers were fed to the crab larvae from DAH 0 - 6 (Z1-Z2 stages). Newly-hatched
Artemia or Artemia meta-nauplii were offered from DAH 6 (Z3 stage) onwards. Rotifers
and Artemia were added daily at 30 - 45 ml-1 and 5 - 10 ml-1 to the rearing tank, respectively
(experiment 1, 2, 3, 4, 7 and 8). For experiment 5 and 6, live feed were feed at the required
prey densities based on the planned treatments. Whenever the crab larvae were fed enriched
live feed, algae- or DIS-enriched live feed were used on alternate days.
In experiments 1, 2 and 3, the effect of different water exchange schemes and the
addition of micro-algae on larval survival and development were evaluated. In experiment 4
to 8, other culture aspects such as Z1 stocking density, live feed density and different
prophylactic treatments were investigated. Water quality management schemes tested in
experiment 1 - 3 are summarized in Table 1. An overview of the experimental design and
culture conditions of all the experiments is presented in Table 2. Cylindro-conical fibreglass
tanks of 30 - 100 l were used in experiment 1 to 6. Experiments 7 and 8 were executed in 1-l
plastic cones. The small-scale experiments (1-30 l) were carried out in a temperature-
controlled room (28 - 30 °C). The experiments in 100-l tanks were executed outdoor under a
transparent roof without temperature control (27 - 31 °C). The source and the disinfection
procedure of the seawater for larval rearing were similar to those used for broodstock
rearing. Formalin at a concentration of 20 µl l-1 was applied every other day as prophylactic
treatment in experiment 1 to 6. Further details on experimental conditions are discussed for
each test separately.
2.3. Larval rearing experiments: objectives and experimental design
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Experiment 1
In this experiment, larval survival and growth in a clear water system with daily
partial water exchange (Clear-Batch) was compared to those in a clear water recirculating
system (Clear-Recirc). In the first rearing system, 30 - 50 % of the culture water was
manually replaced daily. In the recirculating system, all rearing tanks were connected to a
central biofilter. Water was recirculated at a rate of approximately 100 % of the tank volume
every 3 - 4 hours. Live feed and crab larvae were retained in the rearing tanks with the help
of a mesh screen of 70 and 300 µm during the rotifer and Artemia feeding stage,
respectively. Larger mesh screens (250 and 500 - 1000 µm for rotifer and Artemia stage,
respectively) and higher flow rates were used upon daily flushing out uneaten live feed and
waste.
Experiment 2
Here, the Clear-Recirc system was compared with 2 systems where micro-algae were
added. Rearing conditions for the Clear-Recirc system were similar to those described in
experiment 1. In the Algae-Recirc system, micro-algae were added daily to the recirculating
system at a low concentration ranging from 0.1 to 0.2 million cells ml-1. The operation of
the rearing tanks was similar to the Clear-Recirc treatment. In the Green-Batch treatment, a
classical “green-water” system, micro-algae concentrations in the culture tanks were kept at
a ten-fold higher level of 1 - 2 millions cell ml-1. In this system, the culture tanks were
initially only filled to 50 % of their capacity and gradually increased to 100 % by the end of
the Z2 stage by daily adding water and algae. Later on, 10 - 30 % of the rearing water was
replaced daily by clean seawater and/or algae, depending on the density of micro-algae
remaining in the rearing tanks. Upon water exchange, uneaten live feed was also flushed out
through a mesh screen (mesh sizes as described in experiment 1). The same amount of live
feed (30 - 45 rotifers ml-1 and 5 - 10 Artemia nauplii ml-1) was fed in all treatments. In the
systems using algae, Chlorella was used for Z1 - Z3 stages (which is unsuitable as food
source for Artemia); from Z4 onwards, Chlorella was gradually replaced with Chaetoceros.
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Experiment 3
In this experiment, a Green-Batch and a Green-Recirc system were set-up in order to
further evaluate the application of micro-algae on the performance of crab larvae. The first
rearing system was a batch system with addition of high concentrations of algae as
described in experiment 2. The second system consisted of a combination of the Green-
Batch system for early crab stages (Z1 - Z2) and Algae-Recirc system for later stages (Z3
onwards).
Experiments 4, 5 and 6
In these experiments, the effect of Z1 stocking density (50, 100, 150 and 200 l-1,
experiment 4), rotifer feeding density (30, 45 and 60 ml-1) for feeding Z1-Z2 stages
(experiment 5) and Artemia feeding densities (10, 15 and 20 ml-1) for feeding from Z3
onwards (experiment 6) was evaluated. These experiments were run in a Green-Batch
(experiment 4) or a Clear-Recirc system (experiments 5 and 6) as described above.
Experiments 7 and 8
In experiment 7, the effect of prophylactic chemicals on the survival of the larvae was
investigated. Three treatments, consisting of a control (no chemicals used), daily addition of
formalin at 20 µl l-1 and daily addition of Oxytetracycline at 10 mg l-1, were run in 1-l
plastic cones. All cones were placed in a waterbath in order to maintain the rearing
temperature at 30 °C. Water was replaced almost completely everyday. Upon water
exchange, the survival was counted. When counting survival, the bottles were rinsed with
clean water but the biofilm on the walls of the bottles was not removed as a simulation of
large rearing tanks.
Experiment 8 was a replication of experiment 7. In this experiment used bottles were
everyday replaced by new disinfected ones, i.e. the biofilm on the bottle sides was removed.
The survival and development of the crab larvae were compared.
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2.4. Evaluation criteria
The survival rates in the experiments using large (30 to 100-l) containers (experiment
1 to 6) were estimated by volumetric sampling. Depending on the tank volume and the
density of the surviving larvae, triplicate 300- to 1000-ml samples were taken from each
tank. Megalopae (M) (15 - 18 days after hatch (DAH)) and first crabs (C1) (DAH 22) were
counted individually. In experiments 7 and 8 (using small cones) the ival rate
was calculated by individually counting all surviving larvae in each replicate.
Larval development was monitored every three days (every day in experiments 7 and
8) by identifying the average zoeal instar stage of a sample of larvae and assigning it a
value: first zoea (Z1) = 1; second zoea (Z2) = 2, etc. Megalopa stage was assigned a value
of 6. To compare the larval development in each treatment, an average larval stage index
(LSI) was calculated from the average LSI value of all replicate containers in the same
treatment. For large containers (experiment 1 to 6), 5 or 10 larvae (in 30-l and 100-l tanks
respectively) were sampled from each tank to calculate the average LSI. The sampled larvae
were staged under a dissecting microscope. In experiment 8 using small containers, all
larvae were staged visually upon counting daily survival.
2.5. Statistical analysis
One-way analysis of variance (ANOVA) was used to compare data. Homogeneity of
variance was tested with the Levene statistic (P or α value was set at 0.05). If no significant
differences were detected between the variances, the data were submitted to a one-way
ANOVA. The Tukey HSD post-hoc analysis was used to detect differences between means
and to indicate areas of significant difference. If significant differences were detected
between variances, data were transformed using the arcsine-square root (for percentage, i.e.
survival rate) or logarithmic transformations (for LSI value) (Sokal and Rohlf, 1995). P was
set at both 0.05 and 0.01. Whenever differences are significant at P < 0.01, this is also
indicated. All analyses were performed using the statistical program STATISTICA 6.0.
average surv
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3. Results
Experiment 1
Larval performance in experiment 1, comparing the Clear-Batch and Clear-Recirc
systems is presented in Table 3. Survival in the Clear-Recirc system at Z4-Z5 stage on DAH
15 and in the megalopa stage on DAH 18 were significantly higher than those in the Clear-
Batch system (both at P = 0.01). Although slightly higher in the recirculating system, larval
stage index was not significantly different between treatments. The better larval
performance in the Clear-Recirc system was accompanied by significantly lower average
ammonia levels (P < 0.01) and slightly higher nitrite levels (see Table 2).
Experiment 2
Table 4 shows the survival and larval development rate of crab larvae cultured in 3
different rearing systems. On DAH 9, larval survival in the Clear-Recirc system was
significantly lower (P < 0.01) than in both treatments with micro-algae supplementation
(Algae-Recir and Green-Batch treatments). On DAH 12, survival in the Clear-Recirc
treatment was lower (P < 0.05) than in the Algae-Recirc system whereas the Green-Batch
system had intermediate results. The LSI values on DAH 15 show a similar trend although
not significantly different: a slower growth was attained in treatment Clear-Recirc compared
to the rearing systems with micro-algae supplementation.
The average levels of ammonia and nitrite in the Clear-Recirc and Algae-Recirc
systems were significantly lower (P < 0.01) than those in the Green-Batch system (see Table
2). In the Green-Batch system, peaks of ammonia and nitrite concentrations of 3 and 1 mg l-
1, respectively were recorded at the end of the experiment.
Experiment 3
Table 5 presents the larval performance of the crab larvae cultured in 2 rearing
systems in experiment 3. The survival rates and LSI values of both rearing systems on all
sampling days were not significant different (Table 5). However, the survival rates on later
days (from DAH 12 - 22) in the Green-Recirc system tended to be higher than those in the
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Green-Batch system. Also LSI values of the Green-Recirc system in later stages (DAH 9 -
15) tended to be higher than those in the Green-Batch system. The biofilter had a
significantly positive impact on water quality in the second part of the experiment, with
reduced ammonia (P < 0.05) and nitrite (P < 0.01) concentrations as a consequence (see
Table 2).
Experiment 4
Table 6 shows the survival and development rate of crab larvae stocked at 4 different
Z1 densities (50, 100, 150 and 200 l-1) in experiment 4. Survival rates were not significantly
different among treatments. From DAH 6 onwards however, the highest survival was
achieved at a density of 100 Z1 l-1. LSI values also showed little variation between
treatments. Only on DAH 6, larvae in treatment 50 Z1 l-1 had a significantly higher LSI than
larvae in treatment 200 Z1 l-1 (P < 0.05). Also beyond DAH 6, growth tended to be slightly
lower at 200 Z1 l-1.
Experiment 5
Table 7 shows the survival rates and the LSI values of crab larvae fed 3 different
rotifer densities in the Z1-Z2 stages. No significant differences were found for any of the
parameters. However, from DAH 6 onwards, feeding 45 rotifers ml-1 seemed marginally
better than the other prey densities in terms of larval survival. Although not significant,
feeding 30 rotifers ml-1 resulted in the lowest LSI value DAH 6 - 15 and therefore
seems not to be sufficient for optimal larval development. The LSI values of treatments 45
and 60 rotifers ml-1 were very similar.
Table 8 presents the survival and development rate of crab larvae fed Artemia nauplii
at three densities (10, 15 and 20 ml-1) from Z3 stage onwards. Although no significant
differences were observed between treatments, increasing Artemia density tended to
enhance the survival and this tendency became more prominent as crab larvae developed.
LSI values did not vary much between the larvae fed 10 or 15 Artemia ml-1; the larvae fed
s from
Experiment 6
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the highest Artemia density (20 ml-1) tended to develop slightly faster than those in the other
treatments.
Table 10 shows the survival and development rate to the megalopa stage (DAH 22) of
larvae receiving different prophylactic treatments. Unlike in experiment 7, in this
experiment, rearing cones were daily disinfected upon water exchange. From DAH 6 (P <
0.05) or DAH 9 (P < 0.01) onwards, the survival rate of larvae in the treatment using
antibiotics was significantly higher than those in the remaining treatments. Survival rates of
the control and formalin treatments were similar on most days. From DAH 6 onwards, the
LSI values of the formalin treatment were generally higher than for the other treatments (not
always significant). On DAH 15 and 18, the antibiotic treatment resulted in lower LSI
values (P < 0.01).
4. Discussion
4.1. Rearing system
Recirculation
Experiment 7
In Table 9, survival of larvae receiving antibiotics or formalin as prophylactic
treatment is compared with a control receiving no treatment. Antibiotics clearly resulted in
the highest survival; the control treatment had the lowest survival rates. The difference in
survival among treatments was significant (P < 0.01) as early as DAH 3. Only on DAH 4,
the formalin treatment had a higher survival (P < 0.05) compared to the control. A tendency
for slightly higher survival remained however until the last sampling day.
Experiment 8
Water recirculation through a biofilter in the Clear-Recirc system positively affected
larval performance compared to manual partial water replacement in the Clear-Batch system
(experiment 1). The advantages of recirculating systems in commercial fish and crustacean
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larval production have been proven before for other species (Workshop on the use of
recirculation systems in fish and shrimp larviculture, Ghent University, December 2003).
Research into recirculating systems has also been identified as a priority for shrimp culture
(Lawrence and Lee, 1997). In these systems, water exchange is minimized through the use
of biological, chemical, and/or mechanical filtration to maintain continuously good water
quality. As they provide less stress and constant good water quality to the larvae, these
systems are able to maintain a high biological carrying capacity in relatively little space
(Quillere et al., 1993; Twarowska et al., 1997). The results of this experiment show that
recirculating systems are also seem worthwhile to further investigate for crab larviculture in
order to decrease labour requirements and seawater consumption, at the same time
providing a more stable culture medium and thus reducing stress for the larvae. If system
design is kept simple, recirculating systems could also be suitable for large-scale
production.
Role of supplemented micro-algae
In experiment 3 in Chapter 4, the treatments “rotifer only” and “rotifers and algae” are
similar to the Clear-Batch and Algae-Batch treatments in this study. In that experiment,
micro-algae have been proven to aid the development of late zoeae and first metamorphosis.
Also in this study the positive effects of micro-algae addition were clearly noticeable.
Both Algae-Recirc and Green-Batch systems had better survival than the Clear-Recirc
system, and only the Algae-Recirc resulted in higher LSI than the Clear-Recirc system.
Micro-algae have been proven to be beneficial by various modes of action. They could
help to maintain the quality of live feed. As in the cultivation of marine fish larvae,
unconsumed rotifers may reside in the tanks for several days and their nutritional value may
become severely reduced (Makridis and Olsen, 1999). Furthermore, according to these
authors, poorly-fed rotifers were more sensitive to starvation than well-fed rotifers, as their
nitrogen content decreased at a higher rate. Starvation of rotifers may be prevented by
supplementation of micro-algae at a high level as in the Green-Batch rearing system.
It is critical to control and maintain micro-algae populations in aquaculture ponds
since the micro-algae play an important role in stabilizing pond water quality via either
ammonia uptake or oxygen production (Tseng et al., 1991). Since the Clear-Recirc system
already provided optimal water quality, it is unlike that the stabilizing effect on water
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quality is the only factor responsible for the improved performance in the algae-
supplemented system. In batch culture systems this effect would be probably much more
pronounced. A direct comparison between a green and clear water batch system was
however not made in this study.
In a study on the effect of Chlorella on the population of luminous bacteria Vibrio
harveyi, no luminous bacteria were recovered on day 2 and day 3 in flasks with Chlorella,
while those without the micro-algae still harboured luminous bacteria at day 3 (Tendencia
and dela Pena, 2003). Also the diatom Chaetoceros has been shown to produce natural
antibiotics and high concentrations of this marine diatom will eliminate V. vulnificus and
other pathogenic bacteria, which contribute to the propagation of viruses in the shrimp
production environment (Wang, 2003).
In conclusion, micro-algae in mud crab larval rearing seem to play a role in improving
and maintaining live food quality and controlling bacteria levels. In batch culture systems
they might also play an important role in stabilizing water quality.
Choice of system
In experiment 3, the Green-Recirc system (which is a combination of a Green-Batch
system during the rotifer feeding stage and a recirculating system thereafter) seemed to be
better than the Green-Batch system. The Green-Batch system seems more appropriate for
early stages of crab larvae (Z1 - Z2) as it is easier and less stressful for the early zoeae, to
gradually fill up the tanks with fresh seawater, algae and rotifers than flushing out old
rotifers in the recirculation system. In the recirculating system, the young larvae might also
be prone to physical damage and spend a lot of energy trying to swim up against the current.
Early crab larvae are delicate due to their small size (see Chapter 4) with three long spines
on the carapace that are easily damaged when they are entrapped on the mesh screen during
flushing of uneaten feed in the recirculation system. The nutritional effect of micro-algae is
probably also more pronounced during the rotifers feeding stage than during the Artemia
feeding stage. Furthermore, it is not necessary to recirculate water these first days, as the
concentrations of ammonium and nitrite are still low. Using the Algae-Recirc sytem in later
stages is more favorable for reducing the increasing ammonium and nitrite concentrations as
more waste material is produced by the crab larvae. Moreover, as the larvae develop into
more efficient predators, feed is consumed faster, and maintenance of optimal feed quality is
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less of an issue. Many studies successfully applied a similar combined rearing technique due
to its benefit for the larvae and convenience for management, particularly for large rearing
containers. Under green-water culture conditions water is not exchanged for the first three
days. Thereafter, water exchange is slowly increased from 10 - 20 % day-1 for Z2 - Z3 to
between 40 and 50 % day-1 at the end of the rearing cycle (Z4 - M) (Mann et al., 1999b;
Quinitio et al., 2001). In Japan a mesocosm system is used for culturing larvae in larger
tanks (> 10 m3). The tanks are partially filled with green-water at Z1 (20 - 25 % volume),
tanks are then filled up with clean seawater during the course of the Z2-Z3 stages and
during the Z4 and M stages water is exchanged on a flow-to-waste basis (Hamasaki et al.,
2002b).
4.2. Other zootechnics
Although data were not significantly different, stocking at 100 Z1 l-1 seemed to be
best in terms of survival. Treatment 50 Z1 l-1 tended to have lower survival than treatment
100 Z1 l-1, possibly due to higher concentrations of ammonia and nitrite (see Table 2)
released from uneaten feed since all treatments were supplied with an identical ration. For S.
paramamosain, Djunaidah et al. (2001a) found a tendency of increased survival to Z5 in
function of the Z1 stocking density (i.e. survival rates of 27, 39 and 63 % being obtained at
the densities of 50, 75 and 100 Z1 l-1, respectively). Baylon and Failaman (1999) also
reported higher survival and metamorphosis of S. serrata at a density of 50 Z1 l-1 compared
with lower densities of 10 and 25 Z1 l-1. The higher stocking densities tested in our study
(150 and 200 Z1 l-1) might have caused competition for feed and stress (higher risk of
cannibalism) for the larvae, resulting in lower survival (150 Z1 l-1 and 200 Z1 l-1) and
slower development (200 Z1 l-1).
Rotifer density for feeding early larval stages (Z1-Z2 stages)
In experiment 5, the rotifer density of 30 ml-1 tended to give the poorest performance.
A density of 60 rotifers ml-1 was best for larval growth, but did not lead to marked
improvements, and might moreover be economically unrealistic. Therefore, the intermediate
Z1 stocking density
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density of 45 rotifers ml-1 was frequently used for feeding crab larvae in early stages.
Baylon et al. (2004) found that S. serrata Z1-Z2 larvae were capable to ingest 900 (640-
1200) rotifers larva-1 day-1. Based on these data, a density of 45 rotifers ml-1 would exactly
meet the daily live feed requirement of larvae stocked at 50 Z1 l-1 as in experiment 5. Other
studies also indicated that rather high rotifer densities (30 - 80 ml-1) are required for optimal
growth and survival of S. paramamosain (Djunaidah et al., 1998; Zeng and Li, 1999) and S.
serrata (Suprayudi et al., 2002a). For S. paramamosain larvae, feeding 30 and 60 rotifers
ml-1 resulted in a significantly higher survival compared to feeding only 15 rotifers ml-1
(Djunaidah et al., 2001a). These authors found that individual dry weight of Z5 fed 15
rotifers ml-1 was significantly lower than those of Z5 fed with higher rotifer densities.
Practically, feeding 30 rotifers ml-1 at Z1 and increasing gradually to 45 ml-1 at Z2 proved to
be sufficient for a stocking density of 100 Z1 l-1 in our trials in larger rearing tanks (500 -
1,000 l). Increasing the ration by larval stages in this way is compensating the increased
ingestion of crab larvae as they grow (Baylon et al., 2004). For early larvae, feed amount
can however not be reduced to their maximum ingestion potential as they are quite
inefficient predators and therefore might require a minimal density to maximize encounter.
As in our study, most studies investigating the effect of rotifer density, added the live
feed in one single ration. Under these circumstances, theoretic densities are only attained
upon feeding and gradually decrease as larvae consume the prey. Optimal live feed
quantities can however not be separated from feeding frequency. Since zoeal larvae can
consume their optimal ration within 1 hour, Genodepa et al. (2004) suggested they can be
fed once a day. Because of the severe reduction of nutritional value of rotifers with longer
retention times in rearing containers (Makridis and Olsen, 1999) and a minimum prey
density needed for the passive feeding behaviour of zoea larvae (Heasman and Fielder,
1983; Zeng and Li, 1999), the optimal ration and the number of feedings should be further
investigated.
Artemia for feeding later larval stages (from Z3 onwards)
For feeding Z3, at a density of 100 Z1 l-1, a daily feeding ration of 20 Artemia nauplii
ml-1 seemed to result in the best larval performance for S. paramamosain. Especially in later
larval stages (Z4 - Z5), the higher survival compared to lower rations tended to be more
pronounced. In this respect, it might be beneficial to increase the Artemia density by crab
CHAPTER 6 – Rearing techniques
140
stage from 10 to 20 ml-1 from Z3 to Z5. On the other hand, high live feed densities would
increase the chance for early larvae to encounter and capture feed organisms (Zeng and Li,
1999) and therefore would improve the larval performance (Brick, 1974; Heasman and
Fielder, 1983; Quinitio et al., 2001). Optimal rations should therefore be determined for
each larval stage separately. Studies on individual larvae are very useful to determine prey
consumption. According to our previous experiment (Chapter 4), one Z3, Z4, Z5 and
megalopa larva were capable to consume on average 15, 25, 37 and 114 newly-hatched
Artemia day-1, respectively. Therefore, at a stocking of 100 Z1 l-1, the daily feeding densities
of Artemia theoretically should be at least 1.5, 2.5, 3.7 and 11.4 ml-1, for each stage. In a
similar experiment with S. serrata, Baylon et al. (2004) found that there was an increasing
ingestion of Artemia nauplii of 80 - 160 individuals at Z2, 240 at Z3 - Z4 and 320 - 640 at
Z5. These data also show an increase of prey consumption with larval development, but the
absolute values are much higher than in our study. This difference can probably be
explained by the fact that we only counted missing Artemia as ingested; whereas Baylon et
al. (2004) included both missing and those having only missing body parts as consumed. In
the same study, these authors also investigated the effect of prey density on ingestion by
individual S. serrata larvae. For Z1, Z2 and Z3 stages, the number of Artemia nauplii
ingested by the larvae at a lower food density of 2.5 ml-1 was comparable to that at 5 ml-1;
and for Z4 - Z5, a density of 5 ml-1 was comparable to 10 ml-1 (Baylon et al., 2004). In that
study, Artemia was however co-fed with rotifers at a density of 15 - 20 ml-1. If Artemia
were the only food, the optimal Artemia ration would therefore probably be higher than 2.5
to 5 ml-1. In another study on S. serrata, a daily optimum food concentration of 10 Artemia
nauplii ml-1 was established for zoea survival (Brick, 1974).
In conclusion, the optimal Artemia ration for Z3 - Z5 observed in experiment 6 (10 -
20 Artemia nauplii ml-1) seems higher than in most other studies (2.5 - 10 Artemia nauplii
ml-1) (Baylon et al., 2004; Brick, 1974). Maybe the recirculating system used in this study
resulted in a greater loss of prey organisms (e.g., more Artemia were entrapped on the
overflow screen) than in the small batch culture systems used in other experiments. The
small nauplii size of the Artemia strain (Vinh Chau strain) used in our study could be
another cause that led to increased ingestion. In practice, (larger-sized) HUFA enriched
Artemia were normally used in order to reduce the prey amount to 5 - 10 ml-1.
For megalopae of S. paramamosain we found a three fold higher number of ingested
newly-hatched Artemia nauplii compared to Z5 (114 and 37 Artemia, respectively) for a
CHAPTER 6 – Rearing techniques
141
similar prey density (see experiment 1 in Chapter 4). This means megalopae are voracious
predators, capable to chase their prey actively and consume large amounts of feed in a short
time. From this, it could be beneficial when megalopae are fed frequently smaller rations in
order to optimise feed quality and reduce cannibalism. Genodepa et al., (2004) similarly
indicated that in contrast to earlier larval stages, which can be fed once per day, S. serrata
megalopae may need to be fed more often to maximize ingestion. These authors found no
significant differences in the ingestion rate of megalopae fed microbound diets at rations
ranging from 12.5 to 100 % of the standard ration (equivalent to 5 Artemia nauplii ml-1 in
one hour). Baylon et al. (2004) also found a high increase in Artemia ingestion in the first
few days of the planktonic phase of the megalopa stage. Later on, megalopae become more
benthic and prepare for the second metamorphosis to first crab; therefore, Artemia with
jerky swimming are no longer preferable, but minced shrimp or mussel meat are a more
suitable feed.
In general, the optimal Artemia ration for crab larvae should be adjusted depending on
various factors, e.g. species, larval stage, larval status, prey size, rearing system and
zootechnics. As for rotifer feeding, feeding sufficient smaller rations several times
throughout the day might be beneficial.
Prophylactic chemicals
In experiment 7, heavy fungal infection was observed in larvae of the control
treatment, but not in the other treatments. In an experiment to control fungal diseases caused
by Lagenidiales (an order including 5 virulent fungal strains: one Haliphthoros, Sirolpidium
and Atkinsiella strain and two Lagenidium strains) in eggs and larvae of the swimming crab,
Portunus trituberculatus, and the mud crab, S. serrata, Hamasaki and Hatai (1993a, 1993b)
found that a bath treatment with 25 µl l-1 formalin of newly-hatched larvae in the hatching
tank was a practical and effective method for inhibiting the occurrence of these fungal
diseases. As formalin as such was found not to be toxic to crab larvae at that concentration,
it could be considered that the higher mortality in the formalin treatment compared to the
antibiotics treatment, was probably caused by pathogenic bacteria. Laboratory cultures of
crab larvae often suffer severe mortality from disease, particularly from epibiotic bacteria
and larval mycosis (Amstrong et al., 1976; Hamasaki and Hatai, 1993a, 1993b). A study on
S. serrata indicated a significantly higher survival up to DAH 7 (over 90 %) when using
CHAPTER 6 – Rearing techniques
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Oxytetracycline, whereas almost complete mortality occurred in the control treatment
(Mann, 2001). The author considered that potentially up to 80 % of the larval mortality
could be attributed to bacteriological causes.
In experiment 8, the improved hygiene conditions (i.e. new disinfected cones were
used everyday) did not improve survival and development of larvae in the control and
formalin treatments. Oxytetracycline in this experiment proved the best prophylactic
chemical for crab larvae. Antibiotics resulted in a significantly higher survival but seemed
to negatively affect larval development to some extent.
However, antibiotics have not always been used in a responsible manner in
aquaculture. A major consequence of using antibiotics has been the proliferation of resistant
bacteria and the transmission of resistance to other bacterial species (Benson, 1998). The
development of antibiotic resistance by pathogenic bacteria is considered to be one of the
most serious risks to human health at the global level (FAO, 2002). Formalin is more
acceptable than antibiotics as it shows no accumulation in animal tissues (Jung et al., 2001).
Recently however, Japan has strictly banned the use of formalin in aquaculture as it might
cause cancer in humans, reduces oxygen levels in the water and causes algae to die off
(VASEP, 2003). Moreover, in this experiment, formalin did not improve larval survival
sufficiently to make it economically rewarding. Pathogenic bacteria are considered as one of
the most serious causes for the high mortality of early crab larvae. It can be safely assumed
that all inputs (seawater, broodstock, live feed and daily management in hatcheries) into the
culture tank are potential sources of infection (Blackshaw, 2001). Strict hygiene at all steps
is always advised for hatchery activities. However, this advice is not always being followed,
especially in backyard hatcheries. Therefore, other zootechnics should be investigated as
alternatives for the use of chemicals. Ozonation and probiotics could be interesting in this
respect (see Chapter 7).
Cannnibalism
Cannibalism is another important cause of mortality that is strongly linked to system
design and rearing conditions. In a previous experiment in the Clear-Recirc system in 30-l
tanks, a total survival of Z1 to Z5/megalopa of 42 ± 9 % was obtained on DAH 15 (data not
shown). Mortality of the larvae mainly resulted from cannibalism as most of the dead larvae
had missing appendages, but no symptoms of disease or unsuccessful metamorphosis were
CHAPTER 6 – Rearing techniques
143
observed. Especially cannibalism among Z5 and of megalopa on Z5 seemed to be a
problem. Asynchronous moulting exacerbates the problem of cannibalism (Quinitio et al.,
2001). Moulting synchronicity can be improved by improving nutrition through live food
enrichment (Takeuchi et al., 1999). Cannibalism, particularly at the megalopa and crab
stages, is a common problem and accounts for a large percentage of the mortality (30 - 50
%) in Scylla crabs (Dat, 1999b; Heasman and Fielder, 1983; Latiff and Musa, 1995;
Quinitio et al., 2001; Suprayudi et al., 2002a; Zainoddin, 1992). Cannibalism could be
averted by transferring the metamorphosed larvae to separate containers (Davis, 2003),
nursing megalopae in spacious containers (Marasigan, 1998; Rodíguez et al., 1998),
supplying substrata and shelters (Mann et al., 1999b; Marasigan, 1998; Quinitio et al., 2001)
and feeding live HUFA enriched Artemia juveniles instead of small-sized Artemia nauplii.
In experiment 4 in Chapter 5, on average 27 % (maximum 48 %) of Z1 survived to the
first crab stage. In this experiment, megalopa were removed manually from the rearing
bowls immediately after metamorphosis. This survival rate was the best obtained so far and
would be economically viable. However, it is not practical to manually separate out
megalopae in large rearing tanks. Furthermore, S. paramamosain megalopae appear to be
more delicate than those of S. serrata and there is only a short time between first and second
metamorphosis (7 - 10 days). Therefore; it is more convenient to leave the megalopae in the
same culture tank and harvest the crablets after a short period of 10 days. The best practice
seems to be to offer sufficient substrate and HUFA-enriched Artemia juveniles. Optimal
type and amount of substrate have not been sufficiently investigated however and facilities
for culturing Artemia biomass are not always available on research scale.
5. Conclusions and suggestions
The combination of a green-water batch system for early stages and a recirculating
system with micro-algae supplementation for later stages, stocking density of 100 Z1 l-1,
feeding density of 45 rotifers ml-1 for early stages and 20 Artemia nauplii ml-1 for later
stages resulted in the best performance of S. paramamosain larvae.
The optimal ration for crab larvae should however be adjusted depending on various
factors, e.g. species, larval stages, larval status, prey size, rearing system and zootechnics. A
feeding regime with frequent addition of small quantities of feed is worth investigating.
CHAPTER 6 – Rearing techniques
144
Practically, rations of 30 - 45 rotifers ml-1 for Z1 - Z2 and 5 - 10 Artemia (meta) nauplii ml-1
from Z3 onwards can be applied.
Antibiotics improved larval survival but there are some indications that they might
retard growth. Formalin enhanced larval development with intermediate survival. Both
products are not encouraged for commercial mud crab larviculture as they are unsafe and/or
unefficient. Ozonation and probiotics as alternatives to prophylactic chemicals are worth
investigating.
Cannibalism is also an important cause of high larval mortality at later larval stages
and is most practically overcome by supplementation of suitable substrates/shelters and
feeding ongrown Artemia meta-nauplii.
Acknowledgements
This study was supported by the European Commission (INCO-DC), the Flemish
Inter-University Council (Vl.I.R.-IUC) and the International Foundation for Science (IFS).
CHAPTER 6 – Rearing techniques
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Table 1 Overview of larval rearing systems applied in this study based on way of water exchange and micro-algae supplementation
WATER EXCHANGE ALGAE SUPPLEMENTATION
Discontinuous manual partial water renewal
Continuous water treatment through the use of biofilter
No micro-algae supplemented (indoors) Clear-Batch system Clear-Recirc system
Micro-algae supplemented at low levels to provide extra food for live preys (indoors or outdoors)
Algae-Batch system Algae-Recirc system
Micro-algae supplemented at high concentration and self-sustainable under natural sunlight as an extra food for live prey and water conditioning (outdoors)
Green-Batch system
Green-Recirc system (Combination of Green-Batch and Algae-Recir system at early and late larval stages, respectively)
Table 2 Overview of experimental conditions and water quality parameters (mean ± standard deviation) in experiment 1 to 8. For description of rearing systems refer to Table 1
Factor Rearing systemContainer volume
cates
Stocking density (Z1 l-1)
NH4+
(mg l-1) NO2
-
(mg l-1)
Experi- ment (l)
No of repli-
Clear-Batch 0.35±0.14A 0.14±0.11a1 Clear-Recirc 50 0.03±0.07B 0.11±0.09a
0.02±0.04b B 0.04±0.01b B
Rearing system 30 5
Clear-Recirc Al 0.07±0.08b B 0.10±0.08b B 2 Rearing system Green-Batch
100 8 100 1.72±1.30a A 0.57±0.33a A
1.54±1.50a 0.43±0.13A
gae-Recirc
Green-Batch 3 Rearing system Green-Recirc 100 4 100 0.11±0.07b 0.15±0.10B
50 1.50±1.09a 0.53±0.39a
100 1.07±0.62a 0.43±0.35a
150 0.98±0.68a 0.43±0.29a100
200 0.73±0.48a 0.34±0.26a
(30, 45 & 50 rotifers ml-1)
4 Z1 density Green-Batch 3
5 Rotifer density
Clear-Recir 30 5 50 0.06±0.05 0.06±0.06
6 Artemia density (10, 15 & 20 Artemia ml-1)
Clear-Recir 30 5 100 0.04±0.05 0.13±0.09
7 1 4 100 n.d. n.d.
8
Prophylactic treatment (control, formalin & Oxytetracycline)
Clear-Batch 1 4 100 n.d. n.d.
Values within an experiment in the same column followed by the same superscript letter are not statistically different (P ≥ 0.05, regular letters and P ≥ 0.01, capital letters). N.d. = not determined.
CHAPTER 6 – Rearing techniques
146
Table 3 Survival rates and larval stage index (LSI) values of S. paramamosain larvae cultured in 2 different rearing systems. For treatment descriptions refer to Table 1. Experiment 1. DAH = days after hatch Treatment DAH 3 DAH 6 DAH 9 DAH 12 DAH 15 DAH 18 Survival rate (%) Clear-Batch 85±6a 79±9a 70±6a 64±7a 42±6B 32±5B
Clear-Recirc 84±4a 78±8a 72±5a 70±5a 63±9A 47±6A
LSI
Clear-Batch 1.5±0.2a 2.7±0.1a 3.5±0.4a 4.0±0.0a 4.6±0.2a
Clear-Recirc 1.5±0.2a 2.7±0.1a 3.6±0.3a 4.2±0.3a 4.8±0.1an.d. n.d.
Survival rates or LSI values in the same column followed by the same superscript letter are not statistically different (P ≥ 0.05, regular letters or P ≥ 0.01, capital letters). N.d. = not determined.
Table 4 Survival rates and larval stage index (LSI) values of S. paramamosain larvae cultured in 3 different rearing systems. For treatment descriptions refer to Table 1. Experiment 2. DAH = days after hatch Treatment DAH 3 DAH 6 DAH 9 DAH 12 DAH 15 Survival rate (%) Clear-Recirc 74±12a 63±7a 44±6b A 26±11b A 8±7a
Algae-Recirc 74±12a 63±9a 61±7a A 43±7a A 15±8a
Green-Batch 74±11a 67±9a 58±9a A 35±9ab A 13±6a
LSI Clear-Recirc 1.9±0.1a 2.7±0.2a 3.9±0.1a 5.0±0.1a 5.1±0.1b A
Algae-Recirc 2.0±0.1a 2.8±0.3a 4.0±0.1a 5.0±0.1a 5.6±0.2a A
Green-Batch 2.0±0.1a 2.8±0.2a 4.0±0.1a 5.0±0.1a 5.1±0.1ab A
Survival rates or LSI values in the same column followed by the same superscript letter are not statistically different (P ≥ 0.05, regular letters and P ≥ 0.01, capital letters).
Table 5 Survival rates and larval stage index (LSI) values of S. paramamosain larvae cultured in 2 different rearing systems. For treatment descriptions refer to Table 1. Experiment 3. DAH = days after hatch Treatment DAH 3 DAH 6 DAH 9 DAH 12 DAH 15 DAH 22 Survival rate (%) Green-Batch 94±6a 88±9a 80±3a 66±15a 44±20a 9±1a
Green-Recirc 94±6a 89±8a 80±5a 68±11a 56±11a 12±3a
LSI
Green-Batch 1.4±0.3a 2.7±0.1a 3.8±0.4a 5.0±0.0a 5.2±0.2a
Green-Recirc 1.4±0.2a 2.6±0.2a 3.9±0.3a 5.0±0.0a 5.3±0.1an.d. n.d.
Survival rates or LSI values in the same column followed by the same superscript letter are not statistically different (P ≥ 0.05). N.d. = not determined.
CHAPTER 6 – Rearing techniques
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Table 6 Survival rates and larval stage index (LSI) values of S. paramamosain larvae stocked at 4 different Z1 densities (Z1 l-1). Experiment 4. DAH = days after hatch Treatment DAH 3 DAH 6 DAH 9 DAH 12 DAH 15 DAH 22 Survival rate (%) 50 79±9a 56±19a 42±16a 31±17a 28±12a 4±6a
100 80±7a 74±6a 71±10a 56±11a 45±8a 5±4a
150 79±2a 57±12a 45±9a 31±12a 28±10a 5±1a
200 85±5a 53±17a 42±8a 34±3a 30±5a 5±1a
LSI 50 1.7±0.2a 3.0±0.1a A 3.9±0.1a 5.0±0.1a n.d. 100 1.8±0.1a 3.0±0.1ab A 4.0±0.0a 5.0±0.1a
150 1.8±0.2a 3.0±0.1ab A 3.9±0.2a 5.0±0.1a
200 1.8±0.2a 2.7±0.1b A 3.7±0.2a 4.8±0.1a
n.d. n.d. n.d. n.d. n.d. n.d. n.d.
Survival rates or LSI values in the same column followed by the same superscript letter are not statistically different (P ≥ 0.05, regular letters and P ≥ 0.01, capital letters). N.d. = not determined.
Table 7 Survival rates and larval stage index (LSI) values of S. paramamosain larvae fed 3 different rotifer densities (rotifers ml-1) from DAH 0 - 6. Experiment 5. DAH = days after hatch
Treatment DAH 3 DAH 6 DAH 9 DAH 12 DAH 15 Survival rate (%) 30 89±7a 53±10a 30±8a 13±7a 10±7a
45 87±6a 58±9a 35±7a 18±9a 14±8a
60 87±5a 55±7a 32±6a 16±11a 11±10a
LSI 30 1.8±0.2a 2.7±0.1a 3.6±0.2a 3.8±0.2a 4.0±0.4a
45 1.8±0.2a 2.8±0.2a 3.8±0.1a 3.9±0.1a 4.3±0.5a
60 1.8±0.2a 2.8±0.2a 3.8±0.2a 4.0±0.1a 4.4±0.5a
Survival rates or LSI values in the same column followed by the same superscript letter are not statistically different (P ≥ 0.05).
Table 8 Survival rates and larval stage index (LSI) values of S. paramamosain larvae fed 3 different instar-1 Artemia densities (Artemia ml-1) from DAH 6. Experiment 6. DAH = days after hatch Treatment DAH 9 DAH 12 DAH 15 Survival rate (%) 10 26±10a 12±5a 8±3a
15 30±6a 13±7a 10±6a
20 32±8a 19±9a 18±9a
LSI 10 3.1±0.2a 3.7±0.4a 4.3±0.5a
15 3.1±0.1a 3.7±0.2a 4.3±0.5a
20 3.2±0.1a 3.8±0.3a 4.6±0.3a
Survival rates or LSI values in the same column followed by the same superscript letter are not statistically different (P ≥ 0.05).
CHAPTER 6 – Rearing techniques
148
Table 9 Survival rates (%) of S. paramamosain larvae treated daily with prophylactic chemicals from DAH 1 - 9. Experiment 7. DAH = days after hatch Treatment DAH 1 DAH 2 DAH 3 DAH 4 DAH 5 DAH 6 DAH 7 DAH 8 DAH 9 Control 94±2a 79±3a 61±3b B 54±4c B 35±10b B 24±8b B 13±6b B 3±3b B 1±1b A
Formalin 94±3a 81±10a 75±12ab AB 68±11b AB 50±16b AB 38±16b AB 25±11b B 8±7b AB 4±6b A
Antibiotics 97±1a 89±3a 85±5a A 82±4a A 77±5a A 66±6a A 56±3a A 28±12a B 16±11a A
Survival rates in the same column followed by the same superscript letter are not statistically different (P ≥ 0.05, regular letters and P ≥ 0.01, capital letters).
Table 10
Survival rates and larval stage index (LSI) values of S. paramamosain larvae treated daily with prophylactic chemicals. Experiment 8. DAH = days after hatch Treatment DAH 3 DAH 6 DAH 9 DAH 12 DAH 15 DAH 18 DAH 22 Survival rates (%) Control 85±3a 64±7b A 48±8b B 34±13b B 28±11b B 17±7b B 9±5b A
Formalin 84±7a 66±8b A 47±7b B 34±12b B 26±8b B 13±10b AB 11±8ab A
Antibiotics 91±4a 80±2a A 74±4a A 66±8a A 52±6a A 34±3a A 21±5a A
LSI Control 1.8±0.1a 2.6±0.3a 3.4±0.2ab A 4.5±0.3a 5.1±0.1a AB 5.5±0.1b AB 5.8±0.2a A
Formalin 1.8±0.1a 2.8±0.1a 3.7±0.1a A 4.3±0.3a 5.1±0.1a A 5.8±0.1a A 6.0±0.1a A
Antibiotics 1.9±0.0a 2.7±0.2a 3.3±0.2b A 4.3±0.1a 4.9±0.1b B 5.3±0.2b B 5.6±0.2a A
Survival rates or LSI values in the same column followed by the same superscript letter are not statistically different (P ≥ 0.05, regular letters and P ≥ 0.01, capital letters).
CHAPTER 7
The earliest documented attempts for the breeding of a crustacean in captivity were
made by the Japanese scientist Motosaku Hudinaga (Hudinaga, 1942) using mature Penaeus
japonicus females collected from the wild. The demonstration of the technical feasibility of
mass production was successfully achieved in 1953 (Hudinaga and Kittaka, 1967) and this
laid the groundwork to modern Penaeid shrimp culture.
General discussion
1. Challenges and perspectives for mud crab larviculture
Feasibility of mud crab larviculture compared to other crustaceans
Before starting the larviculture of a new aquaculture species, it is important to first
understand some basic biological characteristics of the species in question. Wickins and Lee
(2002) have, based on some basic characteristics (control of breeding, fecundity, duration of
larval life, growth and tolerance to crowding) ranked culture feasibility of a number of
crustacean groups (shrimp, prawn, crayfish, crab and lobster). According to these authors,
feasibility of larviculture of crabs is only just better than of lobsters. Shrimp were regarded
to be the easiest in terms of larval production.
Compared to shrimp, the development of technologies for the larval culture of mud
crabs is much more recent. Although the first research on mud crab larviculture already
dates back from the early 60’s (Ong, 1964), progress was discontinuous and unreliable. In
recent years, a renewed interest in mud crab culture has emerged mainly due to the need of
diversification of cultured marine species in relation to the high risk of epidemic diseases in
shrimp and the increasing demand for high-value aquaculture species in the global market.
In 1991, the first international seminar focusing on mud crab culture and trade was
convened in Sura Thani, Thailand. The seminar stressed that especially attempts to develop
techniques for mud crab seed production have been very limited. The slow pace of progress
was attributed to the general lack of knowledge about certain aspects of the larval and
juvenile stages such as feeding behaviour, nutritional requirements and water quality
requirements. Survival from zoea to first crab stage had been obtained in the laboratory, but
this had not been transferred to commercial practice. Until the most recent international
CHAPTER 7 – General discussion
150
workshop on mud crab rearing, ecology and fisheries held in Can Tho, Vietnam in 2001,
also no commercial hatcheries had been established. In the meantime however, considerable
progress has been achieved and specific problems have been solved one by one, e.g.
maximum survival from newly-hatched zoeae to the first crabs of 48, 15 and 10 % in 1-l
bowls, 100-l tanks and 1000-l tanks, respectively have been achieved.
The slow progress in mud crab larviculture success can be understood from the
delicate nature of the larvae. Davis (2003) claimed that this evolves from the typical “r”
type reproduction strategy of the species. Mud crabs spawn and hatch a large number of
eggs but these larvae are subjected to high mortality. The newly-hatched Scylla larvae are
tiny (hatching from small eggs of 0.3 - 0.4 mm in diameter), are not fully developed yet
(Zeng and Li, 1999), have very little yolk reserve (Cheng and Li, 2001) and need to feed
soon after hatching (Djunaidah et al., 2003; Li et al., 1998; Li et al., 1999; Lumasag and
Quinitio, 1998). They are inefficient feeders in the zoeal stages (Heasman and Fielder,
1983) and therefore require high densities of live food (Zeng, 1998). They are also
extremely susceptible to disease (Mann, 2001) and thus require highly hygienic conditions
(Blackshaw, 2001). They are sensitive to subtle changes in their physical environment (Hill,
1974) and require stable, high quality seawater (Davis, 2003). They are also highly
cannibalistic in the megalopa and crab stages (Dat 1999b; Quinitio et al., 2001; Suprayudi et
al., 2002a) and undergo two energetically demanding and stressful metamorphoses before
reaching the juvenile stage (Hamasaki et al., 2002b). The larvae are thus extremely difficult
to rear in high density conditions and establishing hatchery technology has proven to be
more challenging than for any other commercially important decapod crustacean (Davis,
2003) such as the Penaeid shrimp, Chinese mitten crab (Eriocheir sinensis) (Naihong et al.,
1999) and other Portunidae such as the blue crab (Portunus trituberculatus) with only 4
zoeal stages (Cowan, 1984). However, mud crab larvae are not as difficult to rear as those of
the Palinurid spiny lobsters (mostly Panulirus spp.), which take an extremely long time to
develop (197 - 365 days) (Ong, 1966; Kittika, 1994; Wickins and Lee, 2002).
Challenges for mud crab larviculture
Crustacean larval development occurs within a narrow range of environmental
parameters (Sastry, 1983). The conditions that planktonic marine larvae encounter in the
wild are impossible to reproduce in the laboratory. The ocean acts as a buffer into which
CHAPTER 7 – General discussion
151
harmful chemicals are quickly diluted and changes in temperature, salinity or pH occur
extremely slowly (Davis, 2003). Larvae are able to maintain a minimum distance from one
another and dead or diseased larvae drop out of the surface layers and do not pollute the
water. The larvae are also able to regulate their position in the water column, selecting ideal
conditions of light, salinity, turbulence (Forward et al., 1984) and food availability (Malkiel
et al., 1999). The distribution of plankton in the ocean is not homogenous (Benfield and
Downer, 2001) as oceanographic phenomena tend to concentrate plankton in “fronts” (Clark
et al., 2001) or “patches” (Natunewicz et al., 2001). Crab larvae that are incorporated into
these fronts are vulnerable to predation from larger zooplankton, but in turn have access to
an abundance of suitable-sized plankton as food (Davis, 2003).
Differences between the natural environment and that of a hatchery have been
clearly reviewed by Davis (2003). Predators are excluded from the rearing vessels but the
larvae are crowded together into small volumes, at artificially high densities, increasing
interactions and the risk of disease and cannibalism. Smaller volumes of water are also
much more subjected to changes in physical and chemical parameters. Although live feed is
always made available, the species provided are selected as much for their ease of culture as
for their suitability as feed and usually bear little resemblance to the natural food of the
larvae in the wild. Cultured live feed organisms moreover are often stressed from the rigors
of mass culture and processing, carry bacterial and other contamination and sometimes lack
essential nutrients (Støttrup and McEvoy, 2003).
Perspectives for larviculture of mud crabs
Comparing the conditions of “natural” and “man-made” hatcheries, one can not help
to have a “gloomy” picture about the artificial reproduction of mud crabs. However, “r”
type species can be beneficial in the hatchery operation due their own characteristics. For
example, due to their high fecundity, fewer broodstock animals are needed compared to
shrimp to provide the required number of larvae for a hatchery. Or the low survival rate of
the larvae could be compensated by maintaining more broodstock and stocking higher
numbers of larvae.
An r-selected approach to reproduction emphasizes producing large numbers of
offspring with minimal care given to each offspring by its parents (Andrews and Harris,
1986; Pianka, 1970). Based on this concept, larval rearing of mud crabs in hatcheries could
CHAPTER 7 – General discussion
152
be considered as an “artificial” trade-off to K-selection, in which few offspring (the best,
most active ones are selected at the beginning) are produced, but maximal care (proper
feeding and adequate prophylactic measures) is taken to insure viability of each offspring.
In this light, artificial rearing of mud crab larvae could be commercially viable, although
their quality might be inferior compared to wild seed where a strong natural selection takes
place.
2. Broodstock management (Chapter 3)
Broodstock management is without doubt one of the least understood and researched
areas (Izquierdo et al., 2001), especially for a newly-studied species like the mud crab. The
basis of every hatchery operation is the maintenance and conditioning of a healthy group of
broodstock that could spawn year-round. In most areas, where larval rearing of mud crab,
Scylla spp., is conducted, the source of eggs relies on gonadal maturation and spawning of
wild-caught broodstock in captivity (Mann et al., 1999a). Due to the migratory behavior of
female mud crabs in the wild (Hill, 1994), the knowledge of spawning, breeding and
hatching of eggs under natural conditions is lacking. Most information on these processes
therefore comes from crabs that are held in captive conditions for the purposes of
aquaculture research and production (Mann et al., 1999a).
Fortunately, unlike shrimp (e.g. Penaeus monodon), S. paramamosain broodstock are
still easily available from the wild and they can be readily induced to spawn almost year-
round within a short time (i.e. one week for fully gravid females) after stocking in the
hatchery. Furthermore, pond-reared crabs also produce good quality eggs (Millamena and
Quinito, 2000) and the life cycle has been closed in captivity (Quinitio et al., 2001). When
fully gravid females are selected, there also does not seem to be special nutritional
requirements for maturation and spawning and the animals do not have to pass through a
special conditioning. Based on the average reproductive performance obtained in our study,
the cost for newly-hatched crab larvae is 20 to 40 times cheaper compared to Penaeus
monodon nauplii. In that respect, research on broodstock management was not the first
priority in our study. Nevertheless, data on rearing conditions and main reproductive
characteristics were recorded as a basis to further improve techniques. Chapter 3 firstly
intends to describe the practices that promoted the production of good quality larvae in this
CHAPTER 7 – General discussion
153
study, and secondly, aims to elucidate a number of factors which are important in the
selection and management of broodstock for hatchery production purposes.
Our results indicated that egg quality remained unchanged within 60 days in captivity,
given the low price of crab breeders and the decreasing spawning activity however, it is
recommended to keep the broodstock for not more than 30 days. Males were not needed in
the hatchery as fertilization had happened in the wild and adult females caught from the
wild have usually mated and carry spermatophores for extended periods (Robertson and
Kruger, 1994). Although more difficult to manage, pond systems resulted in the best
reproductive performance. Tank systems could be a more practical alternative if stocking
densities are kept low and a proper substrate is provided. The peak spawning season of S.
paramamosain in South Vietnam seems to be from March to July, which would be the
easiest period to obtain good spawners and hence perform larval rearing. With eyestalk
ablation, the optimal production period for mud crab could be extended from February to
August. Spawning activities seemed to be related to ambient temperatures rather than the
monsoon season. Females of 300 - 500 g produced the highest total number of viable Z1 and
are therefore preferred. Smaller females were sometimes not fertilized, while big females
(over 500 g) tended to have lower relative Z1 fecundities. Although detached eggs could be
incubated artificially, egg incubation by the females themselves was far more reliable and is
therefore recommended for commercial application. Artificial incubation is however useful
for research purposes (e.g. where different treatments need to be tested on the same egg
batch).
Although individual females achieved high fecundities and fertilization rates, overall
reproductive efficiency (i.e. the percentage of females that hatched viable larvae) was rather
low (maximum 31 % in ponds). A possible reason might be that the source of the
broodstock was restricted to inshore regions. Broodstock from offshore water might be more
mature and therefore more efficient.
In conclusion, year-round maturation and spawning of wild breeders of S.
paramamosain for research and pilot production can easily be achieved, especially when
uni-lateral eyestalk ablation is applied. In order to avoid further pressure on this valuable
resource the use of wild breeders should however be discouraged and research efforts
should be directed towards full domestication of the species. In this respect, further research
is warranted on all aspects of maturation and fertilization in captive conditions, investigating
CHAPTER 7 – General discussion
154
dietary requirements and suitable rearing conditions to trigger maturation and spawning in a
more natural way.
3. Optimal feeding for the larvae (Chapter 4)
Rotifers and Artemia as suitable live feed
It is well known that most species of brachyuran crabs have pelagic larvae that are
planktotrophic and have to obtain nutrition from external sources to survive (Lehto et al.,
1998). The supply of a readily available and digestible feed is therefore crucial for larvae at
first feeding. Copepods (Acartia tsuensis and Pseudodiaptomus spp.), which are
nutritionally superior to rotifers and Artemia (Delbare et al., 1996) have been used as live
feeds in aquaculture with some success (Toledo et al., 1998). Copepods are however
difficult to culture consistently at high densities (Delbare et al., 1996). Rotifers and Artemia
are, for the time being, the most practical live foods available to hatcheries (Støttrup and
McEvoy, 2003).
Artemia cysts are commercially available and are very convenient and easy to use.
Therefore, Artemia nauplii are preferred to rotifers, which require continuous culture and
are sometimes subject to crashes. For that reason rotifers are generally replaced by bigger
live prey as soon as possible.
Early zoeae of S. paramamosain might be more rotifer-dependent compared to other Scylla
species
All publications to date have agreed that rotifers are the best live feed for mud crab
(Scylla sp.) larvae at early stages (Z1 - Z2) and Artemia appear to be better than rotifers for
later stages. A comparative study of replacing rotifers with Artemia at every zoeal stage
showed that larvae initially fed with rotifers but then shifted to Artemia at Z2/Z3 or fed a
mixed diet at Z3 gave best over all zoeal survival (Zeng and Li, 1999). Analysing the dry
weight, carbon, nitrogen and hydrogen content of larvae fed rotifers and Artemia revealed
that rotifers can meet larval development requirements at early zoeal stages, but Artemia
should replace rotifers from Z3 onwards as to meet the increasing nutritional demand,
especially around the time of the first metamophosis (Zeng, 1987, cited in Li et al., 1999).
CHAPTER 7 – General discussion
155
However, in a study of Davis (2003), S. serrata larvae benefited from the addition of
Artemia to the diet right from the zoea 1 stage. Larvae were also reared with good results
(up to 40 % to megalopa) on a diet of Artemia nauplii alone (Davis, 2003). This seems
however not possible for S. paramamosain. Our measurements of the diameter of newly-
spawned eggs demonstrated a considerable smaller egg size for S. paramamosain (288 ± 10
µm) than for S. serrata (313 ± 1 and 317 ± 1 µm for eggs spawned from ablated and intact
females, respectively) (Mann et al., 1999a). In comparison with other data sources (Quinitio,
pers. com.), S. paramamosain seems to have the smallest size of newly-extruded eggs
among the four Scylla species. A larger egg diameter probably coincides with an increase in
Z1 size, which results in a higher capability to catch larger prey like Artemia. From this it
becomes clear that differences between the species of the genus Scylla might exist. The right
time to shift to Artemia was therefore re-investigated.
Our results indicated that for S. paramamosain, rotifers were the most appropriate live
feed for the Z1 and Z2 stages. Crab larvae appeared capable of catching instar-1 Artemia
nauplii only from the Z2 stage onwards and the number of prey consumed increased in the
consequent larval stages. Based on our data, Artemia are best introduced at the Z2 stage
already in order to maximize survival and growth. Optimal feeding schedules should
however also take into account the nutritional composition of both live foods and the quality
of the larvae. In this respect, it was noticed that the ability of Z2 to catch instar-1 Artemia
might vary between batches of larvae. In our pilot-scale production trials (0.5 - 4 m3) it was
observed that feeding HUFA-rich rotifers for both Z1 - Z2 with is a good compromise to
make sure all larvae (irrespective of batch quality) can take up sufficient food. Moreover, a
population of crab larvae does not develop synchronously and always consists of a mixture
of stages, especially in large-scale commercial applications. Crab larvae seem to adapt to a
new food source quite easily and therefore an overlap in feeding rotifers and Artemia seems
not really necessary. Although micro-algal cells were not the proper initial feed for early
crab stages, they proved to be useful for stabilizing the quality of the rotifers and to maintain
good water quality.
Alternatives for rotifers
Despite recent advances, rotifer culture is still labour intensive and vulnerable to
periodic and unpredictable crashes (Fu et al., 1997; Suantika, 2001). Therefore, a rotifer-free
CHAPTER 7 – General discussion
156
feeding schedule is always preferable in hatcheries. Although it resulted in lower survival
compared to treatments where rotifers were fed, the results indicated that live umbrella-
stage Artemia were the best replacement for rotifers for feeding Z1 - Z2. In this way,
umbrella-stage Artemia might be a valuable alternative feed in case of rotifer shortage.
Umbrella Artemia could e.g. be supplemented to the diet after a few days of feeding rotifers.
The non-selective feeding at early crab larval stages also indicates there might be
possibilities to develop artificial diets as alternatives (or supplement) for live feed, in order
to reduce the dependency on rotifer cultures.
4. Live feed quality (Chapter 5)
In captivity, rotifers and Artemia nauplii support growth and survival of mud crab
larvae, but this simplified diet is not ideal. The phenomenon of moult death syndrome
(MDS) - high mortality during or after the moult from Z5 to megalopa, has been
encountered repeatedly. Poor nutrition, even if confined to the early larval stages, has been
suggested as a cause for MDS (Hamasaki et al., 2002a, 2002b; Li et al., 1999; Mann et al.,
2001; Marichamy and Rajapackiam, 2001; Quinitio et al., 2001; Suprayudi et al., 2002b;
Zeng and Li, 1999). Inferior nutrition may also be a factor contributing to the highly
variable survival and the high susceptibility to disease often recorded in mud crab
larviculture. The nutritional quality of rotifers and Artemia can be improved by enriching
them with nutrients in a process known as bioencapsulation (Coutteau et al., 1997;
Kanazawa and Koshio, 1994; Rees et al., 1994; Wouters et al., 1997). The effect of
enriching the live food with essential fatty acids (EFAs) contained in formulated emulsions
on survival and growth of mud crab larvae has been tested in this study.
The fatty acid nutrition of crustaceans is unique and can be grouped according to the
following (Tacon, 1987): (i) fatty acids that can be synthesized de novo from acetate are
termed non-essential fatty acids. Animals possess a ∆-9-desaturase-enzyme system that can
Role of essential fatty acids in crustaceans
Lipids are a source of essential fatty acids, which in turn are essential for the
maintenance and integrity of cellular membranes, they are a major source of energy, and are
precursors of the prostaglandin hormones (Tacon, 1987).
CHAPTER 7 – General discussion
157
convert saturated fatty acids into mono-unsaturated forms. The most abundant saturated and
unsaturated fatty acids are palmitic (16:0), stearic (18:0), arachidic (20:0) and oleic acid
(18:1n-9), respectively; (ii) essential fatty acids (EFA) composed of the linoleic acid (18:2n-
6) and linolenic acid (18:3n-3) series of PUFAs (poly unsaturated fatty acids). Crustaceans
cannot synthesize de novo these fatty acid (Kayama et al., 1980); and (iii) highly
unsaturated acids (HUFAs) also composed of the essential linoleic and linolenic series, but
with a chain length of more than 20 carbon atoms and more than 3 unsaturated bonds. They
can be only partially biosynthesized from linoleic acid or linolenic acid. The most common
HUFAs are docosahexaenoic acid (22:6n-3, DHA), eicosapentaenoic acid (20:5n-3; EPA)
and arachidonic acid (20:4n-6; ARA) (Kanazawa et al., 1979a).
The absence of the de novo synthesis of linoleic, linolenic, DHA and EPA was first
recognized in Penaeus japonicus (Kanazawa et al., 1977a, 1977b), P. monodon and P.
merguiensis (Kanazawa et al., 1979b). Chanmugam et al. (1983) reported that the linoleic
acid series (n-6) predominated in the freshwater prawn lipids whereas the linolenic acid
series (n-3) predominated in marine shrimp lipids. The difference between freshwater and
marine species has been widely attributed to a possible deficiency or impairment of the
enzyme ∆-5-desaturase in marine species, thereby rending them incapable of performing the
necessary conversion (Sargent et al., 1989; Drevon, 1992). DHA and EPA are derived from
dietary linoleic and linolenic acid through desaturation and carbon chain elongation
processes with presence of three enzymes (∆-6, 5, 4, desaturase) and the addition of 2
carbon atoms.
DHA and EPA are essential compounds required for cell membrane formation,
osmoregulation, the synthesis of prostaglandins and they also appear to have an activating
role in the immune system (Léger and Sorgeloos, 1992). Feeding crustacean larvae with
DHA and EPA enriched diets leads to improved survival and growth rates (Bengtson et al.,
1991; Kontara et al., 1995; Levine and Sulkin, 1984a).
Effects of DHA, EPA and ARA on mud crab larvae
No real differences in survival in the zoeal stages were found between the different
enrichment treatments tested here. The “nutritional impact” of HUFAs on the zoeal survival
was probably obscured by other more decisive factors such as the batch quality, micro-biota,
zootechnics. The significantly lower metamorphosis success in the low HUFA treatments
CHAPTER 7 – General discussion
158
proved however that HUFA-rich emulsions are needed to attain high survival to the crab
stage. Not that much the total n-3 level, but more particularly the DHA level and the
DHA/EPA ratio seem of crucial importance.
Larval development rate was very much affected by the dietary n-3 HUFA level and
DHA/EPA ratio. During early zoeal stages, significantly faster growth was obtained when
the larvae were fed live feed enriched with an emulsion containing 30 % total n-3 HUFA.
Emulsions with a high (50 %) or low total HUFA content on the other hand, either tended to
decrease survival or resulted in low growth rates. The DHA/EPA ratio of the live feed,
rather than the total HUFA content, became increasingly important during the later zoeal
stages. Overall, an emulsion with moderate total n-3 HUFA content (30 %) and high
DHA/EPA ratio (4) resulted in the best overall performance in terms of survival and larval
development for all zoeal stages. However, the optimal DHA/EPA ratio of live food
enrichment emulsions for early stages (Z1 - Z3) might be lower than 4 and needs to be
verified. During the later stages, also metamorphosis success was strongly correlated with
the DHA/EPA ratio of the live feed and the crab larvae. It is therefore recommended that an
emulsion with approximately 30 % total n-3 HUFA and a DHA/EPA ratio of 4 should be
used to enrich live feed from the Z3 stage to attain high survival to megalopa and first crab
stage. There was also evidence that DHA/EPA requirements might change during
development. Therefore it might be better if this ratio is increased gradually in time (e.g.
emulsions with DHA/EPA ratio of 1 for Z1-Z2 stages, of 2 - 3 for Z3 - Z4 and of 4 for Z5
onwards). Further research remains necessary on the suitable n-3 HUFA level and
DHA/EPA ratio for each larval stage.
Selecting an efficient rearing system
Supplementation of arachidonic acid had no effect on survival or growth during the
zoeal stages. First metamorphosis rate was however improved by the addition of dietary
ARA. Further research on the suitable levels and ratios of ARA in the enrichment diet for
crab larvae is therefore worth pursuing.
5. Rearing systems and other zootechnics (Chapter 6)
When starting larval rearing research of a new species, the rearing system is often
designed as simple as possible in order to focus on more immediate concerns such as first
CHAPTER 7 – General discussion
159
feeding, environmental parameters, etc. Typically, these systems are small in volume (from
a few-hundred ml vials or petri dishes to not more than 5-l containers). In these systems
clear water is normally used and larvae are counted and transferred individually by means of
pipette to clean containers daily. This type of system can be referred to as a clear-water
batch system (Clear-Batch). This system is very fit for fundamental research because the
larvae can be observed carefully and counted one by one. In this respect, a 5000-l tank is a
single unit and statistically it gives no more information than a 3-l pot (Blackshaw, 2001).
For the rearing of Scylla larvae, when antibiotics are used, this system has proven to
produce the highest survival of up to 60 - 90 % to megalopa stage (see Chapter 2) or 48 %
to first crab stage (see Chapter 5, experiment 4). The development of antibiotic resistance by
pathogenic bacteria is however considered to be one of the most serious risks to human
health at the global level (FAO, 2002). Furthermore, upscaling of this system is not practical
due to space requirements needed to treat and store seawater, and the high risk for
horizontal transfer of diseases.
Water quality has been identified to be one of the most important factors affecting
success of marine shrimp hatcheries (Bray and Lawrence, 1992). Due to its simplicity, the
clear-water batch system has been adopted well by researchers and producers for both larval
rearing and growout culture. Therefore most shrimp farmers prefer to set up their facilities
in coastal areas where high quality (oceanic) seawater is not limited. Such areas are however
not easily found anymore, except in remote areas where usually logistic problems are
manifold. Furthermore, these areas may also occasionally be influenced by run-off
containing agricultural chemicals or the presence of endemic or introduced pathogens (i.e.
bacteria and virus) that affect the health of shrimp. Viral diseases have had a significant
economic impact on the shrimp industry worldwide (Kalagayan et al., 1991; Wyban et al.,
1993). The aquaculture industry also faces growing pressure to operate under strict
environmental safety standards (e.g. on effluent discharge).
With respect to the above impediments, closed, recirculating seawater systems offer
an opportunity. The advent of these systems has been identified as a priority for shrimp
culture research (Lawrence and Lee, 1997). Water exchange is minimized in these systems
through the use of biological, chemical, and/or mechanical filtration to maintain proper
water quality. These systems are designed to maintain a high biological carrying capacity in
a relatively little space (Quillere et al., 1993; Twarowska et al., 1997).
CHAPTER 7 – General discussion
160
Considering the limitations of good quality seawater in the Mekong delta, a simple
clear-water recirculation system (Clear-Recirc) for rearing mud crab larvae has been tested.
In this system, the water was recirculated over a central biofilter to cope with the nitrogen
waste produced by the larvae. Larval rearing results in this system tended to be better and
more reliable compared to the batch system due to improved and more stable water quality
and the reduction of stress to crab larvae, which are more sensitive than shrimp.
In tropical areas, outdoor “abandoned and green” ponds or tanks sometimes have an
unexpected higher production than carefully managed clean ones. Reasons for failure of the
latter are most of the time attributed to bad management (e.g. excess feeding or fertilizing,
too high stocking densities, chemical application, etc.). It is true however that these “green”
ponds often are “ecologically balanced”. Where modern aquaculture has rejected the
“green” techniques for a long time, in recent years a renewed interest in these systems has
emerged.
Several recent studies pointed out the potential beneficial roles of micro-algae in
aquaculture rearing systems, such as maintaining the quality of live feed, stabilizing pond
water quality via either ammonia uptake or oxygen production and producing natural
antibiotics. The exact mode of action (e.g. source of micro-nutrients, source of
immunostimulants, water quality conditioner, and microbial conditioner) of “green-water”
(i.e. high concentrations of selected species of micro-algae) in the commercial larviculture
of several species of marine fish remains however unclear and therefore requires further
study (Sorgeloos, 1995).
When applied to crab larvae, the green-water batch system (Green-Batch) obtained
quite high survival rates in some experiments. Results were however variable and
sometimes also culture crashes were experienced due to the die-off of blooming algae
resulting in an increase of ammonium and nitrite in the rearing tanks.
In an attempt to combine the beneficial features of both clear- and green-water rearing
systems, two “mixed systems” were designed. Supplementation of low concentrations of
micro-algae into the indoor clear-water batch system (Algae-Batch) normally resulted in an
algal collapse and the formation of a harmful biofilm due to the low light intensities applied.
Micro-algal supplementation into the outdoor clear-water recirculating systems (Algae-
Recir) on the other hand seemed to improve the survival rates, especially in the later larval
stages. Best results were obtained when operating a green-water batch culture system at the
Z1 and Z2 stages and then gradually start to recirculate the water in later stages. Low
CHAPTER 7 – General discussion
161
densities of micro-algae can still be added during this last phase. This system was called a
green-water recirculation system (Green-Recir). The application of a green-water batch
mode for the early larval stages simplified the daily maintenance of the rearing tanks and
probably provides a type of “mature water” for the sensitive early stage larvae. In this
respect, there is increasing evidence that a process of microbial maturation can control the
composition of the bacterial flora of seawater (Skjermo and Vadstein, 1999). Settling of the
water for a number of days, results to a diverse bacterial flora, dominated by non-
opportunists, that act as a stable buffering system restricting the growth of opportunistic and
potentially pathogenic bacteria (Blackshaw, 2001).
In conclusion it should be mentioned that the selection of optimal rearing systems
largely depends on local conditions and practices should therefore be flexible. For example,
during 2000 - 2002, a project on mud crab S. paramamosain larviculture was carried out at
the Research Institute for Aquaculture III (RIA3) in cooperation with the Bribie Island
Aquaculture Research Centre (BIARC) in Australia and the Gondol Research Institute for
Mariculture (GRIM) in Indonesia. A pilot hatchery was located in Nha Trang province,
Central Vietnam with good access to ocean quality seawater. In this project, a clear water
batch rearing system has been applied in 500 to 4000-l tanks with similar results compared
to our work. Still other places have used flow-through systems with good larval survivals.
Other zootechnics
Even the best-designed rearing system can fail to produce high survival and good-
quality larvae if some other basic requirements are not met. Proper larval rearing densities
and feeding practice for rotifers and Artemia are discussed in Chapter 6. A larval rearing
density of 100 Z1 l-1 systematically produced good results. Feeding rotifers at 45 ml-1 the
first 6 days and Artemia nauplii at 20 ml-1 from day 6 (Z3 stage) proved to be the best
feeding schedule. However in practice, the best procedure for feeding was to divide the
daily ration into smaller portions with several feedings per day after checking the remaining
feed in the water and the ration should be increased by larval stages (i.e. 30 - 45 rotifers
ml-1 for Z1 and Z2 and 5 - 10 (meta) Artemia ml-1 for Z3 onwards) might also be more
practical.
CHAPTER 7 – General discussion
162
Using the rearing systems developed in our study, microbial interactions (especially
for early stages) and cannibalism (especially at megalopa and crab stages) remain the most
important causes of high mortality.
Experiments 7 and 8 in Chapter 6 proved that bacteria are one of the most serious
causes for high mortality of early crab larvae. It can be safely assumed that all inputs
(seawater, broodstock, live feed and daily management in hatcheries) into the culture tank
are potential sources of infection (Blackshaw, 2001). Applying antibiotics gave the best
larval survival in both experiments, but the use of these chemicals is nowadays strictly
regulated in aquaculture. Formalin could increase survival of the larvae slightly, but was not
as effective as antibiotics. Strict hygiene at all steps of the rearing process are therefore
advised. However, this advice is not always followed, especially in backyard hatcheries
where facilities are rather primitive and management is not always optimal. Therefore,
zootechnics should be optimized to overcome these problems and avoid the need for
chemicals (antibiotics and formalin).
Aside from further improving live feed quality (e.g. HUFA enrichment) to meet all
nutritional requirements of the larvae, the biggest challenge for crab larval rearing will
probably be the development of safe and sustainable prophylactic treatments to prevent
microbial interactions. In this respect, the use of ozone and probiotics are two promising
techniques. The microbial flora will need to be controlled in future production systems and
there is evidence that this can be achieved by using a recirculating system in which an ozone
Cannibalism during the megalopa stage accounted in some experiments for as much as
50 % of the mortality. The best solution to overcome this is to supply an appropriate
substrate and to feed larger prey such as juvenile Artemia. Using larger tanks or earthen
ponds to lower the density for advanced larvae might also be useful.
6. Overall conclusions and suggestions for further research
Broodstock rearing of the mud crab S. paramamosain does not pose any special
difficulties. Further research is however needed to accomplish complete domestication of
the species. Another area that also deserves further attention is the development of proper
broodstock diets in order to optimize maturation and mating in captivity and improve larval
quality.
CHAPTER 7 – General discussion
163
treatment is combined with the inoculation of the biological filter with selected nitrifying
and probiotic bacteria (Gatesoupe, 1991; Rombaut et al., 2001).
Mud crab larvae seem to accept inert feeds quite easily. Recently, Genodepa et al.
(2004) have shown that microbound diets incorporated with 14C-labelled rotifers are readily
ingested by S. serrata zoeae and megalopae. Developing formulated feeds to replace
partially the live feed could help to optimize nutrition and also may reduce the risk of
pathogenic infection for crab larvae.
More attention should also be paid to the megalopa and crab stages because these
stages require a high quality diet to metamophose to first crab and grow on to larger-sized
crabs that can be stocked in growout ponds. Cannibalism could be sufficiently reduced by
investigating suitable substrates, transfer of animals to larger containers and supplying
larger size preys like Artemia juveniles or adults.
Using the methodology described in different chapters of this thesis, recent large scale
trials have obtained survival to crab 1 stage of 20 - 30 %. Although several issues still need
to be solved, we may conclude that knowledge of mud crab larval rearing techniques have,
in recent years, evolved to a level comparable to that of shrimp in the early nineties, and
therefore commercial application might be envisaged within a few year time.
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Summary
Mud crab (Scylla spp.) fishing and culture represent a valuable income component of
rural fishery communities in many countries in tropical and sub-tropical Asia. Since wild
stocks are reported to decrease in many countries due to increased exploitation in recent
years, mud crab farming is becoming increasingly popular. In Vietnam, the mud crab Scylla
paramamosain is the second most important cultured species (next to shrimp) in the coastal
zone. Mud crab culture however currently relies almost entirely on wild seed stock. The
main obstacle for the further development of mud crab farming is the establishment of
hatchery-techniques for controlled production of seed.
Overall, the need for development of technologies for mud crab larviculture in
Vietnam is justified for the following reasons: (i) mud crab is a high-value species with an
increasing demand on the global market, (ii) it is a robust species that forms an alternative
for shrimp culture which is facing serious disease problems, (iii) availability of wild mud
crab seed is declining due to overfishing and habitat destruction (iv) mud crab can be
cultured using relatively simple traditional techniques requiring low initial investment, but
generating considerable profit and (v) after the reclassification of the genus Scylla into four
species, S. paramamosain can be considered a “new species” on which very little
information exists (Chapter 1 - Introduction).
Mud crab aquaculture is still an emerging industry, particularly the hatchery phase of
production; therefore very little peer-reviewed literature exists. As a collaborative effort
between some of the main research groups working on larviculture, a paper that describes
the “state of the art” of mud crab larviculture technology has been prepared. This paper
(Chapter 2 - Current status of mud crab Scylla spp. hatchery technology) reviews the
various rearing techniques and conditions currently employed in (mainly experimental)
larviculture of the species to serve as a basis for further reseach on this topic.
Broodstock availability and management are the first concerns for those who wish to
develop a new species for aquaculture. The environmental conditions and broodstock
management techniques used in this study were recorded and evaluated for their effect on
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reproductive performance as a basis for further improving culture techniques and ultimately
fully control domestication and seed production of this species (Chapter 3 - Reproductive
performance of captive mud crab Scylla paramamosain broodstock in Vietnam). From 1996
to 2002, reproductive performance of 786 wild S. paramamosain breeders was recorded.
The period from March to July was most efficient for broodstock rearing and might
correspond to the natural spawning season of S. paramamosain in South Vietnam.
September to February could be the period for gonad maturation in the wild. With eyestalk
ablation, the most favorable period for artificial reproduction could be extended from
February to August. Like marine shrimp, eyestalk ablation in mud crabs improved spawning
success, but did not alter the latency period between purchase and spawning. No negative
effects of ablation on broodstock survival or egg quality were found. Breeders collected
from an inshore region with higher and more stable average salinity levels tended to
perform slightly better than those collected from a region with lower and varying salinity.
Females in the range of 300 to 500 g had the best overall reproductive efficiency and are
preferred as breeders. Rearing broodstock in earthen ponds was more efficient; however,
management of a pond proved more complicated than tank systems. In terms of complete
domestication, broodstock rearing in tanks therefore provides a more practical alternative,
provided larger tanks and more suitable substrate are used. It was noticed that shading the
broodstock tanks was not necessary if shelters for hiding were available. Spawning activity
decreased with prolonged time in captivity. Egg quality criteria such as fertilization rate and
egg diameter did however not vary in function of time in captivity. Although detached eggs
could be incubated artificially, egg incubation by the females themselves was the best
practice. Overall, controlled reproduction of wild mature broodstock females of S.
paramamosain for research and pilot production is not problematic, especially with the
practice of eyestalk ablation. Although individual females had high fecundities and
fertilization rates; spawning and hatching success were however not very high. In this
respect, broodstock captured offshore might be better. Further research should be
undertaken to completely domesticate the species and further document maturation and
fertilization in captive conditions. Therefore dietary requirements and suitable rearing
conditions should be investigated. Life history studies in the wild could provide very useful
information in this respect.
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Chapter 4 (Optimal feeding schedule for mud crab Scylla paramamosain larvae)
identifies the most suitable prey for the early stages of mud crabs. Of the 3 feeds (micro-
algae, rotifers and artificial diets) tested as first feed, rotifers gave the best survival and
growth. The earliest appropriate time to shift from rotifers to Artemia was investigated since
rotifer culture is very laborious and not well mastered by most of hatchery managers in
Vietnam. Crab larvae could start catching newly-hatched Artemia from the zoea 2 stage
onwards and the number of Artemia consumed increased with each larval stage. The ability
of zoea 2 to catch newly-hatched Artemia seemed however to depend on the quality of that
particular batch and varied even between individual larvae. Although micro-algae were not
a proper initial feed for early crab stages, they proved beneficial in improving the nutritional
quality of rotifers, resulting in higher survival in later zoeal stages and a more successful
metamorphosis to the megalopa stage. In an attempt to simplify the feeding schedule, a
series of experiments were carried out where rotifers were replaced by different forms of
Artemia (live and heat-killed umbrella-stage Artemia and frozen or heat-killed Artemia
nauplii). Live umbrella-stage Artemia were the best replacement for rotifers for feeding zoea
1 - zoea 2 larvae compared to other Artemia forms. The unselective feeding behaviour
seems promising to develop artificial diets in order to substitute live feed and to reduce the
dependency on rotifers at the early stages. In a last step, the optimal time to shift from
rotifers to Artemia was investigated. Results showed that rotifers should be replaced by
Artemia already in zoea 2 stage. A transition period to shift from one diet to another seemed
not necessary. Prolonged feeding of rotifers beyond the zoea 2 stage tended to reduce
survival and delay the larval development. Although Artemia are more difficult to capture
for zoea 2 larvae than rotifers, they probably enhance crab larval performance due to their
higher nutritional value. The nutritional value of rotifers and Artemia is however not
consistent and therefore optimal feeding schedules might also depend local conditions and
culture techniques. From the zoea 3 stage onwards, crab larvae can ingest enriched Artemia
meta-nauplii.
In captivity, rotifers and Artemia nauplii support growth and survival of mud crab
larvae, but this simplified diet is not ideal. Inferior nutrition may be a factor contributing to
highly variable survival and the high susceptibility to disease often recorded in mud crab
larviculture. Enriching live feed with highly unsaturated fatty acids (HUFA) has been a
common way to improve the quality of live prey for other species. Chapter 5 (Influence of
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the content of highly unsaturated fatty acids in the live feed on larviculture success of mud
crab Scylla paramamosain) describes the effects of different standard live feed enrichment
emulsions on survival and growth of crab larvae. In most experiments, survival rate in the
zoeal stages was not statistically different among treatments. Larval development rate and
metamorphosis success were however more strongly affected by the dietary treatments. In
this respect, the DHA/EPA ratio in the live feed seems to be a key factor. Enrichment
emulsions with a high (50 %) total (n-3) HUFA content but low DHA/EPA ratio (0.6) or
zero total HUFA content caused growth retardation and/or metamorphosis failure. An
emulsion with moderate total HUFA content (30 %) and high DHA/EPA ratio (4) was the
best in terms of larval development rate during the zoeal stages and resulted in good
metamorphosis. The optimal DHA/EPA ratio of live feed enrichment emulsions for early
stages (Z1 - Z2) could however be lower than 4. Dietary arachidonic acid (ARA) seemed to
improve first metamorphosis, but its exact role needs further clarification. For the larval
rearing of Scylla paramamosain, it is recommended to use enrichment media with a total n-
3 HUFA content of approximately 30 %, with a DHA/EPA ratio of minimum 1. Further
research needs to be performed on the total (n-3) HUFA and DHA/EPA ratio requirements
for each larval crab stage. The role of ARA in metamorphosis also needs to be verified.
In addition to nutritional requirements, establishing proper zootechnics (Chapter 6 -
Improved larval rearing techniques for mud crab Scylla paramamosain) is another crucial
aspect of developing larval rearing technology. Based on the method of water exchange
(discontinuous partial water renewal or continuous treatment through biofiltration) and the
level of micro-algae (Chlorella or Chaetoceros) supplementation (daily supplementation
with low levels of 0.1 - 0.2 million cells ml-1 or maintenance at high levels of 1 - 2 millions
cells ml-1), six different types of rearing systems were tried. The combination of a green-
water batch system for early stages and a recirculating system with micro-algae
supplementation resulted in the best overall performance of the crab larvae. A stocking
density of 100 Z1 l-1 combined with a rotifer density of 45 ml-1 for early stages and Artemia
feeding density of 20 Artemia nauplii ml-1 appeared to produce the best performance of S.
paramamosain larvae. Optimal rations for crab larvae should however be adjusted
depending on various factors such as species, larval stage, larval status, prey size, rearing
system and zootechniques. A practical feeding ration could be 30 - 45 rotifers ml-1 for Z1 -
Z2 and 5 - 10 Artemia (meta) nauplii ml-1 from Z3 onwards. However, antibiotics are not
Summary
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encouraged for mud crab larviculture as they are unsafe. Ozonation and probiotics as
alternatives for prophylactic chemicals are worthwhile to investigate. Cannibalism is also an
important cause of high larval mortality at later larval stages and could be overcome by
provision of suitable substrates/shelters and feeding larger Artemia meta-nauplii.
Although there is still ample room for further improvements, we may conclude that,
on an international level, much progress has been achieved in recent years. Knowledge on
larval rearing technology of mud crab seems to be of a similar level as for shrimp in the
early nineties. This makes us hope that commercial mud crab hatcheries could become
reality in the next few years (Chapter 7 - General discussion).
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Samenvatting Mangrove krab (Scylla spp.) visserij en kweek is een waardevolle inkomenscomponent van
rurale vissersgemeenschappen in vele tropische en subtropische landen in Azië. Aangezien
gerapporteerd wordt dat de natuurlijke stocks in vele landen dalen door de recentelijk
toegenomen exploitatie, wordt kweek meer en meer populair. In Vietnam is de mangrove
krab Scylla paramamosain de tweede meest gekweekte soort (naast garnalen) in de
kustzone. De kweek van mangrove krab steunt echter bijna volledig op het gebruik van wild
zaad. Het belangrijkste probleem voor de verdere ontwikkeling van de kweek van mangrove
krab is de ontwikkeling van broedhuistechnieken voor de gecontroleerde productie van
zaad.
Samenvattend kan het belang van ontwikkeling van broedhuistechnieken voor mangrove
krab gemotiveerd worden door de volgende punten: (i) mangrove krab is een soort met een
hoge waarde en een groeiende vraag op de internationale markt, (ii) het is een sterke soort
die een alternatief vormt voor de kweek van garnalen die geplaagd wordt door
ziekteproblematiek, (iii) beschikbaarheid van wild zaad neemt af door overbevissing en
vernietigen van habitat, (iv) mangrove krab kan gekweekt worden gebruik makende van
relatief eenvoudige traditionale technieken die slechts lage investering vragen, maar toch
aanzienlijke winst opleveren en (v) na de reclassificatie van het genus Scylla in vier species,
kan Scylla paramamosain beschouwd worden als een nieuwe soort waarover erg weinig
informatie bestaat (Hoofdstuk 1 - Introduction).
Mangrove krab aquaculture is nog een industrie in ontwikkeling, zeker de broedhuis-fase
van de productie; daardoor bestaat er erg weinig wetenschappelijke literatuur. Als een
gezamelijk initiatief van enkele van de belangrijkste onderzoeksgroepen werkzaam op
broedhuistechnieken werd daarom een artiekel geschreven die de “state of the art” van
mangrovekrab larvicultuur beschrijft. Dit artiekel (Hoofdstuk 2 - Current stutus of mud
crab Scylla spp. hatchery technology) vat de verschillende kweektechnieken en condities
samen die tegenwoordig gebruikt worden voor de (voornamelijk experimentele) larvicultuur
van deze soort als basis voor verder onderzoek.
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Beschikbaarheid en management van ouderdieren zijn de eerste bekommernissen voor wie
een nieuwe soort wil ontwikkelen voor de aquacultuur. De omgevingscondities en
kweektechnieken gebruikt voor de ouderdieren in deze studie werden beschreven en hun
effect op reproductie geevalueerd als basis voor de verdere verbetering van de
kweektechnieken en uiteindelijk domesticatie en zaadproductie van deze soort volledig te
controleren (Hoofdstuk 3 - Reproductive performance of captive mud crab Scylla
paramamosain broodstock in Vietnam). Van 1996 tot 2002, werden de reproductie
parameters van 786 wilde S. paramamosain ouderdieren gevolgd. De periode van maart tot
juli bleek het meest efficient voor het kweken van ouderdieren en komt waarschijnlijk
overeen met de natuurlijke voortplantingsperiode in Zuid-Vietnam. September tot februari is
mogelijks de periode voor de ontwikkeling van de gonaden in het wild. Door gebruik te
maken van de techniek van oogsteelverwijdering kan de periode voor kunstmatige
voortplanting verlengd worden van februari tot augustus. Zoals bij mariene garnalen,
verhoogt oogsteelverwijdering het aantal dieren dat eitjes aflegt, maar het had geen invloed
op de tijd nodig om eiafleg te bekomen. Er werd geen negatieve invloed vastgesteld van
oogsteelverwijdering op overleving van de ouderdieren of eikwaliteit. Ouderdieren
afkomstig van kustzones met een hogere en meer stabiele saliniteit bleken lichtjes beter dan
deze afkomstig van zones waar de saliniteit lager en meer variabel is. Vrouwtjes van 300 tot
500 gram hadden algemeen de beste voortplantingsefficientie en zijn daarom verkiesbaar als
ouderdieren. Het kweken van ouderdieren in vijvers bleek het meest efficient, maar het
management was moeilijker dan voor tanksystemen. Om complete domesticatie te bereiken
is het kweken van ouderdieren in tanks daarom aangewezen, op voorwaarde dat grotere
tanks met meer substraat gebruikt worden. Er werd ook vastgesteld dat het niet nodig was
de tanks te verduisteren indien schuilplaatsen voorzien werden. Eiafleg nam af naarmate de
tijd in gevangenschap toenam. Criteria voor eikwaliteit zoals bevruchtingsgraad en
eidiameter varieerden echter niet in functie van de tijd in gevangenschap. Hoewel
losgekomen eitjes kunstmatig konden geincubeerd worden, bleek incubatie door de
vrouwtjes zelf de beste techniek. Algemeen kan gesteld worden dat gecontroleerde
reproductie van Scylla paramamosain voor onderzoek en semi-commerciele schaal geen
bijzondere problemen stelt, zeker wanneer gebrijk gemaakt wordt van oogsteelverwijdering.
Hoewel individuele vrouwtjes hoge fecunditeiten en bevruchtingspercentages haddden, was
de algemene eiafleg- en ontluikigsefficientie echter niet hoog. Het gebruik van dieren
gevangen in open zee kan hier misschien een oplossing bieden. Verder onderzoek is nodig
Summary
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om deze soort volledig te domesticeren en maturatie en bevruchting in gevangenschap te
documenteren. Hiervoor moeten de nutitionele vereisten en optimale kweekomstandigheden
verder onderzocht worden. De studie van de levenscyclus in het wild zou hiervoor nuttige
informatie kunnen aanbrengen.
Hoofdstuk 4 (Optimal feeding schedule for mud crab Scylla paramamosain larvae)
identificeerd de meest geschikte prooi voor de vroege larvale stadia van mangrove krab.
Van de drie voeders die als eerste voedsel getest werden (micro-algen, rotiferen en
artificiele voeders), resulteerde rotiferen in de beste groei en overleving. Het vroegste
tijdsstip om van rotiferen naar Artemia om te schakelen werd onderzocht omdat de kweek
van rotiferen erg arbeidsintensief is en voor de meeste broedhuizen erg moeilijk is. Krab
larven konden vanaf het tweede zoea stadium pas-ontloken Artemia vangen en het totaal
aantal dat geconsumeerd werd nam elk stadium toe. De mogelijkheid van zoea 2 larven om
Artemia te vangen bleek echter variabel tussen verschillende groepen en zelfs individuele
larven. Hoewel micro-algen niet geschikt waren als eerste voedsel, bleken ze gunstig voor
het verbeteren van de nutritionele kwaliteit van de rotiferen en resulteerden ze zo in een
hogere overleving in latere larvale stadia en een meer succesvolle metamorphose tot
megalopa. In een poging om het voederschema te vereenvoudigen werd een serie
experimenten uitgevoerd waar rotiferen vervangen werden door verschillende vormen van
Artemia (levende en door middel van warmte afgedode umbrella-Artemia en ingevroren en
door middel van warmte afgedode Artemia nauplii). Levende umbrella-Artemia bleken het
beste vervangvoeder voor rotiferen voor zoea 1 en 2 stadia in vergelijking met de andere
Artemia vormen. Het niet-selectieve voedingsgedrag van de larven toont aan dat mogelijks
artificiele voeders kunnen ontwikkeld worden om levend voedsel te vervangen en de
afhankelijkheid van rotiferen in de eerste stadia te verminderen. In een laatste stap, werd de
optimale tijd om over te schakelen van rotiferen op Artemia nader onderzocht. De resultaten
toonden aan dat rotiferen al moeten vervangen worden door Artemia in het zoea 2 stadium.
Een overgangsperiode om van het ene voeder op het andere over te gaan bleek niet nodig.
Het voeder van rotiferen voorbij het zoea 2 stadium leek de overleving te verminderen en de
larvale ontwikkeling te vertragen. Hoewel Artemia moeilijker te vangen zijn voor zoea 2
dan rotiferen, verbeteren ze waarschijnlijk de productie door hun hogere nutritionele
kwaliteit. De nutritionele kwaliteit van rotiferen en Artemia is echter niet constant en
optimale voederschemas kunnen daardoor afhangen van de lokale condities en de gebruikte
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kweektechnieken. Vanaf het zoea 2 stadium konden krab larven ook aangerijkte Artemia
meta-nauplii opnemen.
In gevangenschap zijn rotiferen voldoende voor de groei en overleving van mangrove krab
larven. Dit gesimplifieerd dieet is echter niet ideaal. Sub-optimale nutritie kan een faktor
zijn die bijdraagt tot de zeer variabele overleving en hoge vatbaarheid voor ziektes die vaak
vastgesteld wordt in de larvicultuur van mangrove krab. Aanrijking van het levend voedsel
met hoog onverzadigde vetzuren (HUFA) is een algemeen gebruikte techniek om de
kwaliteit van het levend voedsel te verbeteren voor andere soorten. Hoofdstuk 5 (Influence
of the content of highly unsaturated fatty acids in the live feed on larviculture success of
mud crab Scylla paramamosain) beschrijft het effect van verschillende standaard
aanrijkingsemulsies voor levend voedsel op groei en overleving van de krab larven. In de
meeste experimenten werd de overleving in de zoea stadia niet statistisch significant
beinvloed. Larvale ontwikkeling en metamorphose succes werden echter sterker beinvloed
door de behandelingen. De DHA/EPA (docosahexaenoic acid/eicosapentaenoic acid)
verhouding in het levend voedsel bleek hierbij doorslaggevend. Aanrijkingsemulsies met
een hoog (50%) total n-3 HUFA gehalte maar een lage DHA/EPAverhouding (0.6) of totaal
deficient in n-3 HUFA veroorzaakten groei achterstand en/of slechte metamorphose. Een
emulsie met een middelmatig totaal HUFA gehalte (30%) en hoge DHA/EPA verhouding
(4) resulteerde in de beste groei in de zoea stadia en gaf een goede metamorphose. De
optimale DHA/EPA verhouding voor aanrijking van levend voedsel voor de vroege larvale
stadia kan echter lager zijn. Arachidon zuur (ARA) in het voer leek de eerste metamorphose
te begunstigen, maar de exacte rol moet verder uitgeklaard worden. Voor de larvicultuur van
Scylla paramamosain wordt aangeraden aanrijkingsmedia te gebruiken met een total n-3
HUFA gehalte van 30 %, met een minimale DHA/EPA verhouding van 1. Verder onderzoek
is echter nodig om de vereisten voor totaal n-3 HUFA gehalte en DHA/EPA verhouding van
elk larvaal stadium te bepalen. De rol van ARA in de metamorphose moet ook verder
bestudeerd worden.
Naast het bepalen van nutritionele vereisten, is het onwikkelen van goede kweeksystemen
(Hoofdstuk 6 - Improved larval rearing techniques for mud crab Scylla paramamosain) een
ander cruciaal aspect in het ontwikkelen van larvale kweektechnieken. Op basis van de
methode van wateruitwisseling (discontinue gedeeltelijke waterverversing of continue
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188
behandeling door biofiltratie) en de hoeveelheid gesupplementeerde micro-algen (Chlorella
of Chaetoceros; dagelijks toegevoegd aan lage concentraties van 0.1 - 0.2 miljoen cellen per
ml of op peil gehouden aan hoge concentraties van 1 - 2 miljoen cellen per ml), werden 6
verschillende kweeksystemen getest. De combinatie van een “groen-water batch” syteem
voor de vroege larvale stadia en een recirculatie-systeem met supplementatie van micr-algen
voor de latere stadia gaf algemeen het beste resultaat. Een stockeringsdensiteit van 100 Z1
per liter gecombineerd met het voederen van 45 rotiferen per ml voor de vroege stadia en 20
Artemia nauplii per ml gaf het beste productieresultaat. Optimale voederniveaus dienen
echter aangepast te worden aan verschillende factoren zoals soort, larvaal stadium,
afmetingen van de prooi, kweeksystem en kweekcondities. Een practisch voederniveau kan
zijn 30 - 45 rotiferen per ml voor Z1 - 2 en 5 - 10 Artemia (meta-) nauplii per ml vanaf Z3.
Het prophylactisch gebruik van antibiotica verbeterde de overleving. Het gebruik van
antibiotica wordt echter ontraden voor mangrove krab larvicultuur angezien dit niet veilig is.
Het gebruik van ozon en probiotica zijn alternatieven die onderzocht kunnen worden.
Kannibalisme is een andere belangrijke oorzaak van mortaliteit bij latere larvale stadia en
kan mogelijks verholpen worden door het voorzien van substraat/schuilpaatsen en het
voederen van grotere Artemia meta-nauplii.
Alhoewel er nog veel ruimte is voor verbeteringen, kunnen we concluderen dat, op
internationaal vlak, er heel veel vooruitgang geboekt is. De kennis van de technologie voor
de kweek van mangrove krab larven lijkt op een zelfde niveau als voor garnalen in de
vroege jaren negentig. Dit laat ons hopen dat commerciele mangrove krab broedhuizen
realiteit worden in de komende jaren (Hoofdstuk 7- General discussion).
Curriculum vitae
Personal data First name NGHIA Family name TRUONG TRONG Date of birth 10 September 1956 Place of birth Can Tho, Vietnam Nationality Vietnamese Married status married, 2 children Address 192, street 30 April, Can Tho City, Vietnam Email [email protected] Education Since 2000 Registered for Ph.D., Ghent University, Belgium. Major subject:
Aquaculture. 1993-1995 M.Sc. in Aquaculture, Laboratory of Aquaculture & Artemia Reference
Center, Ghent University, Belgium. 1976-1980 Aquaculture Engineer studies at College of Aquaculture and Fisheries (CAF),
Can Tho University, Vietnam 1974-1976 B.Sc. studies on Physics, Chemistry and Natural Science at University of
Saigon, Vietnam Professional record Since 2002 Vice Dean of College of Aquaculture and Fisheries (CAF), Can Tho
University, Vietnam 2001-2002 Director of Aquaculture and Fisheries Sciences Institute (AFSI), College of
Agriculture, Can Tho University, Vietnam 1999-2001 Director of Institute for Marine Aquaculture (IMA), College of Agriculture,
Can Tho University, Vietnam 1996-1999 Vice Director of Shrimp Artemia R&D Institute (SARDI), Can Tho
University, Vietnam 1989-1993 Researcher of Artemia-Shrimp R&D Center (ASRDC), Can Tho University,
Vietnam Since 1981 Lecturer of College of Aquaculture and Fisheries, Can Tho University,
Vietnam Scientific activities International cooperation 2001-2007 Coordinator of VL.I.R. (Vlaase Interuniversitaire Raad) IUC: subcomponent
“Distant education: aquaculture as a test case” 2000-2004 Coordinator of EU INCO-DC (International Cooperation with Developing
Countries) program “Culture and Management of Scylla species” 1998-2000 Coordinator of BRITISH COUNCIL LINK “Tropical coastal ecosystems and
sustainable aquatic resource management”
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1997-2000 Coordinator of EU INCO-DC (International Cooperation with Developing Countries) program “Sustainable production of mud crab Scylla species through stock enhancement in mangroves”
1997-2000 Coordinator of VL.I.R. OWN INITIATIVE “R&D on Diversification of Marine Aquaculture in the Mekong Delta”
1997-1998 Aquaculture project specialist of GEC Global Environmental Consultants Ltd. "Vietnam coastal wetlands protection and development", World Bank project Technical assistant of Euro consult in the framework of the project “Rehabilitation of Mangrove Forests-Mekong Delta, FIPI No 2”
1996-2000 Coordinator of VVOB (Belgium) “Extension and Building Capacity on Aquaculture for Local Staff”
1990-2001 Coordinator of NOVIB (Netherlands Organization for International Development Cooperation) “Support for socio-economical development of the poor ethnic community in the coastal area of Vinh Chau district, Soc Trang province, Vietnam”
1989-1993 Coordinator of CANADA FUND “Initiatives for improving the poor community in Soc Trang and Bac Lieu provinces in the Mekong Delta, Viet Nam “Introduction of shrimp Penaeus monodon and P. merguiensis culture in the coastal area with support of technologies and credits”
Local involvement Since 1987 Planning projects and pilot models of aquaculture farming and hatchery of
freshwater fish, prawn, shrimp and mud crab for districts and provinces in the Mekong Delta, Vietnam
1980-1986 Artificial preproduction of freshwater catfish (Pangasius sp.) and sand goby (Oxyeleotris marmoratus)
1980-1983 Master planning for aquaculture in the Mekong Delta, Vietnam Conference/workshop/training participation Sept. 2002 - Jan. 2003 Training course in aquaculture, Laboratory of Aquaculture &
Artemia Reference Center, Ghent University, Ghent, Belgium 6-9 May 2002 Workshop on Integrated management of coastal area, Malaysia 27-30 Apr. 2002 Challenges in aquaculture, Beijing, China 23-27 Apr. 2002 International conference on fisheries and aquaculture in Beijing,
China 1 Sept. - 30 Nov. 2001 Training course in aquaculture, Laboratory of Aquaculture &
Artemia Reference Center, Ghent University, Ghent, Belgium 3-6 Sept. 2001 Larvi’01, held by European Aquaculture Society, Ghent, Belgium 23-26 Aug. 2001 Second workshop Vietnam-Hungary on Small animal production,
Godollo-Szarvas, Hungary 8-10 Jan. 2001 Workshop on mud crab rearing, ecology and fisheries, Can Tho
University, Vietnam 31 Oct. - 3 Nov. 2000 Third World Fisheries Congress, China Society of Fisheries,
Beijing, China 1-4 Dec. 1998 International Forum on the Culture of Portunid Crabs, Boracay, the
Philippines
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11-14 Nov. 1998 The Fifth Asian Fisheries Forum, held by Asian Fisheries Society, Chiangmai, Thailand.
3-7 Sept. 1995 Larvi’95, Gent University, Gent, Belgium Aug. - Oct. 1992 Training course in aquaculture at Lab of Aquaculture and Artemia
Reference Center, Gent University, Gent, Belgium 26-30 Oct. 1992 The third Asian Fisheries Forum, held by Asian Fisheries Society,
Singapore Publications/presentations/reports 1. Nghia, T.T., 1980. Culture for maturation and artificially spawning inducement of the fresh-water catfish
Pagasius micronemus (in Vietnamese). Aquaculture engineer thesis, Can Tho University, 55 pp.
2. Nghia, T.T., Kiem, N., Lai, B., 1985. Artificial reproduction of freshwater catfish Pangasius micronemus. Final report of research period 1980-1984 (in Vietnamese). Faculty of Fisheries, Can Tho University, 33 pp.
3. Nghia, T.T., 1990. Larviculture techniques of the marble sand goby Oxyeleotris marmoratus, Bleeker. Final report of research period 1985-1989 (in Vietnamese). Faculty of Fisheries, Cantho University, 125 pp.
4. Nghia, T.T., 1991. Larviculture techniques and economics of small-scaled Macrobrachium rosenbergii hatcheries in the Mekong Delta, Vietnam. In: Larvi’91. Symposium on Fish and Crustacean Larviculture, 27-30 August 1991. Lavens, P., Sorgeloos, P., Jaspers, E., Ollevier, F. (Eds.). European Aquaculture Society, Special Publication No 15.
5. Nghia, T.T., Quynh, V.D., Quang, N.K., 1992. Introduction of Penaeus merguiensis and Penaeus monodon culture in evaporation ponds of coastal salterns in southern Vietnam. The Third Asian Fisheries Forum, Asian Fisheries Society.
6. Nghia, T.T., Quang, N.K., Thanh, T., Danh, T.C., 1993. Evaluation of using Artemia biomass in the larviculture of Macrobrachium rosenbergii (in Vietnamese). The First Symposium on Artemia culture in Vietnam, Cantho, 16-18 April 1993.
7. Nghia, T.T., Ut, V.N., Quang, N.K., Rothuis, A.J., 1994. Improvement of traditional shrimp culture in the Mekong Delta. NAGA, The Iclarm Quartly, April 1994, pp. 20-22.
8. Ut, V.N., Quang, N.K., Nghia, T.T., Bosteels, T., Rothuis, A.J., 1995. Expansion of improved-extensive shrimp culture in the Mekong Delta. NAGA, The Iclarm Quartly, April 1995, pp. 22-23.
9. Nghia, T.T., 1995. Contribution to the speciation of genus Artemia with special emphasis to Asian populations (M.Sc. Thesis, Gent University, Belgium), 92 pp.
10. Nghia, T.T., Binh, T.V., 1996. Co-operation between research and industry in aquaculture plans. In: IFS (1998) Aquaculture research and sustainable development in inland and coastal regions in South-East Asia. Proceedings of an IFS/EU Workshop. Can Tho, Vietnam 18-22 March 1996, pp. 285-288.
11. Nghia, T.T., Quynh, V.D., Quang, N.K., 1997. Trials of nursing and culturing white shrimp (Penaeus merguiensis) and tiger shrimp (Penaeus monodon) in evaporation ponds of salt fields in the South Vietnam coast (in Vietnamese). In: Book of Technology Research 1993-1997, Can Tho University, pp. 44-50.
12. Nghia, T.T., Ut, V.N., Quang, N.K., 1997. Inprovement of shrimp traditional culture and expansion of improved-extensive shrimp culture model in the Mekong Delta. In: Book of Technology Research 1993-1997, Can Tho University, pp. 51-55.
13. Nghia, T.T., Binh, T.C., Phong, L.N., 1997. Some techniques to improve the seed quality of shrimp Penaeus monodon (in Vietnamese). In: Book of Technology Research 1993-1997, Can Tho University, pp. 57-64.
14. Nghia, T.T., Dat, N.M., 1997. Preliminary results of mud crab (Scylla paramamosain) seed production in the Mekong Delta (in Vietnamese). In: Book of Technology Research 1993-1997, Can Tho University, pp. 65-70.
15. Nghia, T.T., 1997. Improvement on larviculture of the mud crab (Scylla paramamosain) in the Mekong
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Delta. Final report of IFS research grant No. A/2505-1, period September 1996 - September 1997.
16. Nghia, T.T., Dat, N.M., Quynh, V.D., Lavens, P., 1998. Larviculture of the mud crab (Scylla serrata) in the Mekong Delta, Vietnam under controlled conditions. In Books of Abstract of International Conference on Fisheries and food security beyond the year 2000. The Fifth Asian Fisheries Forum, 11-14 November 1998 in Chiangmai, Thailand, p.155.
17. Nghia, T.T., Lavens, P., Wille, M., 1998. Zootechnical and nutritional aspects of mud crab Scylla spp. Larviculture. In: Books of Abstract of International Conference on Fisheries and Food Security beyond the year 2000. The Fifth Asian Fisheries Forum, 11-14 November 1998 in Chiangmai, Thailand, p. 425.
18. Nghia, T.T., Loc, N.H., Quynh. V.D., Lavens, P., Wille, M., 1998. Comparison of a batch and recirculation system for the larviculture of mud crab (Scylla paramamosain) in the Mekong Delta, Vietnam. In: Programm and extended abstracts of International Forum on the Culture of Portunid Crabs, 1-4 December 1998, Boracay, Philippines.
19. Le Vay. L., Ut, V.N., Nghia, T.T., Jones, D.A., 1999. Ecology, fisheries and culture of mud crabs in the Mekong Delta. 7th Coll. Crust. Dec. Med. Lisbon.
27. Hai, T.N., Nghia, T.T., 2004. Effects of rearing densities on development and survival of mud crab (Scylla paramamosain) larvae in green-water system (in Vietnamese). In: Scientific magazine of Can Tho University - Aquaculture section, pp. 187-192.
20. Le Vay. L., Ut, V.N., Nghia, T.T., Jones, D.A., 2000. Sustainable aquaculture and fisheries production of mud crabs (Scylla spp.). In: Responsible aquaculture in the new millennium. Abstracts of contributions presented at the International Conference AQUA 2000. Nice, France, 2-6 May 2000. European Aquaculture Society. Special Publication No 28, Oostende, Belgium, March 2000, p. 393.
21. Nghia, T.T., Wille, M., Sorgeloos, P., 2001. Effects of light, eyestalk ablation and seasonal cycle on the reproductive performance of captive mud crab (Scylla paramamosain) broodstock in the Mekong Delta, Vietnam. In: Book of Abstracts of 2001 Workshop on Mud Crab Rearing, Ecology and Fisheries. Institute for Marine Aquaculture, Can Tho University, Vietnam, 8-10 January 2001, p. 4.
22. Nghia, T.T., Wille, M., Sorgeloos, P., 2001. Overview of larval rearing techniques for mud crab (Scylla paramamosain) with special attention to the nutritional aspects in the Mekong Delta, Vietnam. In: Books of Abstracts of 2001 Workshop on Mud Crab Rearing, Ecology and Fisheries. 8-10 January 2001. Institute for Marine Aquaculture, Can Tho University, Vietnam, p. 13.
23. Nghia, T.T., Wille, M., Sorgeloos, P., 2001. Influence of the content and ratio of essential HUFA’s in the live food on larviculture success of the mud crab (Scylla paramamosain) in the Mekong Delta (Vietnam). In: Hendry, C.I., Van Stappen, G., Wille, M., Sorgeloos, P. (Eds), Larvi’01 - Fish and Shellfish Larviculture Symposium, European Aquaculture Society. Special Publication No 30, Oostende, Belgium, pp. 430-433.
24. Ut, V.N., Le Vay, L., Nghia, T.T., Hanh, T.T.H., Caldwell, B.S., 2001. Effect of substrate and diet in the nursery phase of mud crab (Sctlla paramamosain) production. In: Hendry, C.I., Van Stappen G., Wille, M., Sorgeloos, P. (Eds), Larvi’01 - Fish and Shellfish Larviculture Symposium, European Aquaculture Society. Special Publication, vol. 30, Oostende, Belgium, pp. 610-613.
25. O'Kelly, Le Vay, L., Mardjon M., Nghia, T.T., Jones, D.A., 2001. Larval development of Scylla paramamosain cultured in the laboratory. In: Hendry, C.I., Van Stappen G., Wille, M., Sorgeloos, P., (Eds.). Larvi’01 - Fish and Shellfish Larviculture Symposium European Aquaculture Society. Special Publication 30, Oostende, Belgium.
26. Thao, N.T.T., Nghia, T.T., 2003. Effects of different salinity levels on filtering rate of feed, growth, survival and stress resistance of blood cockle (Anadara granosa Linaeus, 1758) (in Vietnamese). In: Dien, N.H., Chinh, N., Thu, N.T.X., Phung, N.H., Nho, N.T. (Eds.). Books of selected scientific reports - Proceedings of the second national workshop on marine molluscs. Nha Trang, 3-4 August 2001, pp. 137-142.
ISBN 90-5989-036-1