SPAWNING AND LARVAL REARING OF THE MONKEY RIVER PRAWN Macrobrachium lar (FABRICIUS, 1798) (CRUSTACEA: DECAPODA: CARIDEA: PALAEMONIDAE) IN THE FIJI ISLANDS
by
Monal M. Lal
A thesis submitted in fulfilment of the requirements for the Degree of Master of Science in Marine Science
Copyright © 2012 by Monal M. Lal
School of Marine Studies Faculty of Science, Technology and Environment University of the South Pacific Digitally signed by Monal Lal April, 2012 Date: 2011.11.01
20:11:29 +13'00' COLLABORATING INSTITUTIONS ______Frontispiece: male M. lar specimen DECLARATION OF ORIGINALITY ______
Statement by Author
I, Monal M. Lal, declare that this thesis is my own work and that, to the best of my knowledge, it contains no material previously published, or substantially overlapping with material submitted for the award of any other Degree at any institution, except where due acknowledgement is made in the text.
______02/11/2011 Monal M. Lal Date S11020445
Statement by Supervisors
The research in this thesis was performed under my supervision and to my knowledge is the sole work of Mr. Monal M. Lal.
______02/11/2011 Mr. Johnson Seeto Date Principal Supervisor
______02/11/2011 Dr. Timothy Pickering Date External Supervisor
DEDICATION ______
This thesis is dedicated to the Almighty for His guidance and providence and to my mother for her love and support.
i ACKNOWLEDGEMENTS ______
I would like to acknowledge the Australian Centre for International Agricultural Research (ACIAR), who through their USP-ACIAR Scholarship scheme made it possible for me to study for my Master of Science Degree. My sincere thanks and gratitude go to Ms. Cathy Hair for her advice and assistance with getting the project underway.
To my principal and external supervisors Mr. Johnson Seeto and Dr. Timothy Pickering, I thank you very much for your invaluable advice, comments, criticisms, encouragement and feedback throughout the duration of the study. A big vinaka vakalevu to Tim Pickering for securing the Pacific Aquaculture Grant which funded the research and your help with the experimental designs among many other aspects of the study, and the reminders to “focus on your objectives!”. Vinaka vakalevu also to Johnson Seeto, who later told me he knew all along that we would produce PL, when I really did not think it possible.
I am immensely grateful to Mr. Tomohiro Imamura, for teaching me his Rua-Cell System of greenwater culture, without which it would have been extremely difficult to attempt to rear the larvae of M. lar; and for his technical assistance and guidance during the trial phase of the project.
I am also grateful to Mr. Maika Ciqo of the Naduruloulou Research Station who collected the majority of the prawn broodstock used for hatching larvae during the study, Dr. Simon Hodge for assistance with the statistical analyses carried out for the salinity and temperature experiments and Dr. William Camargo for use of bench and floor space in the Seawater Laboratory at USP.
I acknowledge the assistance of Mrs. Liti Muavesi, who provided me with moci at the Nausori Market when required and Mrs. Cherie Soro of Celtrock Holdings for sourcing whole squid which were required as larval feed ingredients. My gratitude
ii also extends to the Frey family; Andreas, Peter and Dawn for the many discussions over dinner and encouragement and support.
I am particularly grateful to Ms. Sarah Myhre and Ms. Kristen Anderson of the PRAISE information service at the University of Hawaii, who were able to source obscure pieces of literature for me that I otherwise would not have had access to. I am also grateful to Mr. Deepak Kissun who shared with me his literature on M. lar and his experiences with culturing the larvae.
I would also like to extend my gratitude to staff of the School of Marine Studies at USP; Mr. Jone Lima, Mr. Shiv Sharma and Mrs. Nanise Bulai for the provision of and assistance with purchasing equipment required for the project. The assistance of staff of the Institute of Marine Resources; Mrs. Shirleen Bala, Mrs. Cherie Morris, Mr. Avinash Singh and Miss. Prerna Chand is also acknowledged.
Finally, to my friends for all your support and encouragement, thank you all; Mr. Viliame Waqalevu, Mr. Kelly Brown, Adi Siteri Tikoca, Miss. Mere Naisilisili for help with image processing of the composite larval photographs, Miss. Laura Williams and Ratu Rusiate and Mrs. Ana Vadiga for the time we shared in the laboratory.
iii ABSTRACT ______
The Monkey River Prawn Macrobrachium lar (Fabricius, 1798) is a large palaemonid prawn indigenous to a number of Pacific Island Countries and Territories (PICTs). Because of its size, relatively fast growth rates and a number of other favourable characteristics, it appears to have good potential for aquaculture except for one major constraint; namely the availability of seed stock for grow-out which is severely limited by the inability to rear the larvae from hatch until metamorphosis into post-larvae (PL).
A greenwater-type technique developed for M. lar larviculture is described here, and was successful in producing PL, representing possibly the first time that this has been reported for this species. The larvae of M. lar developed through 13 zoeal stages before metamorphosing to PL1, moulting within 22 – 31 or more instars at salinities of 30 ± 1.5 ‰ and temperatures of 28 ± 0.8 l of five post-larvae were produced, after 77, 78, 85, 101 and 110 days of culture respectively. Overall percentage survival to PL1 was very low, at 0.08 %.
Larvae were observed to undergo mark-time moulting during development, especially during the later zoeal stages as metamorphosis approached, which lengthened overall culture duration. This is attributed to inappropriate culture conditions, especially in terms of larval nutrition and possibly salinity. A point of difference noted between the feed preferences of M. lar and other Macrobrachium spp. larvae was that the former showed a strong tendency to consume biofloc particles in preference to Artemia nauplii, when both were present in sufficient quantities.
The embryonic development of M. lar appears very similar to that which has been described for other species of Macrobrachium. Ovigerous females of M. lar maintained in the laboratory displayed asynchronous and prolonged hatching, which resulted in relatively low larval yields at the commencement of rearing trials.
iv Embryonic development was found to take approximately 29 days from the time of oviposition until hatch at 28 ± 0.5
Through a series of salinity and temperature tolerance experiments, it has been established that the larvae of M. lar are able to hatch in either freshwater or brackish-water of approximately 10 ‰, but require gradually increasing salinities post-hatch reaching 30 – 35 ‰, which probably needs to be maintained until metamorphosis into PL. Larval stages I – II require a range of 10 – 15 ‰, III – IV require 15 – 25 ‰ and V – VI require 25 – 30 ‰. Stages VII to XIII require 30 –
35 ‰ following which salinity may be reduced after PL1 is reached.
Larval survival and growth were found to be significantly different (Repeated Measures ANOVA and One-Way ANOVA; p <0.05) between larvae maintained within and outside these salinities. Larvae kept in freshwater (0 ‰) die within 4 days unless transferred to brackish-water. A salinity acclimation protocol has also been developed that will be useful in developing a mass culture technique for hatchery production of PL. Results of the temperature tolerance investigations revealed that a temperature range of 30 ± 0.5 Measures ANOVA and One-Way ANOVA; p <0.05).
Given these results, pilot commercial-scale larviculture of M. lar does not appear to be feasible over the short to medium term, and the capture and culture of wild juveniles will have to be relied upon at present for developing pond-based culture of this species. Over the longer term, given that it took time for M. rosenbergii larviculture to become sufficiently practical for successful aquaculture, there is room for similar improvement for M. lar which warrants further research.
Keywords:
Larviculture, Macrobrachium lar, larvae, PL, zoea, greenwater, Pacific Islands, salinity, temperature, survival, growth.
v ABBREVIATIONS AND ACRONYMS ______
ABW Average body weight ACIAR Australian Centre for International Agricultural Research BFT Biofloc Technology CL Carapace length -1 DO2 Dissolved oxygen (usually specified in mgL ) EDTA Ethylene diamine tetraacetic acid disodium salt
(C10H14N2Na2O8.2H20) Fam. Family (taxonomic hierarchy) Fij. Fijian (name of the subject in the Fijian language) FRP Fibre-reinforced Plastic GSL Great Salt Lake, Utah, United States of America ind. Individual(s) JICA Japan International Cooperation Agency min. Minute NACA Network of Aquaculture Centers in Asia-Pacific PICTs Pacific Island Countries and Territories
PL Post-larva(e); PL1 denotes the first post-larval instar, PL2 denotes the second instar and so forth PVC Polyvinyl chloride SMS School of Marine Studies TDR Total daily ration TL Total length
Treflan Antifungal agent used for treating crustacean larvae (C13H16F3N3O4) USD United States Dollar USP University of the South Pacific
vi UNITS OF MEASUREMENT ______mL millilitre mLs-1 millilitres per second (unit of aeration volume) KW kilowatt Ls-1 litres per second (unit of aeration volume) lux unit of luminous output equivalent to lumens (lm) or lm/m2 L litre μm micrometers/micron mgL-1 milligrams per litre mm millimetre cm centimetre Wwatt w/w weight by weight basis
vii CONTRIBUTION OF OTHERS TO THIS THESIS ______
Mr. Maika Ciqo (Naduruloulou Research Station, Naduruloulou, Fiji Islands) Collection of the majority of the M. lar broodstock used in the study (Chapter 2).
Dr. Simon Hodge (University of the South Pacific, Suva, Fiji Islands) Assistance with statistical analyses (Chapter 3).
Miss. Mere Naisilisili(Graphics Department, Institute of Applied Science, University of the South Pacific, Suva, Fiji Islands) Image processing for photographs of M. lar larvae (Chapter 4).
viii PUBLICATIONS ARISING FROM THIS THESIS ______
Published: Imamura, T., Seeto, J., Williams, L., Mow, A.-M., Vadiga, R., & Lal, M. (2009). Freshwater prawn and crab hatchery in Fiji with rotifer culture Rua Cell System. Suva, Fiji Islands: Japan International Cooperation Agency (JICA) and University of the South Pacific.
Lal, M., Pickering, T., & Seeto, J. (2010). Freshwater prawn research breakthrough at USP. USP Beat, 9 5.
Lal, M., Pickering, T., & Seeto, J. (2010). Freshwater prawn research breakthrough at USP. SPC Fisheries Newsletter No. 131 January/April 2010.
Lal, M., Pickering, T., & Seeto, J. (2011). Laboratory larval rearing of Macrobrachium lar in Fiji Islands. Paper presented at the Giant Prawn 2011 and Asian Pacific Aquaculture 2011 Conferences, Kochi, Kerala, India.
In prep.: Lal, M., Pickering, T., & Seeto, J. Morphological development of larvae of the Monkey River Prawn Macrobrachium lar (Fabricius, 1798) (Decapoda: Caridea: Palaemonidae) Zootaxa.
Lal, M., Pickering, T., Seeto, J. & Hodge, S. Salinity and temperature requirements for larviculture of the Monkey River Prawn Macrobrachium lar (Fabricius, 1798) (Decapoda: Caridea: Palaemonidae). Aquaculture.
ix TABLE OF CONTENTS ______
Dedication i Acknowledgements ii Abstract iv Abbreviations and acronyms vi Units of measurement vii Contribution of others to this thesis viii Publications arising from this thesis ix Table of contents X List of tables xvii List of figures xix List of plates xxiii List of inserts xxix
Chapter 1 Introduction 1
1.1 Aquaculture: its current global status and significance 1 1.1.1 Current production 1 1.2 Crustacean aquaculture 3 1.3 Freshwater prawn aquaculture globally 5 1.4 Aquaculture in Pacific Island Countries and Territories (PICTs) 9 1.5 Freshwater prawn culture in Fiji 12 1.6 An overview of the biology of Macrobrachium lar 13 1.6.1 Morphology 13 1.6.2 Nomenclature, classification and taxonomy 21 1.6.3 Distribution 22 1.6.4 Habitat 23 1.6.5 General biology 25 1.6.5.1 Life history 25
x 1.6.5.2 Reproduction 26 1.6.5.3 Feeding 29 1.6.5.4 Behaviour 30 1.6.5.5 Growth 31 1.6.5.6 Parasites and diseases 34 1.7 Reasons to investigate the aquaculture potential of M. lar 35 1.8 History of M. lar research work 38 1.9 Research objectives 40 1.10 Thesis organization 41
Chapter 2 Development of a mass culture technique for larvae of the 42 Monkey River Prawn Macrobrachium lar
2.1 Introduction 42 2.1.1 Freshwater prawn larviculture 42 2.1.2 Larval development types among Macrobrachium prawns 43 2.1.3 Development of a larval rearing technique for M. lar 45 2.1.4 Research objectives 46 2.2 Methodology 47 2.2.1 Water treatment 47 2.2.1.1 Aeration 47 2.2.1.2 Seawater treatment 47 2.2.1.2.1 Primary treatment 47 2.2.1.2.2 Secondary treatment 48 2.2.1.3 Freshwater treatment 48 2.2.2 Rua-cell system of culture 49 2.2.2.1 Marine microalgae culture 50 2.2.2.2 Freshwater microalgae culture 53 2.2.3 Broodstock collection 57 2.2.4 Brooder spawning 59 2.2.4.1 Brooder egg incubation 59
xi 2.2.4.1.1 ‘Early’ orange eggs 60 2.2.4.1.2 ‘Late’ orange/yellow eggs 60 2.2.4.1.3 Grey eggs 60 2.2.4.1.4 Spent females 60 2.2.4.2 Hatch tank preparation 63 2.2.4.3 Brooder stocking and hatching of larvae 65 2.2.5 Broodstock maintenance 67 2.2.5.1 Spent female broodstock 67 2.2.5.2 Partially-spawned broodstock 67 2.2.5.3 Broodstock holding tanks 68 2.2.5.4 Broodstock feeding 69 2.2.6 Larval rearing 71 2.2.6.1 Rearing environment management 71 2.2.6.1.1 Water exchanges 72 2.2.6.1.2 Biofloc management 76 2.2.6.2 Feeds and feeding 78 2.2.6.2.1 Prepared feeds 78 2.2.6.2.1.1 Custard feeds 78 2.2.6.2.1.2 Algamac 3050 flake 81 2.2.6.2.2 Artemia nauplii and meta-nauplii 81 2.2.6.2.3 Feeding rations and schedules 84 2.2.6.2.4 Biofloc and other live feeds 86 2.2.6.2.5 LRT water management 87 2.2.6.3 Larval health management 88 2.2.6.4 Larval observations 89 2.3 Results 92 2.3.1 Broodstock 92 2.3.1.1 Broodstock maintenance 92 2.3.1.2 Larval hatch 93 2.3.1.3 Disease 94 2.3.2 Larval rearing 95
xii 2.3.2.1 Larval observations 95 2.3.2.1.1 Growth 95 2.3.2.1.2 Behaviour and feeding 97 2.3.2.1.3 Disease 100 2.3.2.1.4 Aberrant larvae/deformities 102 2.3.3.2 Larval rearing trials 103 2.3.3.3 Larval rearing salinity and temperature 107 2.4 Discussion 111 2.4.1 Broodstock maintenance 111 2.4.1.1 Stocking density for holding broodstock 111 2.4.1.2 Larval hatch 111 2.4.1.3 Disease 114 2.4.2 Larval rearing 115 2.4.2.1 Larval observations 115 2.4.2.2 Larval rearing trials 116 2.4.2.2.1 Survival 116 2.4.2.2.2 Growth 117 2.4.2.2.3 Feeds and feeding 118 2.4.2.2.4 Salinity 120 2.4.2.2.5 Culture system 121 2.5 Conclusion 122
Chapter 3 Determination of salinity and temperature optima for 124 larval rearing of Macrobrachium lar
3.1 Introduction 124 3.1.1 Previous investigations of the salinity and temperature 125 requirements of Macrobrachium species. 3.1.2 Previous work on investigating culture conditions for the larvae 130 of M. lar 3.1.3 Research objectives 132
xiii 3.2 Methodology 133 3.2.1 Methodology development 133 3.2.1.1 Salinity tolerance study 133 3.2.1.2 Temperature tolerance study 135 3.2.2 Larval mass cultures 135 3.2.3 Salinity tolerance experiments on M. lar larvae 136 3.2.3.1 Collection and acclimation of larvae 136 3.2.3.2 Acclimation set up 136 3.2.3.3 Coarse-resolution tolerance tests 142 3.2.3.4 Fine-resolution tolerance tests 145 3.2.3.5 Experiment maintenance 145 3.2.3.6 Larval examination and data collection 146 3.2.3.7 Data analyses 146 3.2.4 Temperature tolerance tests 147 3.2.4.1 Collection and acclimation of larvae 147 3.2.4.2 Temperature tolerance tests 148 3.2.4.3 Larval examination and data collection 150 3.2.4.3.1 Larval survival 150 3.2.4.3.2 Larval growth 150 3.2.4.4 Data analyses 151 3.3 Results 152 3.3.1 Salinity tolerance tests 152 3.3.1.1 Larval survival during coarse-resolution tolerance tests 152 3.3.1.2 Larval growth during coarse-resolution tolerance tests 155 3.3.1.3 Larval survival during fine-resolution tolerance tests 158 3.3.1.4 Larval growth during fine-resolution tolerance tests 162 3.3.1.5 Salinity acclimation protocol for larvae 165 3.3.2 Temperature tolerance tests 166 3.3.2.1 Larval survival at treatment temperatures 166 3.3.2.2 Larval growth at treatment temperatures 168 3.4 Discussion 170
xiv 3.4.1 Salinity tolerance experiments 170 3.4.2 Temperature tolerance tests 172 3.4.3 Further work 173 3.5 Conclusion 173
Chapter 4 Larval development stages of the Monkey River Prawn 175 Macrobrachium lar
4.1 Introduction 175 4.1.1 Larval development in Macrobrachium prawns 175 4.1.1.1 Patterns of larval development 175 4.1.1.2 Growth processes 179 4.1.2 Important terms and concepts used in describing the 184 morphological development of Macrobrachium larvae 4.1.2.1 Morphological terms 184 4.1.2.2 Zoea and prezoea larvae 185 4.1.2.3 Post-larva 186 4.1.2.4 Variability in morphological development 187 4.1.2.5 Larval dispersal, settlement and colonisation strategies 188 4.1.3 Description of larval development of Macrobrachium lar 190 4.1.4 Research objectives 190 4.2 Methodology 191 4.2.1 Embryonic development study 191 4.2.2 Larval development study 194 4.2.2.1 Rearing of larvae 194 4.2.2.2 Larval microscopy 194 4.2.2.3 Determining larval developmental stages 195 4.2.2.4 Specimen drawings 199 4.2.2.5 Specimen photograph processing 199 4.2.3 Larval development staging guide 200 4.3 Results 201
xv 4.3.1 Embryonic development of M. lar larvae 201 4.3.2 Observations on the larval development of M. lar 253 4.3.3 Descriptions of larval development stages 255 4.3.4 Guide to identifying the larvae of M. lar 273 4.4 Discussion 276 4.4.1 Embryonic development of M. lar larvae 276 4.4.2 Larval development of M. lar 276 4.4.3 Larval development characteristics 277 4.5 Conclusion 279
Chapter 5 General conclusion and recommendations 281
5.1 Review of objectives 281 5.2 Outlook for pilot-scale hatchery production 283 5.3 Study constraints and limitations 284 5.4 Recommendations for future research 284
References 287
Appendices 306
Appendix 1.1 Glossary of anatomical terms for caridean prawns 306 Appendix 2.1 Daily record and LRT population estimation sheets 310 Appendix 2.2 Composition data for Multivitamin capsule 311 Appendix 2.3 Algamac 3050TM composition and nutritional data 312
xvi LIST OF TABLES ______
Chapter 1
Table 1.1 List of Macrobrachium species that are farmed or believed to 6 have aquaculture potential. Table 1.2 Aquaculture production by region: quantity and percentage of 9 world production. Table 1.3 Aquaculture production in the Pacific Islands region by country 10 in 2007. Table 1.4 Aquaculture production in the Pacific Islands region by 10 commodity in 2007.
Chapter 2
Table 2.1 Prepared custard feed ingredients. 79 Table 2.2 Feeding sieve mesh sizes used for particular larval stages 80 Table 2.3 Criteria used for determining condition index in evaluating M. 91 rosenbergii larval quality. Table 2.4 20 ‰ regime trials. 104 Table 2.5 25 ‰ regime trials. 104 Table 2.6 30 ‰ regime trials. 104
Chapter 3
Table 3.1 Larval salinity and temperature optima investigations of other 127 species of palaemonid prawn. Table 3.2 Summary of One-Way ANOVA results on the effect of larval 160 survival at fine-resolution test salinities. Table 3.3 Salinity acclimation protocol for larviculture of M. lar. 165 Table 3.4 Results of Repeated Measures ANOVA for the effect of 167
xvii temperature on survival of M. lar larvae. Table 3.5 Summary of One-Way ANOVA results on the effect of larval 167 survival against treatment temperatures. Table 3.6 Results of Repeated Measures ANOVA for the effect of 169 temperature on growth of M. lar larvae. Table 3.7 Summary of One-Way ANOVA results on the effect of larval 169 growth against treatment temperatures.
Chapter 4
Table 4.1 Developmental characteristics and rearing conditions of 177 Macrobrachium spp. Table 4.2 Egg development of M. lar at 28 ± 0.5 203 Table 4.3 Age of first appearance and size ranges of M. lar larvae. 254 Table 4.4 Summary of readily discernable features characterising the 270
larvae and PL1 of M. lar.
xviii LIST OF FIGURES ______
Chapter 1
Figure 1.1 Volume of world capture fisheries and aquaculture production. 1 Figure 1.2 a Catch trends by valuable marine species groups. 2 Figure 1.2 b Catch trends by major inland species groups. 2 Figure 1.3 World aquaculture production by major species groups in 2008. 3 Figure 1.4 Trends in world aquaculture production: average annual growth 4 rate for major species groups during 1970–2008. Figure 1.5 External features of a caridean prawn. 13 Figure 1.6 Diagnostic carapace features for prawns of the infra-order 14 Caridea. Figure 1.7 Diagnostic characters for prawns of the infra-order Caridea. 15 Figure 1.8 Anterior portion of the carapace of M. lar showing the 16 rostrum, antennal and hepatic spines. Figure 1.9 Dactylus and distal propodus of third pereiopod of M. lar, 17 showing the notch situated close to the apex of the dactylus. Figure 1.10 Current taxonomic hierarchy for Macrobrachium lar.21 Figure 1.11 Distribution map of M. lar.22 Figure 1.12 Generalised growth curve of M. lar along with those of other 33 species of Macrobrachium.
Chapter 2
Figure 2.1 Broodstock collection sites 1 and 2 at Waisere Creek. Inset: Fiji 57 Islands with the location of Waisere Creek on Viti Levu. Figure 2.2 Daily feed regime. 84 Figure 2.3 Larval feeding schedule. 85 Figure 2.4 Visual criteria used for determining condition index in 90 evaluating M. rosenbergii larval quality.
xix Figure 2.5 Graph of 30 ‰ trial number 20 showing the day of first 105 appearance of larval developmental stage. Figure 2.6 Graph of 30 ‰ trial number 20 showing larval stage intermoult 106 durations. Figure 2.7 Graph of larval survivorship during 30 ‰ regime trial number 107 20. Figure 2.8 a Graph of 30 ‰ trial number 20 salinity and temperature trends 109 during days 1 to 65. Figure 2.8 b Graph of 30 ‰ trial number 20 salinity and temperature trends 110 during days 66 to 96.
Chapter 3
Figure 3.1 Survivorship performance of zoea I M. lar larvae during 153 coarse-resolution salinity testing. Figure 3.2 Survivorship performance of zoea III M. lar larvae during 153 coarse-resolution salinity testing. Figure 3.3 Survivorship performance of zoea V M. lar larvae during 154 coarse-resolution salinity testing. Figure 3.4 Survivorship performance of zoea VII M. lar larvae during 154 coarse-resolution salinity testing. Figure 3.5 Effect of coarse resolution test salinities on survival of M. lar 155 larvae. Figure 3.6 Growth performance of zoea I M. lar larvae during coarse- 156 resolution salinity testing. Figure 3.7 Growth performance of zoea III M. lar larvae during coarse- 156 resolution salinity testing. Figure 3.8 Growth performance of zoea V M. lar larvae during coarse- 157 resolution salinity testing. Figure 3.9 Growth performance of zoea VII M. lar larvae during coarse- 157 resolution salinity testing.
xx Figure 3.10 Survivorship performance of zoea I M. lar larvae during fine- 158 resolution salinity testing. Figure 3.11 Survivorship performance of zoea III M. lar larvae during fine- 159 resolution salinity testing. Figure 3.12 Survivorship performance of zoea V M. lar larvae during fine- 159 resolution salinity testing. Figure 3.13 Survivorship performance of zoea VII M. lar larvae during 160 fine-resolution salinity testing. Figure 3.14 Effect of fine-resolution test salinities on survival of M. lar 161 larvae. Figure 3.15 Growth performance of zoea I M. lar larvae during fine- 163 resolution salinity testing. Figure 3.16 Growth performance of zoea III M. lar larvae during fine- 163 resolution salinity testing. Figure 3.17 Growth performance of zoea V M. lar larvae during fine- 164 resolution salinity testing. Figure 3.18 Growth performance of zoea VII M. lar larvae during fine- 164 resolution salinity testing. Figure 3.19 Effect of fine resolution test salinities on growth of M. lar 165 larvae. Figure 3.20 Effect of treatment temperatures on larval survival. 166 Figure 3.21 Effect of treatment temperatures on larval growth. 168
Chapter 4
Figure 4.1 The generalised structure of the crustacean cuticle. 180 Figure 4.2 Critical points during the moult cycle of a decapod crustacean 183 larva, showing the Reserve Saturation Point (RSP) and Point of No Return (PNR). Figure 4.3 Conceptual model of the export strategy of larval dispersal as 189 given by the example of a generalised grapsid crab.
xxi Figure 4.4 Embryonic development of Macrobrachium olfersii. 193 Figure 4.5 Larval body measurements. 196 Figure 4.6 External characteristics of an M. lar larva. 197 Figure 4.7 Rostrum and carapace armature on an M. lar larva. 198 Figure 4.8 Pleopod morphology details. 198 Figure 4.9 Graph of 30 ‰ trial number 20 showing larval stage intermoult 254 durations. Figure 4.10 Lateral and dorsal views of M. lar zoea I. 255 Figure 4.11 Lateral view of M. lar zoea II. 257 Figure 4.12 Lateral view of M. lar zoea III. 258 Figure 4.13 Lateral view of M. lar zoea IV. 259 Figure 4.14 Lateral view of M. lar zoea V. 260 Figure 4.15 Lateral view of M. lar zoea VI. 261 Figure 4.16 Lateral view of M. lar zoea VII 262 Figure 4.17 Lateral view of M. lar zoea VIII. 263 Figure 4.18 Lateral view of M. lar zoea IX. 264 Figure 4.19 Lateral view of M. lar zoea X. 265 Figure 4.20 Lateral view of M. lar zoea XI 266 Figure 4.21 Lateral view of M. lar zoea XII. 267 Figure 4.22 Lateral view of M. lar zoea XIII. 268
Figure 4.23 Lateral view of M. lar PL1. 269
xxii LIST OF PLATES ______
Chapter 1
Plate 1.1 Adult male M. lar.19 Plate 1.2 Adult female M. lar.19
Chapter 2
Plate 2.1 1200 L FRP diatom culture tank. 51 Plate 2.2 a Diatom biofloc seen at low power. 53 Plate 2.2 b Closer view of organisms seen in the diatom biofloc 53 Plate 2.3 1200 L FRP freshwater microalgae culture tank. 54 Plate 2.4 Freshwater microalgal floc harvested from the culture tank. 56 Plate 2.5 a Freshwater microalgal floc seen at low power. 56 Plate 2.5 b Desmodesmus sp. alga. 56 Plate 2.6 Measuring TL of male brooder prawn. 61 Plate 2.7 Weighing brooder prawn. 61 Plate 2.8 Acclimation and release of brooders into the hatch tank. 61 Plate 2.9 Broodstock maintained in 1000 L holding tank. 61 Plate 2.10 a ‘Early’ orange-berried female. 62 Plate 2.10 b ‘Late’ orange-berried female. 62 Plate 2.10 c Grey-berried female. 62 Plate 2.10 d Spent female. 62 Plate 2.11 1000 L polyethylene holding tank for ovigerous female 63 broodstock. Plate 2.12 Mixing freshwater and seawater for hatch. 64 Plate 2.13 Tank prepared to receive berried broodstock. 64 Plate 2.14 Brooder carrying eggs that are ready to hatch. 66 Plate 2.15 Closer view of grey egg mass. 66 Plate 2.16 Hatch tank covered with a spawn in progress. 66
xxiii Plate 2.17 LRT after a spawn. 66 Plate 2.18 Partially-spawned female. Note a few remaining eggs near the 68 retracted pleopods. Plate 2.19 LRT filled to operating capacity with healthy microalgal 73 bloom. Plate 2.20 Green water being filtered through a 50 μm2 mesh net into the 74 water preparation tank. Plate 2.21 LRT being drained during a water exchange. 76 Plate 2.22 Egg custard feed being screened through a 150 μm2 feeding 80 mesh. Plate 2.22 Colurella sp. rotifers. 86 Plate 2.23 Removal of Artemia cysts from an LRT. 88 Plate 2.24 Areas of melanisation on the shell of an M. lar specimen. 94 Plate 2.25 Recently moulted larva disentangling itself from its exuvium. 96 Plate 2.26 Zoea VIII larva reaching out to capture a biofloc particle. Note 98 the faecal trail underneath the telson and uropods. Plate 2.27 The same larva in Plate 2.26 above shown manipulating the 98 biofloc particle towards the mandibles. Plate 2.28 Zoea X larva with full foregut, midgut and hindgut regions. 100 Plate 2.29 Larval disease indications on a zoea X larva. 101 Plate 2.30 a Deformed zoea I larva. Note the single eye and bent telson. 102 Plate 2.30 b Normal zoea I larva. 103
Chapter 3
Plate 3.1 50 L jerry cans used for storage of the mixed test salinity 137 solutions. Plate 3.2 Drip bag and burette units. 139 Plate 3.3 Salinity tolerance test acclimation tanks. 140 Plate 3.4 Salinity tolerance test acclimation tanks showing drain valves. 140 Plate 3.5 Salinity tolerance test acclimation tanks showing the placement 141
xxiv of the drain valves, retaining screens, air diffusers and drip bag delivery lines. Plate 3.6 1 L tolerance test jar. 143 Plate 3.7 Salinity tolerance test water bath system set up. 144 Plate 3.8 Salinity tolerance test water bath system in operation. 144 Plate 3.9 Temperature experiment LRTs. 148 Plate 3.10 LRT equipped with Aqua One 200 W immersion heater. 149
Chapter 4 Colour Plates Section 204
Plate 4.1 a, b Eggs of M. lar after approximately 14 days of development at 204 28 Plate 4.2 a, b Eggs of M. lar after approximately 18 days of development at 204 28 Plate 4.3 a, b Eggs of M. lar after approximately 21 days of development at 204 28 Plate 4.4 a, b Eggs of M. lar after approximately 23 days of development at 205 28 Plate 4.5 a, b Eggs of M. lar after approximately 25 days of development at 205 28 Plate 4.6 a, b Eggs of M. lar after approximately 27 days of development at 205 28 Plate 4.7 a, b Eggs of M. lar hatching after 29 days of development at 28 206 Plate a shows the larva stretching the outer membrane of the egg during hatch and b shows the newly hatched larva. Plate 4.8 M. lar first zoea larva. 207 Plate 4.9 Rostrum of the first zoea larva. 208 Plate 4.10 First zoea hepatopancreas showing lipid globules. 208 Plate 4.11 First zoea non-articulating sixth abdominal somite join with 209 telson. Plate 4.12 First zoea telson. 209
xxv Plate 4.13 M. lar second zoea larva lateral view. 210 Plate 4.14 M. lar second zoea dorsal view. 210 Plate 4.15 Rudimentary uropod development visible. 211 Plate 4.16 Partial articulation between the telson and sixth abdominal 211 somite. Plate 4.17 Supra-orbital spine is now present. 212 Plate 4.18 Pterygostomian spine is now present. 212 Plate 4.19 Eyes are now stalked. 213 Plate 4.20 M. lar third zoea larva dorsal view. 214 Plate 4.21 M. lar third zoea larva lateral view. 214 Plate 4.22 Uropods now emergent and rudimentary endopods are seen 215 developing inside the telson. Plate 4.23 Three segments present in the antennal flagellum. 215 Plate 4.24 a First tooth appears on the dorsal carina of the rostrum. 216 Plate 4.24 b Closer view of the first rostral tooth. 216 Plate 4.25 M. lar fourth zoea larva dorsal view. 217 Plate 4.26 M. lar fourth zoea larva lateral view. 217 Plate 4.27 Second tooth appears on the dorsal carina of the rostrum. 218 Plate 4.28 Buds of the fifth pereiopods are apparent. 218 Plate 4.29 Chromatophores on appendages of the fourth zoea larva. 219 Plate 4.30 Uropod endopods are now emergent. 219 Plate 4.31 M. lar fifth zoea larva dorsal view. 220 Plate 4.32 M. lar fifth zoea larva lateral view. 220 Plate 4.33 Four segments present in the antennal flagellum. 221 Plate 4.34 a The fifth pereiopod (walking leg) has now developed 221 Plate 4.34 b Enlarged view of the fifth pereiopod. 222 Plate 4.35 The telson is now almost completely rectangular. 222 Plate 4.36 M. lar sixth zoea larva lateral view. 223 Plate 4.37 One tooth still present along the dorsal carina of the 224 rostrum, however 2 setae have emerged in front of the first tooth.
xxvi Plate 4.38 Pleopod buds emergent. Buds for the third, fourth and fifth 224 pairs are seen here. Plate 4.39 M. lar seventh zoea larva lateral view. 225 Plate 4.40 a Pleopod buds have elongated. A bud for the second pair of 226 pleopods is now emergent for this larva. Plate 4.40 b Closer view of pleopods. Pleopods which have emerged 226 earlier (usually the third and fourth pairs) now start becoming biramous. Plate 4.41 Protrusion develops in front of the first dorsal rostral tooth 227 where the next tooth will emerge. Plate 4.42 Eight segments present in the antennal flagellum. The usual 227 range for this stage is six to eight segments. Plate 4.43 M. lar eighth zoea larva lateral view. 228 Plate 4.44 Second tooth appears on the dorsal carina of the rostrum. 229 Total tooth count is three for this stage. Plate 4.45 The antennular flagellum starts to develop and now has two 229 segments. Plate 4.46 Eight segments present in the antennal flagellum. 230 Plate 4.47 Pleopod pairs three to five are now biramous and 230 developing setae. Plate 4.48 M. lar ninth zoea larva lateral view. 231 Plate 4.49 Third tooth appears on the dorsal carina of the rostrum. 232 Total tooth count is four for this stage. Plate 4.50 Three segments present in the antennular flagellum. 232 Plate 4.51 Nine segments present in the antennal flagellum. 233 Plate 4.52 All pleopods are now biramous and possess setae. 233 Plate 4.53 Buds of the appendices internae seen developing on the third 234 and fourth pairs of pleopods. Plate 4.54 M. lar tenth zoea larva lateral view. 235 Plate 4.55 Fourth tooth appears on the dorsal carina of the rostrum. 236 Total tooth count is five for this stage.
xxvii Plate 4.56 Four segments present in the antennular flagellum 236 Plate 4.57 Ten segments present in the antennal flagellum. 237 Plate 4.58 Chelae appear at the ends of the second pair of pereiopods. 237 Plate 4.59 M. lar eleventh zoea larva lateral view. 238 Plate 4.60 Fifth tooth appears on the dorsal carina of the rostrum. 239 Plate 4.61 Sixteen segments present in the antennal flagellum. 239 Plate 4.62 Chelae on second pair of pereiopods now more prominent. 240 Plate 4.63 Appendices internae now fully formed. 240 Plate 4.64 M. lar twelfth zoea larva lateral view. 241 Plate 4.65 Seventh tooth appears on the dorsal carina of the rostrum. 242 Plate 4.66 Nine segments present in the antennular flagellum. 242 Plate 4.67 Nineteen segments present in the antennal flagellum. 243 Plate 4.68 Chelae on second pair of pereiopods more developed than 243 previously and possess setae. Plate 4.69 Eight setae present on rear margin of fifth pair of pleopods. 244 Plate 4.70 M. lar thirteenth zoea larva lateral view. 245 Plate 4.71 Eighth tooth appears on the dorsal carina of the rostrum. 246 Plate 4.72 Thirteen segments present in the antennular flagellum. 246 Plate 4.73 Twenty-nine segments present in the antennal flagellum. 247 Plate 4.74 Chelae now very prominent and used by the larva in feeding. 247 Plate 4.75 Eleven setae present on rear margin of fifth pair of pleopods 248 Plate 4.76 M. lar first Post-Larva. 249 Plate 4.77 Dorsal carina rostral tooth count remains unchanged at eight. 250 Plate 4.78 First tooth appears on the ventral carina of the rostrum 250 Plate 4.79 Telson now triangular. 251 Plate 4.80 Second chelipeds significantly larger than other pereiopods. 251 Plate 4.81 Natatory exopods now highly reduced in comparison to the 252 endopods.
xxviii LIST OF INSERTS ______
Guide to identification of the larval stages of Macrobrachium lar 274 (Fabricius, 1798)
xxix Monal Lal MSc THESIS: CHAPTER ONE ______
CHAPTER ONE
INTRODUCTION
1.1 Aquaculture: its current global status and significance
1.1.1 Current production
Globally, fish provides over 1.5 billion people with almost 20 %, and another 3 billion people with at least 15 % of their average per capita intake of animal protein. In 2008, capture fisheries and aquaculture combined provided the world with approximately 142 million tonnes of fish (Figure 1.1), of which 115 million tonnes was used as human food (FAO, 2010).
Figure 1.1 Volume of world capture fisheries and aquaculture production. X axis indicates years. Source: (FAO, 2010).
Aquaculture plays an important role in the global supply of food, providing approximately 46 % of total world food fish supply in 2008, which was an increase from 43 % in 2006 (FAO, 2010).
With the recent rapid increases in global population demand for aquatic food products 1 Monal Lal MSc THESIS: CHAPTER ONE ______and the levelling off of yields from capture fisheries, it appears that sustaining fish supplies from capture fisheries alone will not be able to meet the growing demand for aquatic food (FAO, 2009).
Aquaculture is a fast-expanding and diversifying food producing sector, and it continues to grow at an average annual rate of 8.3 % worldwide. This is in stark contrast to world capture fisheries production which has almost stopped growing since the mid-1980’s as shown in Figures 1.2 a and b below (x axis indicates years). The total value of the world aquaculture harvest in 2008 was estimated at $98.4 billion US dollars (FAO, 2010).
Figure 1.2 a Catch trends by valuable marine species groups. Source: (FAO, 2010).
Figure 1.2 b Catch trends by major inland species groups. Source: (FAO, 2010). 2 Monal Lal MSc THESIS: CHAPTER ONE ______
Aquaculture has also been demonstrated to be a more efficient and less wasteful protein production sector than pigs, poultry or beef (Hall et al., 2011).
1.2 Crustacean aquaculture
Crustaceans comprised a relatively small volume (9.5 %) of the total world aquaculture production in 2008, ranked third behind freshwater fish and molluscs respectively at 5 million tonnes. In terms of value however, they ranked second at 23.1 % of total world production at $22.7 billion USD as shown in Figure 1.3.
Figure 1.3 World aquaculture production by major species groups in 2008. Source: (FAO, 2010). 3 Monal Lal MSc THESIS: CHAPTER ONE ______
Historically, aquaculture production of crustaceans has continued to increase during the period from 2000 to 2008, having an annual growth rate of almost 15 % (Figure 1.4) (FAO, 2010).
Figure 1.4 Trends in world aquaculture production: average annual growth rate for major species groups during 1970 – 2008. Source: (FAO, 2010)
Despite the fact that cultured crustaceans still account for less than 50 % of the total global production volume for this species group (FAO, 2010, 2009), the bulk of which is supplied by capture fisheries; of the portion that is produced by aquaculture, 73.3 % comprise of shrimps and prawns (FAO, 2010).
Freshwater aquaculture in particular constitutes a significant proportion of total world aquaculture production, with a contribution of 59.9 % by quantity and 56 % by value. Crustacean aquaculture in freshwater amounted to approximately 1.9 million tonnes (38.2 % of total production) in 2008, behind the yield of 2.4 million tonnes (47.7 %) grown in brackish water (FAO, 2010). For the high-island countries of the south-west Pacific, freshwater aquaculture (tilapia, carp, prawns and milkfish) is one of the
4 Monal Lal MSc THESIS: CHAPTER ONE ______production sectors expected to benefit strongly from projected increases in temperature and rainfall under climate change scenarios to the year 2100 (Pickering et al., in press).
1.3 Freshwater prawn aquaculture globally
The dominant commodity in freshwater crustacean aquaculture is the Giant River Prawn Macrobrachium rosenbergii. According to the figure reported for 2008 by the FAO (FAO, 2010), China (which is the main producer for this species) harvested 128,000 tonnes, accounting for 61.5 % of the total global production for the species. The total production figure for the previous year (2007) amounted to 221,174 tonnes (New, 2010; FAO, 2009).
The farming of a number of other freshwater prawn species apart from M. rosenbergii has expanded in the last two decades. These include the Oriental River Prawn Macrobrachium nipponense in China, the Monsoon River Prawn M. malcolmsonii in India and the Amazon River Prawn M. amazonicum, with the total annual quantities of the former two species harvested in 2007 being 192,397 and 4,100 tonnes respectively (New, 2010).
Worldwide, there are approximately 200 species of freshwater prawns of the genus Macrobrachium which are circumtropical in distribution and native to all continents except Europe (Kutty & Valenti, 2010; Nandlal, 2010).
Of these, there are some 29 species known from literature that are either under various stages of domestication for aquaculture or are already cultured commercially. These are listed overleaf in Table 1.1.
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Table 1.1 List of Macrobrachium species that are farmed or believed to have aquaculture potential. Source: modified from Kutty & Valenti (2010). Species Indigenous locationa Adult Culture habitatb statusc M. acanthurus Atlantic Americas: from North FW/BW E (Wiegmann, 1836) Carolina to Caribbean Islands and South Brazil M. amazonicum South America: Brazil, FW/BW A/C (Heller, 1862) Colombia, Venezuela M. americanum Pacific Americas: from Baja FW E Bate, 1868 California (Mexico) to North Peru, including Cocos and Galápagos Islands M. australiense Indo-West Pacific FW/BW E Holthuis, 1950 M. birmanicum Indo-West Pacific: East India, FW/BW E (Schenkel, 1902)* Bangladesh, lower Ganges Basin and lower Irrawaddy basin, Burma M. carcinus Atlantic Americas: from Florida FW/BW E (Linnaeus, 1758) to the Caribbean Islands and South Brazil M. choprai Indo-West Pacific: Ganges and FW E (Tiwari, 1949) Brahmaputra river systems M. dayanum Indo-West Pacific: Pakistan, FW E (Henderson, 1893) India, Bangladesh M. dux Eastern Atlantic: West Africa FW E (Lenz, 1910) from Rio Muni to Zaire M. equidens Indo-West Pacific: Madagascar FW/BW E (Dana, 1852) to South China, New Caledonia M. felicinum West Africa: Nigeria FW E Holthuis, 1949 M. gangeticum South Asia, India FW E Bate, 1868 M. idae Indo-West Pacific: Madagascar FW/BW E (Heller, 1862) to the Philippines, Indonesia M. idella Indo-West Pacific: East Africa, FW/BW E (Hilgendorf, 1898) Madagascar, India M. jelskii Atlantic America: from Costa FW/BW E (Miers, 1877) Rica and Trinidad to Venezuela and Brazil M. kistnense Indo-West Pacific: India FW E (Tiwari, 1952) M. lanchesteri Indo-West Pacific: Thailand, FW/BW E (De Man, 1911) Malaysia, India M. lamarrei Indo-West Pacific: India, FW/BW E (H. Milne-Edwards, 1837) Bangladesh 6 Monal Lal MSc THESIS: CHAPTER ONE ______
M. lar Indo-West Pacific: Vanuatu, FW/BW E/A (J. C. Fabricius, 1798) Wallis and Futuna M. macrobrachion Eastern Atlantic: Senegal to FW/BW E (Herklots, 1851) North Angola M. malcolmsonii Indo-West Pacific: India, FW/BW A/C (H. Milne-Edwards, 1844) Pakistan, Bangladesh M. mirabile Indo-West Pacific: East India, FW/BW E (Kemp, 1917) Bangladesh, Myanmar, Thailand, Malaysia, Indonesia M. nipponense Northern and South-East Asia: (De Haan, 1849) China, Japan, Korea, Vietnam, FW C Myanmar and Taiwan M. nobilii Indo-West Pacific: India FW E (Henderson and Matthai, 1910) M. olfersii Western Atlantic: Florida and FW E (Wiegmann, 1836) Texas, Mexico to South Brazil. Absent from the West Indies M. rosenbergii Indo-West Pacific: East India, FW C (De Man, 1879) Bangladesh, Burma, Thailand, Malaysia, Indonesia M. rude Indo-West Pacific: East Africa, FW/BW E (Heller, 1862) Madagascar, India, Bangladesh M. villosimanus Indo-West Pacific: Northeast FW E (Tiwari, 1949) India, Bangladesh M. vollenhovenii Eastern Atlantic: Cape Verde FW/BW/ E (Herklots, 1857) Islands and Senegal to South SW Angola a According to Holthuis (1980), except for M. felicinum, M. gangeticum, M. kistnense and M. nobilii. b FW = freshwater, BW = brackishwater, SW = saltwater. c Highest status of culture achieved to date. C = commercial culture established; A = artisanal culture existing; E = experimental or research and development (current or past). * Invalid taxon. Now known as Macrobrachium malcolmsonii malcolmsonii (H. Milne-Edwards, 1844).
7 Monal Lal MSc THESIS: CHAPTER ONE ______
Out of these species, with the exception of M. rosenbergii, M. nipponense and M. amazonicum, the species which show the most promise for reaching commercial status are M. malcolmsonii in the Indian sub-continent, M. vollenhovenii in West Africa and M. carcinus in Central America (Kutty & Valenti, 2010).
According to New (2010) and FAO (2009), annual global production of all species of freshwater prawns is in the neighbourhood of 460,000 tonnes, with a total value of $1.86 billion USD. Based on these figures, it is apparent that freshwater prawn farming has become a major and expanding contributor to global aquaculture both in terms of volume and value.
8 Monal Lal MSc THESIS: CHAPTER ONE ______
1.4 Aquaculture in Pacific Island Countries and Territories (PICTs)
Aquaculture production in Pacific Island Countries and Territories (PICTs) is considerably smaller in comparison to regions in other parts of the world. Table 1.2 lists production in the Oceania region (which includes Australia and New Zealand) as accounting for around 0.3 % of the total global production in 2008.
Table 1.2 Aquaculture production by region: quantity and percentage of world production. Source: FAO (2010).
9 Monal Lal MSc THESIS: CHAPTER ONE ______
PICTs which have active aquaculture industries are listed in Table 1.3, along with the volumes produced and the values of production.
Table 1.3 Aquaculture production in the Pacific Islands region by country in 2007. Source: Ponia (2010). Country Value Volume (USD thousands) (Tonne) American Samoa 10 – Cook Islands 2,473 186 Federated States of –– Micronesia (FSM) Fiji Islands 2,244 323 French Polynesia 173,598 2,464 Guam 1,391 162 Kiribati 17 – Marshall Islands 128 – Nauru 15 8 New Caledonia 28,835 1,843 Northern Mariana Islands 205 14 Palau 24 2 Papua New Guinea (PNG) 1,725 191 Samoa 33 10 Solomon Islands 74 108 Tonga 180 – Vanuatu 495 31 Total 211,646 5,342
The dominant aquaculture commodities produced in PICTs are listed in Table 1.4 below.
Table 1.4 Aquaculture production in the Pacific Islands region by commodity in 2007. Source: Ponia (2010). Commodity Value Portion of (USD million) aquaculture sector (%) Pearls 176 82.9 Crustaceans 31 14.6 Seaweed 1 0.5 Finfish 2 0.9 Edible molluscs 0.6 0.3 Ornamental 0.5 0.2 Other 1.3 0.6 Total 212.4 100
10 Monal Lal MSc THESIS: CHAPTER ONE ______
Crustaceans are ranked as the second most valuable commodity after pearls, and the category consists of production from the following species; Blue Shrimp Litopenaeus stylirostris, Giant Tiger Shrimp Penaeus monodon, White Shrimp Litopenaeus vannamei, Giant Freshwater Prawn Macrobrachium rosenbergii, Monkey River Prawn Macrobrachium lar and Red Crawfish Cherax quadricarinatus (Ponia, 2010). The Monkey River Prawn is cultivated on a very small scale in the Cook Islands, Vanuatu and French Polynesia based on juvenile capture (Pickering, unpubl).
The small scale of PICT aquaculture production is due to a number of reasons; including the small sizes of many PICTs which limits the amount of area available for land-based aquaculture, limited domestic markets, transport problems and a lack of infrastructure (Pickering & Forbes, 2002; Adams et al., 2001).
Despite this, a number of positive attributes of PICTs are favourable towards development of an aquaculture industry. These include the presence of high-value species for culture, proximity to major seafood markets in Asia, a relatively inexpensive labour force and a tradition of working with marine resources (Adams et al., 2001).
Aquaculture is also a relatively new development in the Pacific region, with the earliest attempts at modern aquaculture dating back to less than 40 years ago. It has however been traditionally practised in a few countries (Nandlal, 2010; Pickering & Forbes, 2002; Adams et al., 2001). Progress to develop the industry has been slow (Pickering & Forbes, 2002), but despite this significant industries now exist in the Fiji Islands, Cook Islands, New Caledonia and French Polynesia (Adams et al., 2001).
11 Monal Lal MSc THESIS: CHAPTER ONE ______
1.5 Freshwater prawn culture in Fiji
A number of “inland water” prawn species have been documented from Fiji, belonging to the families Penaeidae, Alpheidae, Atyidae and Palaemonidae. Shokita et al. (1984) report a total of 26 species belonging to these families as being found in the Fiji Islands.
Of the family Palaemonidae, two species of the genus Palaemon and twelve of the genus Macrobrachium have been reported in Fiji. The species of Macrobrachium include M. equidens, M. grandimanus, M. australe, M. caledonicum, M. lar, M. latidactylus, M. lepidactyloides, M. placidum, M. gracilirostre, M. aemulum, M. latimanus and M. rosenbergii (introduced) (Nandlal, 2010; Choy, 1984; Shokita et al., 1984). However, Marquet et al. (2003) list M. caledonicum as being endemic to New Caledonia, and it is easy to mis-identify M. australe as M. caledonicum, so the Fiji record of M. caledonicum in Shokita et al. (1984) followed by Nandlal (2010) needs to be confirmed.
All of the species of Macrobrachium listed above are indigenous with the exception of M. rosenbergii, and occur naturally in river systems and inland waters where they constitute freshwater prawn fishery resources both in Fiji and other PICTs.
In Fiji, the species which support a substantial artisanal or subsistence fishery include M. lar (Fij. uradina), M. equidens (Fij. kadikadi or sasakadi), M. grandimanus, M. australe and Palaemon spp. (Fij. “moci”), primarily due to their relatively larger size in comparison with members of the other species which are found in the country; with the exception of moci which are sold in larger quantities to make up for their small size.
These fisheries usually involve women and employ push nets, hands, fine spears and traps to capture the animals. Catches are usually sold at local municipal markets, non- market outlets or consumed at home (Nandlal, 2005, 2010; Richards et al., 1994; Shokita et al., 1984).
12 Monal Lal MSc THESIS: CHAPTER ONE ______
1.6 An overview of the biology of Macrobrachium lar
1.6.1 Morphology
Morphological features
M. lar possesses a number of features which are useful when attempting to identify the species. A general description of the external features of prawns belonging to the infra- order Caridea is outlined in Figure 1.5, and features found on the carapace are described in Figure 1.6. A glossary of relevant anatomical terms is included in Appendix 1.1.
Figure 1.5 External features of a caridean prawn. Source: Raabe & Raabe (2008)
13 Monal Lal MSc THESIS: CHAPTER ONE ______
Figure 1.6 Diagnostic carapace features for prawns of the infra-order Caridea. Source: Chan (1998)
As with other caridean prawns (see Figure 1.7), the first two pairs of pereiopods (walking legs) bear chelae (claws), whereas the third and remaining two pairs of pereiopods do not, the pleurae of the second abdominal somite overlap those of the first and third somites and ovigerous females bear eggs underneath the abdomen. Male specimens also possess on the endopods of their second pair of pleopods a projection called the appendix masculina located adjacent to the appendix interna (Chan, 1998).
14 Monal Lal MSc THESIS: CHAPTER ONE ______
Figure 1.7 Diagnostic characters for prawns of the infra-order Caridea. Source: Chan (1998)
The rostrum (see Figure 1.8) is well developed, compressed and toothed; being short in fully developed males. The dorsal carina of the rostrum bears 7 – 10 teeth, with 1 – 2 of these being completely post-orbital. The ventral carina bears 2 – 4 teeth with the first
15 Monal Lal MSc THESIS: CHAPTER ONE ______tooth being located about mid-way along the length of the ventral carina (Short, 2004; Holthuis, 1980; Kubota, 1972).
Figure 1.8 Anterior portion of the carapace of M. lar showing the rostrum, antennal and hepatic spines. A.s. = antennal spine and h.s. = hepatic spine Source: Short (2004).
The carapace bears two pairs of spines; the antennal spine on the anterior margin below the orbit and the hepatic spine which is positioned obliquely below the antennal spine (Figure 1.8). There is also a short, well developed branchiostegal groove from the anterior margin of the carapace to the hepatic spine (Kubota, 1972).
The mandible bears a three-jointed palp, and exopods are present on all maxillipeds. The first two pairs of pereiopods (walking legs) bear chelae, and the second chelipeds (claw- bearing legs) are enlarged in adult specimens. The last three pairs of legs are simple (Kubota, 1972).
The shape of the dactylus and distal propodus of the last three pairs of pereiopods is also distinctive, and has been described by Holthuis (1950) as “biunguiculate” and Tiwari and Pillai (1973) as a “conspicuous notch” (see Figure 1.9).
16 Monal Lal MSc THESIS: CHAPTER ONE ______
Figure 1.9 Dactylus and distal propodus of third pereiopod of M. lar, showing the notch situated close to the apex of the dactylus. Source: Short (2004).
Adult males of M. lar are highly distinctive, and easily distinguished by their short, sinuous rostrum and long, robust second pereiopods with widely gaping fingers each bearing a large incisor tooth on the cutting edge (Short, 2004).
Short (2004) also reports that the species is most easily confused with M. tolmerum, M. aemulum and M. auratum sp. nov., however there are a number of characters which distinguish M. lar from these species. These include the merus being clearly longer than the carpus of the second pair of pereiopods and the fingers of the second pair of pereiopods being long and greater than half the length of the manus in developed male specimens (Short, 2004).
17 Monal Lal MSc THESIS: CHAPTER ONE ______
Sexual dimorphism
Adult male and female M. lar are sexually dimorphic, and possess a number of characteristics which separate the sexes. Photographs of male and female specimens of M. lar are presented overleaf in Plates 1.1 and 1.2 respectively. Adult male M. lar are typically larger than females. Kubota (1972) reports that males can attain a size of over 16 cm (standard length), while few females reach 14 cm.
Adult male specimens also have greatly enlarged second pereiopods, which can be as long as 30 to 36 cm from the base of the coxa to the tip of the dactylus in very large specimens. These pereiopods can make up to 20 % of the weight of the animal, whereas females have much shorter and slimmer second pereiopods which can make up to only 11 % of the weight of the animal for large specimens (Short, 2004; Kubota, 1972; Holthuis, 1950).
Differences also exist in the shape and structure of the chelae of the second pair of pereiopods. The dactyli of the chelae are more curved in large males and possess one or two teeth, whereas the dactyli of females are less curved and have small single projections instead of teeth (Kubota, 1972).
Perhaps the most definitive method of determining the sex of M. lar is by looking for the presence or absence of the appendix masculina on the second pair of pleopods (see Figure 1.7). This characteristic is applicable to all members of the sub-family Palaemoninae (Kubota, 1972) and infra-order Caridea (Chan, 1998).
The overall shape of the cephalothorax and abdomen are also useful in determining sex in mature specimens. Males usually possess a larger cephalothorax and slimmer abdomen than females, which tend to have a smaller cephalothorax and wider abdomen to house the brood chamber where eggs are held when they become ovigerous (Nandlal, 2010; Kubota, 1972).
18 Monal Lal MSc THESIS: CHAPTER ONE ______
Plate 1.1 Adult male M. lar.
Plate 1.2 Adult female M. lar. 19 Monal Lal MSc THESIS: CHAPTER ONE ______
Colour
The body colour of M. lar is a deep olive or reddish brown to olive grey or blue grey black without specks or blotches and often variegated with swirls of orange brown, blue grey and light olive grey. Adult specimens are countershaded with the dorsal surfaces being darker than the ventro-lateral surfaces. The abdominal condyles are usually a light cream to orange colour, with the posterior portion of the abdomen often being darker than the anterior section. The terga are also usually darker than the pleura (Short, 2004; Tayamen & Brown, 1999; Kubota, 1972).
Younger animals are typically lighter in colour than the adults, generally being a pale yellow brown. The rostrum is olive grey to olive brown, with the dorsal teeth and plumose setae being orange brown (Short, 2004; Kubota, 1972).
The first chelipeds and pereiopods range from a blue grey to dark brown colour. The second chelipeds are olive to dark brown or black, and sometimes marbled with irregular brown, olive or blue-grey blotches. The fingers of the second chelipeds are a dark reddish brown, with a pink mark on the manus at the base of the dactylus (Short, 2004; Kubota, 1972).
Size
The Monkey River Prawn is considered a large species of freshwater prawn. There are various reports in literature which differ on the size ranges that this species is able to attain. Holthuis (1980) reported the maximum total length being 18.1 cm, whereas Kubota (1972) found standard lengths of 14 cm and 16 cm for females and males respectively.
Another account by Short (2004) in his study states maximum recorded sizes of developed males and females as 6.1 cm (carapace length), 19.5 cm (total length) and 4.4 cm (carapace length), 14.5 cm (total length) respectively. He also reports that the minimum size of an ovigerous female collected was 1.4 cm (carapace length). 20 Monal Lal MSc THESIS: CHAPTER ONE ______
1.6.2 Nomenclature, classification and taxonomy
Macrobrachium lar is commonly known as the Monkey River Prawn, which is also the FAO name for the species (Chan, 1998; Holthuis, 1980). The species also has a number of local names in the areas where it occurs. Examples of these local names in PICTs include Tahitian Prawn in Hawaii (Kubota, 1972), Giant Jungle Prawn (Short, 2004), oura-pape in Tahiti, ulaula in Wallis and Futuna, koura in the Cook Islands and uradina in the Fiji Islands (Nandlal, 2010; ITIS, 2004; Short, 2004; Holthuis, 1980).
Synonyms that have been reported in literature for the Monkey River Prawn are detailed in ITIS (2004), Short (2004), Holthuis (1980) and Kubota (1972). The current taxonomic hierarchy for Macrobrachium lar is reproduced below in Figure 1.10.
Kingdom Animalia Phylum Arthropoda Sub-phylum Crustacea Brünnich, 1772 Class Malacostraca Latreille, 1802 Sub-class Eumalacostraca Grobben, 1892 Super-order Eucarida Calman, 1904 Order Decapoda Latreille, 1802 Sub-order Pleocyemata Burkenroad, 1963 Infra-order Caridea Dana, 1852 Super-family Palaemonoidea Rafinesque, 1815 Family Palaemonidae Rafinesque, 1815 Sub-family Palaemoninae Rafinesque, 1815 Genus Macrobrachium Bate, 1868 Species Macrobrachium lar (J. C. Fabricius, 1798) Figure 1.10 Current taxonomic hierarchy for Macrobrachium lar. Source: http://www.itis.gov/servlet/SingleRpt/SingleRpt
Positive taxonomic identification of Macrobrachium lar is accomplished by examining a number of morphological features. An exhaustive description of these features can be found in Short (2004), however the simplest features for use in gross examination and rapid diagnosis of live specimens include the rostrum dentition along with features of the dactylus and distal propodii of the third, fourth and fifth pairs of pereiopods. The presence and positions of the antennal and hepatic spines is also useful for performing identifications.
21 Monal Lal MSc THESIS: CHAPTER ONE ______
1.6.3 Distribution
The Monkey River Prawn is by far the most widespread species of the genus, with a broad Indo-West Pacific distribution from eastern Africa to the Ryukyu Islands (Japan) and Marquesas Islands (Short, 2004; Suzuki, 2001). A distribution map for the species is presented in Figure 1.11.
Figure 1.11 Distribution map of M. lar. Source: Short (2004).
M. lar is present in the majority of PICTs. It is indigenous to archipelagos such as the Fiji Islands, French Polynesia, Wallis and Futuna, the Solomon Islands and Vanuatu (Nandlal, 2010; Short, 2004); which have freshwater bodies suitable for habitation.
It was introduced intentionally to Hawaii in 1956, 1957 and 1961 to improve forage conditions for game fish and for recreational purposes (Atkinson, 1977, 1973; Kubota, 1972; Maciolek, 1972; Brock, 1960). Since that introduction, the species has successfully established itself on all the major islands which make up the Hawaiian island chain by means of marine larval dispersal, even to the extent that it is displacing indigenous species such as Macrobrachium grandimanus (Atkinson, 1977, 1973).
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1.6.4 Habitat
Short (2004) notes that adult M. lar show a preference for well-oxygenated water and are usually found in small freshwater streams or creeks which are connected to the sea. Individuals are usually seen near riffles or waterfalls, particularly where there is abundant shelter such as large rocks, fallen timber or large tree roots. Studies carried out by Kubota (1972) reported similar findings. Juveniles are found in estuaries, lowland fresh waters and inshore marine areas (Short, 2004; Kubota, 1972).
In Australia, the species is largely restricted to permanently flowing rivers and creeks ranging in elevation from near sea level to foothills up to 150 m. The Monkey River Prawn also occurs on offshore continental islands and archipelagos. On small oceanic islands, the species often occupies habitats which extend far upstream to inland headwaters (Short, 2004). Factors affecting the distribution of M. lar in a Hawaiian stream are discussed in detail by Kubota (1972).
Short (2004) reports that adults of M. lar are found on fine to coarse silt, sand or bedrock substrates, in areas which experience low to high flow velocities. A preference for high water clarity is also noted, at depths ranging from 0.3 to 1.5 m. An account by Holthuis (1950) recorded two juvenile specimens at a depth of 55 m in the Bay of Bima, Sumbawa, Indonesia, in reef habitat on a substrate of mud with patches of fine coral sand (Short, 2004).
Fringing vegetation type in habitat areas varies from anthropogenically disturbed to pristine rainforest, monsoonal forest or open riparian. Recorded physicochemical tolerances for the species after Short (2004) are as follows:
Temperature: 20.7 – 28.2
DO2: 6.1 – 9.0 ppm pH: 6.0 – 7.1 ppm Hardness: < 20 – 40 ppm
23 Monal Lal MSc THESIS: CHAPTER ONE ______
An interesting report by Short (2004) states that “the two Giant River Prawns M. lar and M. rosenbergii are mostly allopatric (occurring in separate, non-overlapping geographic areas) in Australia, although they do occur together in the McIvor River in North-East Queensland. M. rosenbergii occurs across the seasonally wet or ‘monsoonal’ north mostly in long, slow flowing rivers, whereas M. lar is most abundant in the wet tropics of North-East Queensland in high gradient, rainforest streams. The McIvor River is near the border of these two climatic zones, and M. lar shows a preference for running water in rocky areas whereas M. rosenbergii is found in backwaters with fallen timber”.
In areas of suitable habitat, individuals of M. lar exhibit gregarious behaviour and can be found at relatively high densities exceeding 30 – 40 individuals per m2 of stream bottom (pers. obs., Waisere Creek, Fiji Islands). Pond grow-out trials by Nandlal (2010) and Barbier et al. (2006) in Vanuatu and Wallis and Futuna reflect this. Nandlal (2010) suggests that it may be possible to increase stocking densities for rearing juveniles from 5 individuals per m2 to near 40 per m2. Current work by Alo et al. (2011) in Vanuatu has found that optimal stocking densities for M. lar in ponds is between 15 and 25 individuals per m2, which is comparatively higher than the stocking density of 5 – 8 individuals per m2 for semi-intensive M. rosenbergii culture currently practised in Fiji (Nandlal & Pickering, 2006a).
As M. lar is a large sized prawn, it is of importance to artisanal and subsistence fisheries in almost all areas where it occurs. Known regions where this species supports local fisheries include Mauritius, Indonesia, Western New Guinea, the Phillipines, Tahiti, the Fiji Islands, Guam, the Mariana Islands (Holthuis, 1980, 1950), Vanuatu (Barbier, et al., 2006; Nandlal, 2005), the Solomon Islands, Wallis and Futuna, the Cook Islands, Samoa and American Samoa (Nandlal, 2010; Ponia, 2010).
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1.6.5 General biology
1.6.5.1 Life history
M. lar is a diadromous species, with the adults occupying freshwater habitats in the upper reaches of streams and creeks usually far inland, larvae adopting an oceanic existence in the plankton and post-larvae and early juveniles being found in estuarine and inshore marine areas as well as lowland fresh waters (Short, 2004).
Ovigerous females are typically found in the upper reaches of streams in Fiji (Shokita et al., 1984), and some females are known to either release larvae in the adult habitat areas or undertake breeding migrations downstream from their habitat areas to release larvae closer to the sea (Nandlal, 2010; Mather et al., 2006; Kubota, 1972).
Kubota (1972) mentions that “breeding migration for females is probably not a hard and fast rule, but dependant on the terrain and distance of the stream, as well as the age and size of the female”. He also mentions that in areas where water velocities are sufficiently fast or barriers to migration are present, females probably do not migrate.
Newly hatched larvae are carried by river currents to the sea. After metamorphosis, newly settled PL appear to remain in the marine environment for a short while before they travel to freshwater sources and eventually begin migrating upstream to recruit to adult habitat areas (Kubota, 1972). Recruitment is likely to occur year-round as documented by Kubota (1972) in Hawaii, with seasonal peaks occurring at certain times of the year (Pickering, unpubl.).
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1.6.5.2 Reproduction
Sexual maturation
Kubota (1972) reports that sexual maturation in female M. lar took place between 5 and 9 months post-settlement, with the smallest ovigerous females being 5.2 to 6.6 cm in standard length. The onset of spermatogenesis in males was not reported.
Male Monkey River Prawns are able to continue mating after they become sexually mature, however females need to undergo a pre-mating moult before being able to be ready to mate (Kubota, 1972). This is similar to the maturation pattern of other palaemonids such as M. rosenbergii (Brown et al., 2010; Nandlal & Pickering, 2006b). It has been observed that the larger and more aggressive males were the individuals which copulated with females that had completed their pre-mating moult (Kubota, 1972).
Courtship and mating
Kubota (1972) and Nandlal (2010) observed courtship and mating behaviours in M. lar in the laboratory, and found that it was very similar to that of M. rosenbergii. The male initiates a courtship display, which involves raising its body on the last three pairs of pereiopods, waving the antennae and raising and extending the enlarged chelate second pair of pereiopods while making intermittent jerking movements.
This display lasts for 10 to 20 minutes before the female accepts and is held by the male using the second pair of pereiopods while the ventral portion of her cephalothorax is cleaned using another pereiopod. This procedure continues for around 10 to 15 minutes before copulation takes place which lasts for a few seconds.
The female is overturned and positioned ventral side up while the male presses down from above, bringing his penes (genital pores) at the base of the fifth pair of pereiopods into close contact with the cleaned area of the female’s cephalothorax. With a sudden vigorous vibration of the pleopods and trembling of the body, the spermatophores are 26 Monal Lal MSc THESIS: CHAPTER ONE ______ejected and deposited in a single gelatinous mass on the female’s ventral median thoracic region. This mass covers the oviducts of the female. Following copulation, the female is protected by the male for a time until she is ready for oviposition.
Oviposition
In preparation for oviposition, the female cleans her abdominal area using the first pair of pereiopods. Kubota (1972) observed that many females underwent oviposition within 30 hours of mating. In females that were mature but did not mate, unfertilized eggs were still positioned under the abdomen but dropped off within 2 to 3 days.
The process of egg deposition on the pleopods and in the brood chamber underneath the abdominal somites takes 20 minutes, and is very similar to that of M. rosenbergii. The female bends her abdomen forward underneath the body and extends the pleopods to open the brood chamber. The eggs are extruded through the respective genital pore first on one side between the fourth, third, second and first pleopods in succession (progressing towards the anterior end of the animal), before the process is repeated on the other side. Following oviposition, the female spends a great deal of time cleaning and aerating the egg mass (Kubota, 1972).
Embryonic development
The eggs of M. lar once deposited are slightly ellipsoidal in shape and range in size from 0.63 – 0.72 × 0.57 – 0.63 mm in the long and short axes respectively. They increase in size to 0.83 – 0.93 × 0.57 – 0.63 mm close to the time of hatch (Kubota, 1972).
Atkinson (1977, 1973) reported egg sizes of 0.85 – 0.92 × 0.58 – 0.62 and 0.22 – 0.25 × 0.15 – 0.17 mm. The size of a brood varies depending on the size of the female, and was found to be in the range of 924 to 38,800 eggs for females of 5.2 cm and 14.7 cm standard length respectively (Kubota, 1972).
27 Monal Lal MSc THESIS: CHAPTER ONE ______
Incubation period is dependant on temperature, and ranges from 27 to 31 days at water temperatures of 22 – 25 , the female aerates the egg mass by continuous movements of the pleopods and dead eggs and debris are removed using the first pair of chelate pereiopods (Kubota, 1972). Descriptions of embryonic development are provided by Kubota (1972).
Larval hatch and development
During the hatching process, the female lifts her tail at infrequent intervals and vigorously beats her pleopods to release a stream of larvae into the water column (Atkinson, 1973). Hatch typically occurs in the evening, and peak downstream larval drift has been confirmed to take place during these hours, between 1900 and 2300 (Luton et al., 2005; Kubota, 1972).
Atkinson (1973) reports that the female may delay hatching by decreasing or stopping the rate of beating of the pleopods if unsatisfactory conditions prevail at the time the larvae are ready to hatch. These conditions include those of temperature (above 26 unsuitable illumination i.e. constant dark or constant light. If these conditions persist, the female may only release 200 to 300 larvae each day or under extreme circumstances eat the eggs.
The larval development of M. lar appears to be extended, and encompasses at least eleven zoeal stages over a period of 89 days (Atkinson, 1977, 1973).
28 Monal Lal MSc THESIS: CHAPTER ONE ______
1.6.5.3 Feeding
M. lar is a generalistic benthic omnivore, as similar studies on other Macrobrachium spp. such as M. grandimanus, M. ohione, M. rosenbergii and M. carcinus have determined (Brown et al., 2010; Kubota, 1972; Maciolek, 1972).
Kubota (1972) reports that the stomach contents of M. lar contained for the most part organic detritus in various stages of microbial decomposition. Identifiable stomach contents included fish scales and vertebrae, mandibles, setae and exoskeletal material of crustaceans and insect body parts.
Plant material also makes up a significant component of the diet of wild M. lar. Identifiable vascular material of plant origin in the stomachs of M. lar included guava pulp (Psidium guajava), leaf hairs of the Hibiscus (Hibiscus tiliaceus) and algae (primarily blue-green algae; Cyanophyceae) (Kubota, 1972).
Larned et al. (2001) found that M. lar is an important frugivorous (fruit-eating) detritivore in Hawaiian stream ecosystems, where it plays a crucial role in nutrient cycling and accelerating detrital processing. Particular fruits that M. lar was observed feeding on included mangos and guavas.
M. lar utilizes the first pair of pereiopods as the chief means by which it captures food items, as do other species of Macrobrachium. As these pereiopods are chelate, food items are picked up and transferred directly to the mouth. The enlarged second pair of pereiopods is also used if live prey is captured or manipulation of large food items is required. The third pair of maxillipeds also holds food items while they are masticated by the mandibles (Kubota, 1972).
29 Monal Lal MSc THESIS: CHAPTER ONE ______
1.6.5.4 Behaviour
The Monkey River Prawn prefers to shelter in dark places under cover such as piles of fallen timber or large boulders during the day, generally near flowing water. At night, adults are usually very shy of light and quickly move away from the beam of a torch light (Short, 2004).
Activity is also linked to ambient light levels, and the amount of water in their habitat. During periods of overcast weather, individuals may be seen walking around near vegetation on stream beds (pers. obs., Waisere Creek, Fiji Islands). Foraging for food is restricted to night time, which is typical of many Macrobrachium spp. (Nandlal, 2010).
M. lar is a strong swimmer and adept crawler, which makes it well-adapted for life in the turbulent, fast flowing, high-elevation rocky streams and pools which it inhabits. They are also known to climb waterfalls and steep slopes, and are able to walk short distances over land to neighbouring streams or pools (Nandlal, 2010; Kubota, 1972).
Cleaning behaviour
M. lar, like many other species of Macrobrachium, regularly clean various body parts including the cephalothorax, antennae and antennules along with the surfaces of the eyes, gills and carapace undersides to ensure they are free of detritus and epibionts.
The egg masses of ovigerous females are also kept clean to ensure they are free of detritus and epibionts. The first pair of pereiopods is used to perform this function, and is indispensable to the animal (Kubota, 1972). Kubota (1972) found that in individuals where the first pair of pereiopods was removed, death resulted within seven days.
Migration and territoriality
Tagging and clipping studies carried out by Kubota (1972) found that M. lar displayed some intra-stream movement, with males tending to remain within a certain area. Mark 30 Monal Lal MSc THESIS: CHAPTER ONE ______and recapture studies carried out by the same author also suggested territorial behaviour exhibited by dominant males. The breeding migration undertaken by some females is discussed earlier in section 1.6.5.2 on reproduction.
M. lar exhibits hierarchical competitive behaviour when individuals are concentrated together. Kubota (1972) reports on a laboratory study where the largest individual (usually a dominant male) kept in an aquarium occupied the portion of the tank with the most cover and seized the most food. He also reports that this competition for space was also evident when many animals were placed in a tank without any cover. The smallest individuals were often killed but seldom eaten.
Territoriality has also been reported in other Macrobrachium spp. such as M. rosenbergii (Karplus & Sagi, 2010), M. amazonicum (Moraes-Valenti & Valenti, 2010), M. carcinus (Kutty & Valenti, 2010; Kubota, 1972) and M. grandimanus (Kubota, 1972).
1.6.5.5 Growth
The life cycle of decapod crustaceans involves growth through three phases of life; the larva, juvenile and adult (Anger, 2001). Development in M. lar and other decapod crustaceans is metamorphic (hemimetabolous and anamorphic), and growth occurs through the processes of moulting and ecdysis (Anger, 2001; Hickman Jr. et al., 2001); where the old exoskeleton is shed and replaced with a new, larger one to allow for somatic growth (Brown et al., 2010).
Growth is a continuous process largely regulated by the moult cycle, which consists of a number of stages; viz. intermoult, premoult and postmoult (Brown et al., 2010). Somatic tissue growth occurs during the intermoult period, until the next moult event. Moult frequency depends on a number of factors including the age and sex of the individual, ambient water temperatures, food, dietary mineral availability and the individual’s position in the social hierarchy (Brown et al., 2010; Nandlal, 2010).
31 Monal Lal MSc THESIS: CHAPTER ONE ______
The moulting process in M. lar is very similar to that of M. rosenbergii, and is described in detail by Kubota (1972). Young prawns tend to grow more rapidly than mature individuals and males have been shown to attain larger sizes than females, which invest more energy in egg production after reaching sexual maturity (Brown et al., 2010; Kubota, 1972).
Kubota (1972) observed that moulting in adult and juvenile M. lar maintained in the laboratory occurred at intervals of 50 – 68 days and 25 – 40 days respectively at water temperatures of 22 – 23 individuals ceased active feeding. In the case of females that were about to undergo a pre- mating moult, the ripe ovary containing eggs occupied the bulk of the cephalothoracic cavity and pressed on the stomach which had atrophied to accommodate the extra volume.
Following a moult, individuals of M. lar remained soft for 2 days before solidification of their exoskeletons. In the laboratory, newly moulted individuals seek shelter to avoid being cannibalised (pers. obs.; Kubota, 1972).
Kubota (1972) was able to obtain information on the growth rates of M. lar in the wild from tagging experiments. A comparison of the growth rate of M. lar with those of M. rosenbergii, M. carcinus and M. vollenhovenii is given in Figure 1.12.
32 Monal Lal MSc THESIS: CHAPTER ONE ______
Figure 1.12 Generalised growth curve of M. lar along with those of other species of Macrobrachium. Source: Kubota (1972).
Based on this information, he estimated that male M. lar reached a standard length of 6.4 cm in 6.8 months, while a 7.0 cm female would be aged between 9 and 11 months old from the time of settlement. More recent work by Nandlal (2010) based on pond grow- out determined an approximate growth rate of 0.2 g/day, with individuals reaching 20 – 40 g in weight in 120 days.
Alo et al. (2011) in their ongoing study in Vanuatu report that M. lar growth performance in ponds results in prawns which attain an average weight of 35 g after a period of 5 – 6 months. They also mention that growth rate in this species appears to slow down (marking the end of the exponential growth phase) after the animals reach an average body weight of 40 g.
33 Monal Lal MSc THESIS: CHAPTER ONE ______
1.6.5.6 Parasites and diseases
A number of disease conditions have been documented for the Giant Freshwater Prawn M. rosenbergii. These include a Macrobrachium Hepatopancreatic Parvo-like Virus (MHPV), Macrobrachium Muscle Virus (MMV), idiopathic muscle necrosis (IMN), white muscle disease (WTD/WMD) caused by the pathogens MrNV (Macrobrachium nodavirus) and XSV (extra-small virus) and a type of White Spot Syndrome Baculovirus (WSBV) (Pillai et al., 2010; Johnson & Bueno, 2004).
Other conditions that are known to occur include bacterial diseases such as Black Spot/Brown Spot Shell Disease, various bacterial necroses and Rickettsial Disease. Various other conditions affect the hatchery phase of culture and include fungal infestations, Mid-cycle Disease (MCD), metazoal parasitic infestations and fouling by epibionts (Pillai et al., 2010; Johnson & Bueno, 2004).
By comparison, relatively few disease conditions have been reported for the Monkey River Prawn. Kubota (1972) mentions that M. lar along with other freshwater prawns serve as carrier hosts for the Rat Lungworm Angiostrongylus cantonensis,which is a causative agent of the disease eosinophilic meningitis (meningoencephalitis) in humans.
Wild individuals of Macrobrachium spp. are also known to be hosts for parasitic isopods and trematode worms, although reports of metazoan parasites in cultured populations are rare (Pillai et al., 2010).
Mention is made of a “black spotted” disease by Kubota (1972) and Maciolek (1972) in individuals caught in a stream in Hawaii, although the etiology was unclear. Possible causative agents suggested were three fungi; viz. Dermocystidium, Lagenidium and a third unidentified species. This disease appears to be very similar to the Black Spot/Brown Spot Shell Disease described for M. rosenbergii, although for this condition bacteria of the genera Vibrio, Pseudomonas and Aeromonas are implicated as the causative agents (Pillai et al., 2010).
34 Monal Lal MSc THESIS: CHAPTER ONE ______
1.7 Reasons to investigate the aquaculture potential of M. lar
Shokita et al. (1984) in their report on inland water prawns present in Fiji identified three species which appeared to have potential for aquaculture. These were M. lar, M. australe and M. equidens. However, mention is also made of the fact that mass artificial seed production of any of these species had yet to succeed.
Out of these three, M. lar was put forth as the leading candidate owing to the large size it attains relative to the other two species. It is in fact the largest palaemonid prawn that is native to any of the “high” PICTs (Mather et al., 2006). It is this and a number of other attributes which make the Monkey River Prawn an attractive candidate for aquaculture development.
A number of further reasons for assessing the culture potential of the species include:
1. Possibility of threats posed by introduced/invasive species
Among freshwater species introduced to Fiji are two species of Tilapia – Oreochromis mossambicus and O. niloticus, and the Giant Freshwater Prawn Macrobrachium rosenbergii. According to Jenkins et al. (2010), feral tilapia was included in a list of several possible threats to indigenous river fishes which, along with de-forestation along riverbanks and unsustainable agriculture land-use practices turning clear waters muddy, were associated with the absence of as many as ten of the indigenous fish species in some of the rivers they studied.
There is currently no evidence that individuals of M. rosenbergii escaping from ponds during culture have been able to establish themselves in local streams and other waterways in Fiji, although this has occurred in South America (New, 2010). Ironically however, there are reports that introductions of M. lar into the Hawaiian Islands from Guam, Tahiti and the Marquesas Islands have become so well established that the species is displacing a number of both native and endemic species such as Macrobrachium grandimanus (Atkinson, 1973; Kubota, 1972; Maciolek, 1972). 35 Monal Lal MSc THESIS: CHAPTER ONE ______
A better understanding of reproduction in captivity for indigenous freshwater species in the Pacific such as M. lar is desirable, in order to support any future fishery restoration or re-stocking efforts.
2. Disease threats
To date, there has been no published evidence of serious infectious and parasitic diseases affecting cultured populations of Macrobrachium rosenbergii in Fiji. Despite this, there have not been any health assessments that have been carried out on pathogens and parasites in cultured populations (Arthur et al., 2004), so the disease status of Fijian M. rosenbergii is largely unknown. A number of important disease conditions have been reported for M. rosenbergii in other countries, which are mentioned earlier in section 1.6.5.6.
As culture of M. rosenbergii develops and intensifies, new disease conditions are likely to be reported in the future along with more serious cases of those that have already been documented. For this reason, the availability of a secondary species for culture such as M. lar would be an advantage for the local prawn culture industry should a serious disease concern arise in the M. rosenbergii population. Given that M. lar is a native species, healthy culture stock could easily be sourced from wild populations provided that they remain unaffected by disease from cultured M. rosenbergii populations.
3. Overfishing, destructive fishing and other anthropogenic threats to M. lar in Fiji
Nandlal (2005) reports that in modern times, natural stocks of M. lar have declined in many places due to over-exploitation, illegal fishing and habitat modification as a result of an increase in sediment load, pesticides, fertilizers or introduced exotic fish species.
A compounding factor is that the status of local stocks of freshwater prawns in general is largely unknown, with stocks in small streams near major urban centres possibly being severely depleted (Richards et al., 1994). Lewis (1985) in (Richards et al., 1994) reported 36 Monal Lal MSc THESIS: CHAPTER ONE ______that the increasing use of chemicals which kill prawns of all species and sizes, was at that time a major source of concern. It is not known whether this illegal method of fishing is still in practice, however anecdotal reports suggest that it is.
Locally, M. lar is highly prized as a food source by inland villagers and urban dwellers alike who purchase it from municipal markets around the country. Annual sales of this species at municipal markets Fiji-wide have been estimated at 170 – 200 tonnes, according to records of the Ministry of Fisheries and Forests of the Government of Fiji (Ponia, 2004).
There is anecdotal evidence that wild stocks in a number of heavily-fished areas are being threatened by overexploitation. This is largely due to the use of improper fishing practices such as the use of chemicals or Derris sp. root to stun or kill the prawns in streams or creeks. This unfortunately kills many juveniles and sub-adults along with the adults that are targeted, often eliminating whole populations in that particular locality along with other non-target organisms.
Declining wild stocks have also been linked to pollution and habitat alteration (Mather et al., 2006). Haynes (1999) studied the populations of M. lar (among other macroinvertebrates) in two adjacent streams, one of which had been subjected to logging activity over a 3 year period. She found that populations of M. lar were more abundant in the stream that had not seen any logging activity.
5. Aquaculture development opportunities in Fiji and other Pacific Island Countries
If the constraint of seed stock supply can be overcome, then Macrobrachium lar is ideally placed to become an important aquaculture commodity in the Pacific Islands region, due to a number of favourable biological and other characteristics which include its large size, acceptability among locals as a prized food source (Nandlal, 2005) and ability to withstand desiccation for short periods of time (Chan, 1998).
37 Monal Lal MSc THESIS: CHAPTER ONE ______
Unfortunately, hatchery techniques to close the life cycle of M. lar in captivity and produce PL for pond stocking have not yet been developed. This means that any PICTs wishing to create freshwater aquaculture employment and income-generating activities through prawn farming will need to involve the already domesticated species M. rosenbergii, as has already been done in Fiji.
For any PICTs that do not wish to introduce M. rosenbergii for aquaculture, for the reasons discussed in the FAO Code of Conduct for Responsible Fisheries 5: Aquaculture Development document (FAO, 2000, 1997); successful domestication of M. lar will provide an indigenous alternative.
1.8 History of M. lar research work
Past research efforts on M. lar can be traced back some 39 years ago, with the majority of these studies focusing on attempting to rear larvae from hatch till the post-larval stage, in order to close the life-cycle in captivity and domesticate the species for aquaculture.
The first attempt at completing the larval development of M. lar in captivity recorded in published literature was by Kubota (1972), who was able to rear larvae till the fifth zoeal stage. His work also appears to be the earliest study to focus exclusively on the distribution, ecology and general biology of M. lar. Further attempts at rearing the larvae in captivity were made by Atkinson (1977, 1973), Muranaka in Hanson & Goodwin (1977), Takano (1987b), Nandlal (2010), Sethi and Roy in Kutty & Valenti (2010) and Sethi et al. (2011).
The outcomes of these studies have provided some indications as to why larval rearing so far has proved to be unsuccessful; and these include conditions of improper salinity, temperature, disease and other water conditions (Kubota, 1972), as well as nutrition (Atkinson 1977, 1973; Kubota, 1972).
Apart from larviculture efforts, other areas to assess the potential of M. lar culture have been investigated. One of these was an ACIAR-funded mini-project in 2005 co- 38 Monal Lal MSc THESIS: CHAPTER ONE ______implemented by SPC and the Vanuatu Department of Fisheries entitled “Monoculture of the freshwater prawn, Macrobrachium lar, in Vanuatu and integrated prawn-taro farming in Wallis and Futuna” (Barbier et al., 2006; Nandlal, 2005). The results of this study showed that small-scale capture-based culture efforts for M. lar were feasible.
A study on the genetic structure of wild populations of M. lar (Mather et al., 2006), as well as collection and identification of juveniles for capture-based culture trials (Alo et al., 2011; Young Uhk, 2006) have also been carried out.
The Secretariat of the Pacific Community (SPC) is an inter-governmental organization in the Pacific region that provides technical and policy advice and assistance as well as training and research services to its PICT member countries; and regards M. lar as a species of interest for further research under its Fisheries, Aquaculture and Marine Ecosystems Division (www.spc.int).
The SPC currently has a region-wide project concept for a coordinated approach to the domestication of M. lar for aquaculture. This project is working in a few key areas in a number of countries to address issues that have been identified. These key areas include follow-on post-larval and juvenile capture and grow-out trials (in Vanuatu and Fiji), larviculture research to close the larval lifecycle (in Fiji), genetic analysis to find out more about larval dispersal (in Fiji, Vanuatu and New Caledonia), feed formulations (in Vanuatu and Fiji), marketing (in Vanuatu) and training and extension activities (in Vanuatu and New Caledonia) (Alo et al., 2011; Lal et al., 2011).
This thesis is the outcome of work done as a component of the SPC-coordinated project “Larviculture research to close the larval life cycle of M. lar in Fiji”.
39 Monal Lal MSc THESIS: CHAPTER ONE ______
1.9 Research objectives
The overall goal of this study is to identify opportunities for aquaculture of the Monkey River Prawn M. lar, based upon commercial-scale hatchery production of post-larvae for pond stocking. This requires the development of techniques to hatch larvae under laboratory conditions and rear them through to the post-larval stage for the first time in captivity. Three specific research questions are addressed by the study:
i) Is it possible to devise a technique to successfully rear the larvae of M. lar from hatch to post-larva in captivity?
ii) Is it possible to describe all larval developmental stages from hatch to metamorphosis, and elucidate the precise number of larval stages?
iii) What are the optimal conditions of salinity and temperature required for successful culture of M. lar larvae?
These research questions led to the formulation of a set of objectives to answer them. These primary objectives were as follows:
i) To develop and describe a new larval rearing technique by which larvae of M. lar will be able to be successfully reared from hatch, through all larval developmental stages until they metamorphose into post-larvae.
ii) To simply describe the embryonic development stages of M. lar with the view of determining time till hatch of ovigerous females maintained in captivity.
iii) To rear and describe larvae of M. lar through all larval developmental stages until metamorphosis into post-larvae.
iv) To produce a simple larval development guide for identifying the developmental stages of M. lar larvae.
40 Monal Lal MSc THESIS: CHAPTER ONE ______
v) By means of a series of controlled experiments, determine the optimal ranges of salinity and temperature required by the larvae of M. lar for successful laboratory-scale culture.
1.10 Thesis organization
This thesis is organised into five chapters. Chapter 1 presents an overview of the current status of aquaculture on global and regional scales with particular emphasis on the Pacific region and PICTs. Reasons are put forth about the need for this study to be carried out and the history of M. lar aquaculture research is also reviewed, with a summary of the study objectives presented at its conclusion.
Chapter 2 describes the development of a mass culture technique for larvae of M. lar and analyses the results of a trial which successfully produced post-larvae. Chapter 3 reports on the results of a series of controlled experiments which were conducted to determine the optimal ranges of salinity and temperature required for successful culture of larvae of the Monkey River Prawn. Chapter 4 describes the egg and larval development stages of this species and includes a simple guide for the identification of the larval stages.
Chapter 5 summarises the overall findings of the study, outlines recommendations for further research opportunities and in light of these findings, provides a new assessment of the potential of M. lar as a candidate for aquaculture development.
41 Monal Lal MSc THESIS: CHAPTER TWO ______
CHAPTER TWO
DEVELOPMENT OF A MASS CULTURE TECHNIQUE FOR LARVAE OF THE MONKEY RIVER PRAWN Macrobrachium lar
2.1 Introduction
2.1.1 Freshwater prawn larviculture
As the Giant Freshwater Prawn M. rosenbergii is the most researched and highly commercialised of the approximately 230 species of the genus which have been described worldwide (Holthuis & Ng, 2010), methodologies developed for rearing the larvae of this species have been used as a benchmark for larviculture of other Macrobrachium species.
Larval rearing systems for Macrobrachium prawns largely fall into two types; the ‘greenwater’ and ‘clearwater’ systems. The greenwater system developed by Takuji Fujimura in Hawaii in the 1950s for M. rosenbergii, involved deliberate encouragement of phytoplankton development (mainly Chlorella spp.) in the larval rearing tank (LRT) (Valenti et al., 2010). This practice was believed to improve water quality and increase larval survival rates, however high pH levels and algal blooms or crashes often caused larval mortality (Valenti et al., 2010).
The greenwater system of larviculture is used extensively in Asia and elsewhere for larviculture of penaeid shrimp (Treece & Yates, 2000), as the naupliar larval stages rely on phytoplankton as their primary food source.
Clearwater systems involve rearing larvae under relatively ‘clean’ conditions, whereby live and inert feeds are offered at controlled rates. Today, very few prawn hatcheries use greenwater systems, as commercial experience has shown that clearwater systems are easier to manage and thus more efficient (Valenti et al., 2010).
Clearwater systems may be divided into a number of different types, depending on their method of water usage. These include flow-through, static (open) and recirculation
42 Monal Lal MSc THESIS: CHAPTER TWO ______
(closed) systems. Greenwater-type systems are primarily static systems, to prolong phytoplankton retention times in the LRT.
Flow-through systems rely on large quantities of water of suitable quality, which is regularly exchanged with LRT water to remove toxic substances produced by the larvae and decomposing uneaten feed. Static or batch culture systems use less water than flow through systems but regular water exchanges still need to be performed (Valenti et al., 2010; Correia et al., 2004; Valenti & Daniels, 2004).
Recirculation systems are closed systems which do not require regular inputs of clean water. The LRT water is instead continuously recycled through filtration media and processed to remove toxic waste substances (Valenti & Daniels, 2004). This is important where water resources are scarce e.g. in inland hatcheries where water needs to be transported great distances or artificial seawater is used. Recirculation systems provide several advantages over open-type systems including lower water consumption rates, stable water quality, lower labour requirements and enhanced feasibility of establishing inland hatcheries; however higher levels of skilled labour and capital investment are also required (Valenti et al., 2010; Valenti & Daniels, 2004).
2.1.2 Larval development types among Macrobrachium prawns
There are three types of larval developmental patterns recognised in freshwater prawns of the genus Macrobrachium on the basis of criteria such as the number and size of eggs, larval development duration, environmental parameters for larval development and the number of zoeal stages (de Grave et al., 2008; Alekhnovich & Kulesh, 2001; Jalihal et al., 1993; Shokita, 1985).
The first type of developmental pattern has been described as being “typical” (Alekhnovich & Kulesh, 2001) or “prolonged/normal” (Jalihal et al., 1993), and is characterised by species which produce numerous, small eggs that hatch into free- swimming larvae. Development occurs at salinities between 10 and 30 ‰ and at temperatures of 24 – 30 !
43 Monal Lal MSc THESIS: CHAPTER TWO ______first stage zoea larvae and a prolonged development period with up to or more than 10 larval stages which may exceed 3 months (Jalihal et al., 1993; Alekhnovich & Kulesh, 2001). M. lar fits into this type of developmental pattern; along with M. rosenbergii, M. nipponense, M. acanthurus, M. formosense, M. malcolmsonii, M. carcinus, M. intermedium, M. idella, M. olfersii, M. novaehollandiae, M. equidens, M. amazonicum, M. americanum, M. grandimanus, M. lanceifrons, M. nilotocum and M. lanchesteri (Jalihal et al., 1993).
The second type of developmental pattern has been termed “semi-abbreviated” (Alekhnovich & Kulesh, 2001) or “partially-abbreviated” (Jalihal et al., 1993), and contains species which produce fewer, larger eggs that hatch into first stage larvae which are also free-swimming but roughly twice the size of those belonging to species which undergo the typical or prolonged/normal type of development. The number of larval stages is reduced, with some researchers reporting between 5 and 9 (Alekhnovich & Kulesh, 2001) and others reporting less than 10 or up to 3 (Jalihal et al., 1993). Larval development occurs in either freshwater or at salinities between 6 and 12 ‰, at temperatures of 25 – 33 Species which undergo this type of development include M. lamarrei, M. lamarrei lamarrei, M. australiense, M. unikarnatakae, M. canarae, M. sankollii, M. borellii, M. nattereri, M. malayanum, M. tiwarii, M. johnsonii, M. shokitai, M. kistnensis, M. potiuna, M. niphanae, M. asperulum, M. koreanum, M. pilimanus and M. ferreirai (Jalihal et al., 1993; Shokita et al., 1991).
The third type of development is termed “abbreviated” (Alekhnovich & Kulesh, 2001) or “completely abbreviated” (Jalihal et al., 1993) and is a pattern which involves no free- swimming larvae. Only freshwater species have so far been reported to undergo this type of development, and the larvae pass through between 1 and 4 zoeal stages before metamorphosis within 2.1 – 15 days. The larvae at hatch are morphologically fairly advanced, subsist on their yolk sac and adopt a benthoplanktonic existence (Alekhnovich & Kulesh, 2001; Jalihal et al., 1993). Examples of species which belong to this development type category are M. dayanum and M. hendersodayanum (Jalihal et al., 1993).
44 Monal Lal MSc THESIS: CHAPTER TWO ______
Among the species which exhibit the typical or prolonged/normal type of development, there are comparatively few which, like M. lar, require salinities for successful development that approach fully marine conditions.
These species include M. equidens which requires approximately 33 ‰ (Ngoc-Ho, 1976 in Shokita, 1985), M. grandimanus which requires 17.5 – 35 ‰ (Shokita, 1985), M. intermedium whose lifecycle has not yet been closed in captivity but with the first 8 zoeal stages being reared at 35 ‰ (Williamson, 1971), M. acanthurus which was reared between 23.5 and 35 ‰ by Dobkin (1971), M. vollenhovenii which has a requirement for between 16 and 24 ‰ (Willführ-Nast et al., 1993) and M. americanum which requires between 20 and 30 ‰ for its early larval development after which salinity is reduced to between 15 and 20 ‰ (Holtschmit & Pfeiler, 1984). Holthuis (1980) states that M. latidactylus and M. latimanus may also have a marine larval development phase.
2.1.3 Development of a larval rearing technique for M. lar
The earliest attempt at larviculture of M. lar in published literature dates back to 1972, with the work of W. T. Kubota (1972). Subsequent efforts by Atkinson (1977, 1973), Takano (1987b), Sethi and Roy (Kutty & Valenti, 2010), Nandlal (2010) and Sethi et al. (2011) progressed to mid- or late-stage larvae, but did not produce PL in the laboratory.
There exists in the literature a brief mention of some work by M. S. Muranaka in Hanson and Goodwin (1977), which apparently resulted in the successful production of M. lar PL in captivity, however it appears that this work was never published.
A number of reasons have been put forth to explain the difficulty of rearing the larvae of M. lar under laboratory conditions. These include a pelagic larval development period which is prolonged to maximise larval dispersal (Atkinson, 1973; Short, 2004; Mather et al., 2006), the provision of inappropriate nutrition to the larvae – especially material not of plant origin (Atkinson, 1977, 1973; Kubota, 1972), rearing in an inadequate/inappropriate culture environment, e.g. inappropriate conditions of salinity
45 Monal Lal MSc THESIS: CHAPTER TWO ______
(Nandlal, 2010; Atkinson, 1973; Kubota, 1972) and the larvae being inherently “delicate” and thus difficult to rear (Atkinson, 1973).
Considering these previous experiences, the research for this chapter was carried out to develop a new larviculture technique designed with the aim of successfully rearing the larvae of M. lar to PL. Although there has been much debate over whether clearwater or greenwater-type systems are superior, a greenwater system was chosen for this study. The primary reason for this was to provide the larvae with a wide range of food resources in the event that the prepared feeds offered proved to be nutritionally inadequate. Investigations by Cohen et al. (1976) and Lober & Zeng (2009) found that the addition of microalgae improved the culture performance of M. rosenbergii larvae, which further supported the choice of a greenwater-type system.
2.1.4 Research objectives
Research carried out for this chapter was aimed at answering the following questions:
i) Is it possible to devise a technique to successfully rear the larvae of M. lar from hatch to post-larva in captivity?
ii) Is it possible to describe all larval developmental stages from hatch to metamorphosis, and elucidate the precise number of larval stages?
In order to fully answer question i) and answer in part question ii), the following specific objective was formulated:
i) To develop and describe a new larval rearing technique by which larvae of M. lar will be able to be successfully reared from hatch, through all larval developmental stages until they metamorphose into post-larvae.
The description of the larval developmental stages of M. lar is addressed in Chapter 4 of this thesis.
46 Monal Lal MSc THESIS: CHAPTER TWO ______
2.2 Methodology
All experimental work was carried out at the Seawater Laboratory of the University of the South Pacific at the Laucala Campus in Suva, Fiji. The following section describes various aspects of the methods and procedures used in attempts to mass rear larvae of M. lar.
2.2.1 Water treatment
2.2.1.1 Aeration
Aeration at the Seawater Laboratory is provided by means of two regenerative (low pressure) Regenair® R5325A-2 air blowers coupled to Baldor® 1.85 HP motors that are operated alternately every 12 hours. These supply air to the overhead air distribution system in the laboratory. Air output is approximately 0.5 Ls-1 at individual 5 mm PVC outlets.
2.2.1.2 Seawater treatment
2.2.1.2.1 Primary treatment
Seawater for experimental work at the Seawater Laboratory was obtained adjacent to the School of Marine Studies jetty. A pair of EIM high-capacity (80 m3hr-1) submersible pumps mounted beside the jetty supply seawater to the laboratory. These pumps are used on an alternating basis at high tide to obtain seawater of the highest available salinity.
Incoming seawater is passed through an SD-36S Culligan Water Conditioner® sand filter (12 m3hr-1 capacity) which removes all particles larger than 75 – 100 μm3. This filtered seawater is then pumped to food-grade polyethylene and fibre-reinforced plastic (FRP) storage tanks outside the laboratory. Storage was at ambient temperatures with continuous aeration provided at approximately 0.5 Ls-1.
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2.2.1.2.2 Secondary treatment
Stored seawater was pumped into two dedicated 1000 L square polyethylene treatment tanks using a New Pondy 100 W NSM 2522 portable submersible pump with a capacity of 80 Lmin-1. This pump was used for all water transfers in the laboratory for routine procedures such as larval rearing water preparation or water exchanges along with food- grade polyethylene hoses with a diameter of 25 mm. The pump was rinsed with freshwater and disinfected using a weak chlorine solution (containing at least 5 % available chlorine) when necessary.
This seawater was treated with 4 g of EDTA (C10H14N2Na2O8•2H20) per 1000 L used as a chelating agent, then filtered by pumping it through a Filterpure® 5 μm wound polypropylene cartridge filter placed inside the treatment tank for approximately 3 hours. All treated seawater was usually used within a few hours of being prepared and had an -1 4+ average salinity of 30 ± 2 ‰, pH of 7.8 ± 0.2, DO2 > 6.5 mgL and average NH and
NH3 concentrations no higher than 1.5 and 0.1 ppm respectively.
2.2.1.3 Freshwater treatment
Freshwater was obtained from the municipal water supply via a fire hose outlet and stored in three dedicated 1000 L square polyethylene freshwater treatment tanks, after passing it through a 5 μm wound polypropylene cartridge filter plumbed in-line with the treatment tank. This freshwater was chelated by addition of 4 to 6 g of EDTA per 1000 L, depending on the degree of turbidity of the water.
Following this, a 5 μm wound polypropylene cartridge filter was connected to a submersible water pump, placed inside the treatment tank and run for 12 to 15 hours. This -1 4+ treated freshwater had an average pH of 7.1 ± 0.3, DO2 > 6.5 mgL and average NH and NH3 concentrations no higher than 1.5 and 0.1 ppm respectively. After treatment, this freshwater was stored in 1500 L FRP tanks at ambient temperatures with continuous aeration provided at approximately 0.5 Ls-1.
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2.2.2 Rua-Cell system of culture
Given that the Monkey River Prawn is a diadromous species with larvae requiring full- strength seawater to complete their development, a technique had to be developed which allowed larvae to be cultured in an environment where the salinity of the culture medium changed from being freshwater-dominated upon hatch, to fully marine after a few zoeal moults up until metamorphosis into post-larva.
The Rua-Cell System was developed by Mr. Tomohiro Imamura, a Japan International Cooperation Agency (JICA) crustacean hatchery expert attached to the Seawater Laboratory at USP in 2009 (Imamura et al., 2009). The technique was initially used for enhancing hatchery production of Macrobrachium rosenbergii post-larvae, and the procedures used here are essentially the same as those used for that technique with a few exceptions. These are further explained in the larval rearing procedures in section 2.2.7.
The Rua-Cell System is a ‘green water’ type of culture technique where microalgae (both freshwater and marine) are maintained in the same culture vessel as the larvae. A critical component of the technique is the formation of biofloc in the larval rearing tank (LRT) as a source of larval feed. Comparatively smaller volumes of water are exchanged daily using this technique to maintain viable populations of microalgae in the LRT than in a ‘clear water’ type of technique, and closer observations of larval health are necessary to detect and control larval parasites or diseases as a result of this. LRTs are illuminated constantly and larvae fed a range of three types of custard feeds, viz. Egg, Squid and Shrimp Custards together with enriched Artemia nauplii.
The sections below detail various aspects of the Rua-Cell System and the procedures involved in growing larvae of the Monkey River Prawn Macrobrachium lar.
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2.2.2.1 Marine microalgae culture
A mixed species culture of various marine microalgae was grown outdoors using the open batch culture method. The product of this culture was referred to as ‘brown water’ (Imamura et al., 2009). Various species of diatoms in particular were found to propagate, with a number of Nitzschia spp., Navicula spp. and Skeletonema spp. being dominant. Cells of these various genera were naturally present in the seawater used for the cultures after treatment of the seawater had been carried out, and were the seedstock for the starter cultures.
It was found that it was much easier and less time-consuming to mass culture marine diatoms using this method than to maintain monospecific/axenic cultures of different diatom species.
Treated seawater (as described earlier in section 2.2.1.2) was filled into circular 1200 L FRP culture tanks that had been cleaned and sun-dried using a portable submersible pump (Plate 2.1). This seawater was filtered through a 50 μm2 mesh net as it was filled into the culture tank and treated with EDTA (C10H14N2Na2O8•2H20) at a rate of 1 g per 100 L of tank volume. The tank was then left overnight with aeration provided via a single 5 mm diameter air line at a rate of approximately 0.5 Ls-1 before nutrient chemicals were added the following morning.
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Plate 2.1 1200 L FRP diatom culture tank.
Industrial-grade nutrient chemicals viz. sodium nitrate (NaNO3), sodium di-hydrogen phosphate (2-hydrate NaH2PO4•2H2O) and sodium metasilicate (Na2SiO3•5H2O) were added at the rates of 10 g per 100 L, 1 g per 100 L and 1 g per 100 L respectively.
Each nutrient salt was weighed separately using a Tanita KD-17C top-pan balance and then dissolved using a sufficient amount of treated freshwater (see section 2.2.1.3), before being added to the culture tank. The brown water culture media had an average salinity of -1 4+ 30 ± 2 ‰, pH of 7.8 ± 0.2, DO2 > 6.5 mgL and average NH and NH3 concentrations no higher than 1.5 and 0.1 ppm respectively. Temperature was not regulated and ambient levels varied from 22 to 26" # three additional evenly spaced open-ended 5 mm diameter air lines at a rate of approximately 0.5 Ls-1 at each air line after the nutrient chemicals were added. Each air line was weighed down with a cleaned stone weight.
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A light brown to gold coloured bloom was typically seen in the tank after 4 to 5 days. When weather conditions were favourable with strong sunlight incident on the culture tanks for several hours each day, blooms were sometimes seen after 3 days. During adverse weather or extended cloudy periods, blooms were sometimes only seen after 7 to 8 days or not at all. When this occurred, the culture would be discarded, the tank cleaned and culture-restarted.
A total of four tanks were operated in rotation, to ensure that a constant supply of a minimum of 500 L of brown water was available every day whilst a larviculture run was in progress. When starting a new culture, if a mature culture was ready for harvest in a neighbouring tank, 20 to 40 L was harvested, screened through a 50 μm2 mesh net and used to inoculate the new culture after the nutrient chemicals had been added.
Each culture was maintained for approximately 5 to 8 days, at which point the microalgae were usually observed to start ‘crashing’. When this was seen, the entire culture was either harvested or discarded, depending on the requirements of the larviculture run. Any biofloc (refer to Plates 2.2 a and b) seen to form along the walls and floor of the tank was also periodically harvested and offered as food to the M. lar larvae. This is explained further under section 2.2.7.2.4.
Cell counts of the microalgae varied from approximately 1 × 102 to 3 × 104 cellsmL-1; 1 × 102 to 3 × 103 cellsmL-1 and 1 × 103 to 3 × 105 cellsmL-1 for the Nitzschia spp., Navicula spp. and Skeletonema spp. respectively. All cell counts were carried out using an M-36 double-ruled haemocytometer under an Olympus CH-2 binocular compound light microscope.
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Plate 2.2 a Diatom biofloc seen at low Plate 2.2 b Closer view of organisms power. seen in the diatom biofloc. R = rotifers.
2.2.2.2 Freshwater microalgae culture
A mixed species culture of freshwater microalgae was grown outdoors using the open batch culture method. The product of this culture was referred to as ‘green water’. Various species of green microalgae were found to propagate with what appeared to be a single Desmodesmus sp. (see Plate 2.5 b) and several Chlorella spp. being dominant in the cultures. It was found that it was considerably easier and less time-consuming to mass culture freshwater microalgae using this method than to maintain monospecific/axenic cultures of various species.
The source of the ‘stock’ green water was a series of three 5 tonne tanks containing Nile Tilapia Oreochromis niloticus. A single tank containing a healthy microalgal bloom was selected for inoculating the mass cultures after samples were removed from all the tanks for microscopy. If cells of the desired species (Desmodesmus sp. and Chlorella sp.) were present in sufficient quantities in the samples, approximately 400 to 500 L of the water from a single tank was pumped using a portable submersible pump into a circular 1200 L FRP culture tank through a 50 μm2 mesh net.
Treated freshwater (as described earlier in section 2.2.1.3) was filled into the culture tanks to top up to the final culture volume through a 50 μm2 mesh net (Plate 2.3).
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Plate 2.3 1200 L FRP freshwater microalgae culture tank.
Industrial-grade nutrient chemicals viz. sodium nitrate (NaNO3) and sodium di-hydrogen phosphate (2-hydrate NaH2PO4•2H2O) were added at the rates of 10 g per 100 L and 1 g per 100 L respectively after the culture tank had been filled. Each nutrient salt was weighed separately and then dissolved using a sufficient amount of treated freshwater (see section 2.2.1.3) before being added to the culture tank. The green water culture -1 4+ media had an average pH of 7.5 ± 0.3, DO2 > 6.5 mgL and average NH and NH3 concentrations no higher than 1.5 and 0.1 ppm respectively. Temperature was not regulated and ambient levels varied from 22 to 26
Vigorous aeration was provided to the culture tank via four evenly spaced open-ended 5 mm diameter air lines at a rate of approximately 0.5 Ls-1. Each air line was weighed down with a cleaned stone weight. A dark green coloured bloom was typically seen in the tank after 2 to 3 days. When weather conditions were favourable with strong sunlight incident on the culture tanks for several hours each day, blooms were sometimes seen after 1 day.
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During adverse weather or extended cloudy periods, cultures crashed. When this occurred, the culture would be discarded, the tank cleaned and culture-restarted.
A total of four tanks were operated in rotation, to ensure that a constant supply of a minimum of 1000 L of green water was available every day whilst a larviculture run was in progress. Each culture was maintained for approximately 5 to 8 days, at which point the microalgae were usually observed to start ‘crashing’. When this was seen, the entire culture was either harvested or discarded, depending on the requirements of the larviculture run. Any biofloc (refer to Plates 2.4, 2.5 a and 2.5 b) seen to form along the walls and floor of the tank was also periodically harvested and offered as food to the M. lar larvae. This is explained further under section 2.2.7.2.4.
Cell counts of the microalgae varied from approximately 5 × 104 to 12 × 105 cellsmL-1 and 4 × 104 to 6 × 105 cellsmL-1 for the Desmodesmus sp. and Chlorella spp. respectively. All cell counts were carried out using a double-ruled haemocytometer.
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Plate 2.4 Freshwater microalgal biofloc harvested from the culture tank.
Plate 2.5 a Freshwater microalgal Plate 2.5 b Desmodesmus sp. alga. biofloc seen at low power.
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2.2.3 Broodstock collection
Adult male and ovigerous female individuals of M. lar to be used as broodstock were collected at two sites in Waisere Creek, in the Vugalei District of Tailevu Province, Viti Levu. The approximate coordinates for these sites are 17$%R'()'P+,)-.//R))/)P E and 17$%R'/'(P+,)-./(R$$.)P0 !
Waisere Creek is a relatively small waterway into which a number of smaller streams drain and has direct connection with the sea along the eastern coastline of Viti Levu by flowing into a small estuary north of Viwa Island (see Figure 2.1). According to a number of villagers residing at Visa Village, which is close to the broodstock collection sites chosen, M. lar is abundant in Waisere Creek throughout the year.
Figure 2.1 Broodstock collection sites 1 and 2 at Waisere Creek. Inset: Fiji Islands with the location of Waisere Creek on Viti Levu. Site 1: 17$%R'()'P+, 178//R))/)P0 Site 2: 17$%R'/'(P+,)-./(R$$.)P0. Source: Map: Dept. of Lands and Surveys, Fiji, 1983. 4 ed.. Inset: http://maps.google.com/
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Broodstock were collected using a push net 80 cm wide and 100 cm in length with a 5 mm2 mesh size. The net was swept across the stream bottom adjacent to the stream banks whilst submerged vegetation alongside the banks was disturbed to move the prawns into the net.
Once captured, the prawns were immediately transferred to three 80 L plastic tubs half- filled with stream water that were aerated continuously at a rate of approximately 50 – 60 mLs-1 using Sonpar CP 900 portable battery-powered aerators connected to 25 mm corundum air diffusers. Stream water parameters ranged from 21 to 24 1 2 > 6.7 mgL-1 and pH 7.1 to 7.6. Measurements were made using a YSI 85 salinity, temperature and conductivity meter and YSI pH100 EcoSense pH Meter. Male prawns were maintained in a separate tub from female prawns. Berried (ovigerous) females were handled with extra care, and the abdomen was bent forward underneath the body when out of the water to protect the eggs. No more than 25 animals were placed in a single tub, and approximately 50 animals were captured on every collection trip. A small quantity of leaf litter from the stream bed was placed into each tub to reduce the amount of light incident on the animals and each tub was also covered with its lid to prevent spillage or the prawns jumping out. The broodstock prawns were transported to the Seawater Laboratory at the School of Marine Studies (SMS) at the University of the South Pacific (USP) by road as soon as sufficient numbers had been collected.
Upon arrival at the laboratory, all broodstock were positively identified as Macrobrachium lar according to Short (2004), measured for total length (tip of the rostrum to tip of the telson), carapace length (tip of the rostrum to the edge of the dorsal rear margin of the carapace), cheliped length for males only (posterior margin of basis where it joins the coxal segment to the distal tip of the pollex – see Plate 2.6) and wet weighed (Plate 2.7). All broodstock, particularly ovigerous females, were also disinfected prior to stocking in either the larval hatching tanks or broodstock holding tanks. This was accomplished by exposing the animals to a 30 ppm solution of 38 % formalin for 10 minutes whilst they were in their transportation tubs. This solution was aerated at a rate of approximately 80 – 100 mLs-1 using a single airstone whilst the broodstock were being disinfected and 100 % of the water volume was exchanged twice once disinfection was
58 Monal Lal MSc THESIS: CHAPTER TWO ______complete to eliminate any remaining formalin residue. Any leaf litter collected for the animals during collection was discarded prior to disinfection.
2.2.4 Brooder spawning
Once broodstock had been weighed and measured, they were sorted before being disinfected, acclimated and stocked (Plates 2.8 and 2.9).
2.2.4.1 Brooder egg incubation
Berried (ovigerous) females were sorted according to the stages of their egg mass development. Four broad categories of egg mass development were recognized, viz. ‘early’ orange eggs, ‘late’ orange/yellow eggs, grey eggs and spent eggs. Up to five females bearing eggs of the same or similar developmental stage were stocked in floating 21 mm PVC pipe-framed cages 450 × 450 × 300 mm in length, width and depth respectively covered with 2 mm2 mesh polyester net material (see Plate 2.11).
These cages were placed inside a 2500 L rectangular FRP tank as well as a round 1000 L polyethylene tank. Both tanks were filled to a depth of 50 cm with treated freshwater and heated to 28 ± 0.5 / 23 ) 23 respectively, both regulated by thermostat probes attached to separate control units. Aeration was provided via four evenly spaced 5 mm diameter air lines attached to 4 cm corundum air diffusers at a rate of approximately 200 mLs-1 at each outlet. Critical water -1 parameters remained at DO2 > 6.5 mgL and pH 7.2 to 7.6.
Females bearing immature eggs (‘early’ orange and ‘late’ orange/yellow stages) were held in these egg incubation tanks until their eggs developed to the grey egg stage, at which point they were transferred to hatching tanks.
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2.2.4.1.1 ‘Early’ orange eggs
‘Early’ orange eggs were found to be a bright orange colour (Plate 2.10 a), where the bulk of the egg mass consisted of yolk matter. It was later observed that eggs up to 10 days old (post-extrusion) retained this colour when the brooder females were held at 28 ± 0.5
2.2.4.1.2 ‘Late’ orange/yellow eggs
‘Late’ orange/yellow eggs were found to be a dull orange colour up until around day 16 of incubation when they changed to an ochre or pale yellow colour (Plate 2.10 b). At this stage a clear area had developed on one side of the egg and eyespots had started becoming apparent by the time the eggs turned yellow. Females bearing eggs of this colour were stocked in a separate floating PVC-framed cage either in isolation or together with females bearing grey coloured eggs.
2.2.4.1.3 Grey eggs
Grey eggs were found to be a dark grey to brown in colour (Plate 2.10 c), indicating eggs approximately 20 days old post-extrusion when the brooder females were held at 28 ± 0.5 0! 4 !# ! incorporated into the larval hepatopancreas. These eggs were ready to hatch in a matter of days and so the brooder females were transferred directly to the hatch tanks.
2.2.4.1.4 Spent females
Spent females had an empty brood chamber containing no or very few eggs after having spawned (Plate 2.10 d). These females were stocked into a 1000 L broodstock holding tank (Plate 2.9) along with any male broodstock collected. Spent females removed from hatch tanks were also transferred to the broodstock holding tank, where they could mate with a male brooder following a moult.
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61 Plate 2.6 Measuring TL of male brooder prawn. Plate 2.7 Weighing brooder prawn.
Plate 2.8 Acclimation and release of brooders. Plate 2.9 Broodstock maintained in 1000 L. into the hatch tank. holding tank. Monal Lal MSc THESIS: CHAPTER TWO ______
Plate 2.10 a ‘Early’ orange-berried Plate 2.10 b ‘Late’ orange-berried female. female.
Plate 2.10 c Grey-berried female. Plate 2.10 d Spent female.
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Plate 2.11 1000 L polyethylene holding tank for ovigerous female broodstock.
2.2.4.2 Hatch tank preparation
Circular 1000 L flat-bottomed polyethylene tanks were used as hatching tanks which also doubled as larval rearing tanks (LRTs). Approximately 300 L of brackish (10 ‰) water was prepared for the hatch tanks in a dedicated 1000 L square polyethylene water preparation tank.
All hatching was carried out at a salinity of 10 ± 0.5 ‰, and treated seawater and freshwater (as described earlier in sections 2.2.1.2 and 2.2.1.3 respectively) were mixed in the water preparation tank using a portable submersible pump (Plate 2.12). A 50 μm mesh net was used to screen both the incoming treated freshwater and seawater into the water preparation tank as well as the mixed brackish water pumped to the hatching tank.
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The hatch tanks were heated using dedicated 1 KW titanium immersion heaters regulated by thermostat probes attached to control units. Temperature was maintained at 28 ± 0.5 -1 1 , -./6( 12 > 6.5 mgL 4+ and average NH and NH3 concentrations no higher than 1.5 and 0.1 ppm respectively.
Gentle aeration was provided via four evenly spaced 5 mm diameter air lines attached to 40 mm corundum airstones at a rate of approximately 15 – 30 mLs-1 at each outlet. Air delivery was regulated by individual plastic screw valves installed on each air line. Each air line was weighed down with a cleaned stone weight. A fluorescent light tube fitting containing a single 4 feet Osram 36 W ‘warm white’ tube and a single 4 feet Eurolux 36 W ‘cool white’ tube was suspended over each tank using 4 mm nylon ropes approximately 300 mm from the surface of the water when the tank was filled to its maximum volume (which was set at 900 L). This provided a total light output of approximately 6700 lux at the surface of the water. These lights were kept off for the duration of the hatch event and turned on the morning after. The appearance of the prepared hatch tank when it was ready to receive broodstock females is shown in Plate 2.13.
Plate 2.12 Mixing freshwater and Plate 2.13 Tank prepared to receive seawater for hatch. berried broodstock.
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2.2.4.3 Brooder stocking and hatching of larvae
Ovigerous broodstock bearing grey coloured eggs (Plates 2.14 and 2.15) were typically stocked in the hatch tanks during the afternoon or early evening shortly after collection. At the time of stocking hatch tanks at the beginning of a larval rearing run, ovigerous broodstock bearing grey coloured eggs that had been caught earlier and being maintained in the broodstock maintenance tanks were also transferred to the hatch tanks. No more than 20 ovigerous females were stocked in a single hatch tank. The onset of hatch typically began several hours after the females were stocked.
Broodstock were provided PVC pipes 60 mm in diameter cut into approximately 300 mm lengths as artificial habitat. These pipes also had 8 mm diameter holes drilled into their walls in a random pattern to allow for better circulation of water. The animals were not fed whilst in the hatch tanks.
After all broodstock had been acclimated and released, the hatch tank was covered with a square sheet of 3 mm ply board to which black polyethylene sheets had been stapled (see Plate 2.16). This was to darken the tank to provide a more comfortable environment for the broodstock and encourage a better hatch rate. A space of approximately 50 mm was allowed between the lip of the tank and the bottom of the ply board by placing 60 × 60 × 1500 mm wooden spacers over the tank to allow ventilation of the surface of the water. This was done to avoid the hatch tank water heating up beyond the desired limits.
Typically, a single hatch tank was set up at a time, with broodstock being transferred to another hatch tank after having spent up to 36 hours in the first tank. This duration of hatch was found to be optimal through a number of trials conducted earlier to allow sufficient numbers of larvae to be hatched synchronously in the tank and to avoid the later problem of rearing larvae of mixed age and/or size. When sufficient numbers of ‘grey-berried’ broodstock were available, two hatch tanks were operated concurrently.
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The hatch tank was examined 4 to 5 hours after stocking broodstock for newly hatched larvae. This was done by scooping a quantity of tank water into a 1.2 L larval counting bowl and examining it under torchlight for the presence of first stage zoea larvae. When a sufficient number of larvae were observed in the counting bowl (more than 5 individuals), a pre-mixed 20 L bucket containing green microalgae from the green water culture and seawater was added to the tank. The salinity of this mixture was adjusted to match that of the hatch tank water. This was to ‘seed’ the tank with microalgae, allowing the cells to start multiplying as the larval culture run began. The tank was then re-covered and left overnight for hatch to continue. Plate 2.17 shows the hatch tank (now functioning as a larval rearing tank) the morning after a hatch, with broodstock still present in their PVC pipe habitats in the tank.
Plate 2.14 Brooder carrying eggs that Plate 2.15 Closer view of grey egg mass. are ready to hatch.
Plate 2.16 Hatch tank covered with a Plate 2.17 LRT after a spawn. spawn in progress.
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2.2.5 Broodstock maintenance
2.2.5.1 Spent female broodstock
Broodstock females were removed from the hatch tanks following a 36 hour hatch interval and sorted. All females that were in a spent condition with over 90 % of their egg mass having hatched were transferred to an 80 L plastic tub containing water of the same salinity and temperature as that of the hatch tank. These females were acclimated to freshwater and the temperature of the broodstock maintenance tank over a period of 4 to 6 hours and then transferred there.
2.2.5.2 Partially-spawned broodstock
Some broodstock females were found to have hatched only a portion of their egg mass and these were called partially-spawned brooders (see Plate 2.18). These broodstock typically had 5 to 30 % of their egg mass remaining, depending on the size of the individual. It was found that some of the larger females had more ‘complete’ hatches than smaller females. These broodstock were transferred on to the next hatching tank, together with any newly caught grey-berried females and grey-berried females held in the brooder egg incubation tanks.
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Plate 2.18 Partially-spawned female. Note a few remaining eggs near the retracted pleopods.
2.2.5.3 Broodstock holding tanks
All broodstock apart from ovigerous females were maintained in a 2500 L rectangular FRP tank as well as a square 1000 L polyethylene tank. Both tanks were filled to a depth of 500 mm with treated freshwater and maintained at 26 ± 0.5 .
Aeration was provided at a rate of approximately 200 mLs-1 at each outlet through 4 cm corundum air diffusers via six and three evenly spaced 5 mm diameter air lines, for the 2500 L and 1000 L tanks respectively. Air delivery was regulated by individual plastic -1 screw valves installed on each air line. Water parameters remained at DO2 > 6.5 mgL and pH 7.2 to 7.6.
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Broodstock were provided PVC pipes 60 mm in diameter cut into approximately 300 mm lengths as artificial habitat and shelters for newly-moulted individuals. These pipes also had 8 mm diameter holes drilled into their walls in a random pattern to allow for better circulation of water. The number of pipes provided was proportional to the number of animals maintained in each tank.
Male prawns were collected so that mating could take place in the tanks with newly- moulted non-ovigerous females. These were stocked at a ratio of one male to ten females. Male broodstock were selected for their size (larger animals with large black chelipeds were preferred) and the absence of any signs of disease. At any given time during a larval rearing run, approximately 100 brooder animals (both male and female) were maintained in the laboratory, at least 30 to 40 of which were ovigerous females.
Any females that were observed to come into berried condition in the broodstock holding tank were transferred into the brooder egg incubation tanks. The broodstock holding tank was siphoned twice daily with a length of 110 cm PVC pipe with a diameter of 21 mm attached to a length of flexible polyethylene hose to remove exuvia, faeces and uneaten feed. 100 % of the water in the tank was exchanged once daily, and any moribund or dead individuals were removed.
2.2.5.4 Broodstock feeding
Broodstock held in both the egg incubation tanks and maintenance tanks were fed a varied diet of formulated freshwater prawn pellets with a 32 % crude protein content (Crest Chicken Limited), freshwater mussel (Fij. kai) Batissa violacea flesh, Variegate Venus clam Ruditapes variegatus (Abbott & Dance, 2000) flesh, peanut worm (Fij. ibo) Siphonosoma australe and wild yam (Fij. kaile) Dioscorea pentaphylla flesh.
Live freshwater mussels and Variegate Venus clams were maintained in the laboratory in 20 L buckets containing treated freshwater and treated seawater respectively. These buckets were supplied with aeration at a rate of approximately 200 mLs-1 through two 5
69 Monal Lal MSc THESIS: CHAPTER TWO ______mm diameter air lines, at the ends of which were fitted 40 mm corundum air diffusers. Air delivery was regulated by individual plastic screw valves installed on each air line. The water in each bucket was exchanged daily, which also served to depurate the bivalves before they were fed to the broodstock. Freshwater mussels were purchased from the Nausori Municipal Market, and the Variegate Venus clams and peanut worms were hand collected from the Nasese mudflats near the USP Lower Campus.
Both the mussel and clam flesh were offered by opening the bivalves and severing the anterior and posterior adductor muscles with a knife to ensure they remained open. The shells were then introduced to the tank and removed when the tank was cleaned after the flesh had been eaten. The wild yam tubers were washed with freshwater to remove dirt and any root fibres that remained attached, before being cut into approximately 1 cm3 pieces which were offered to the broodstock.
The total daily ration (TDR) was set at 5 % of the approximate total biomass of the broodstock maintained in each respective tank, with 20 to 30 % of the TDR being offered to the animals between 0700 and 0800 and the remainder being offered between 1930 and 2100. If the prawns were observed to finish the evening portion of the TDR before 2300, an extra offering of up to 2 % of the approximate total biomass was made. The TDR was typically comprised of approximately 60 % formulated freshwater prawn pellet, and 40 % freshwater clam flesh, which was substituted with either Variegate Venus clam flesh or peanut worms when these were available. These latter feeds were a priority offering for non-ovigerous female broodstock to enhance ovarian maturation and subsequent egg quality.
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2.2.6 Larval rearing
The tanks used for hatching larvae served as the larval rearing tanks (LRTs). For the larviculture run which was successful in producing post-larvae of M. lar, a total of four LRTs were used.
The morning following a hatch event, the ply board and black polyethylene sheets were removed and the fluorescent light units installed over the tank turned on. This was typically carried out by 0800 at the latest. The broodstock were left in the tank for hatch to continue until 36 hours had elapsed since their introduction, after which they were moved to the next tank to be prepared for hatch after being sorted.
In the meantime, an estimate of the number of larvae initially hatched was made and recorded. This was carried out by mixing the tank water gently using a larval counting bowl to distribute the larvae evenly in the tank, scooping 1 L of water from the tank and then counting individual larvae as they were gently poured back into the tank through the spout of the counting bowl. A total of five counts were performed, and the average of these counts was multiplied by the tank volume to determine the number of larvae hatched. Another count was made immediately before the broodstock were removed from the tank, to produce an estimate of the final number of larvae hatched in the tank.
2.2.6.1 Rearing environment management
After the initial hatch estimate had been made, the tank volume was increased by 100 L. This strategy was employed to slowly increase the salinity of the culture medium to match that of full strength seawater, and so the tank volume was increased by 100 L every day until the final culture volume of 800 L was reached.
This approach was developed from the results of the salinity tolerance study (described in chapter 3), which determined the salinity tolerance ranges for the larvae of M. lar,
71 Monal Lal MSc THESIS: CHAPTER TWO ______allowing for the development of a protocol for increasing the salinity of the culture media without negatively affecting larval survival and growth rates.
After this final culture volume of 800 L had been reached, 12 to 25 % (~100 to 200 L) of the water was exchanged daily, depending on the requirements of the larvae and the quality of the water in the LRT.
Aeration volume was progressively increased as the larvae developed. Gentle aeration was employed at a rate of approximately 15 – 30 mLs-1 at each airline for early stage larvae viz. zoeae I to IV (see Plates 4.8, 4.13, 4.14, 4.20, 4.21, 4.25 and 4.26 in Chapter 4 for photographs), so as not to damage them by excessive turbulence. This rate was increased to approximately 150 – 200 mLs-1 for mid and late stage larvae (zoeae V to X) in order to keep feed and biofloc particles in suspension where they could be accessed by the larvae. This also prevented circulating matter from settling on the tank floor which would lead to accelerated decomposition and poor water quality.
2.2.6.1.1 Water exchanges
Water to be added to the LRTs was prepared in two dedicated square 1000 L polyethylene tanks, with fresh quantities of water prepared every day. An assessment of the amount of water to be exchanged in each LRT was made after the larvae were examined each morning. If the larvae and microalgal blooms were found to be in good health (Plate 2.19) with the biofloc volume present being sufficient, then between 10 to 15 % of the culture water was exchanged in the LRT. However if the larvae required treatment for epibionts or disease, or if the biofloc volume in the LRT was above optimal levels and/or the microalgal bloom appeared to be too heavy or had ‘crashed’, then a higher volume of water between 30 and 90 % of the LRT volume was exchanged.
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Plate 2.19 LRT filled to operating capacity with healthy microalgal bloom.
Each water preparation tank was aerated at a rate of 330 mLs-1 using a single 25 mm diameter × 150 mm glass-bonded silica diffuser unit, and heated to maintain 28 ± 0.5 -1 DO2 was maintained > 6.5 mgL and pH varied from 7.2 to 7.6. The salinity, temperature and pH of the mixed replacement water were monitored using a salinity, temperature and conductivity meter and pH Meter.
For preparing the replacement water for the LRTs, the required amounts of brown water and green water were pumped out of their respective mass culture tanks using a dedicated portable submersible pump into the mixing tanks through 30 mm diameter flexible food- grade polyethylene hoses. Both the brown and green water were filtered through a 50 μm2 mesh net upon addition to the water preparation tank and also when the mixture was pumped into the LRTs (Plate 2.20). Fresh quantities of replacement water were mixed daily, whenever a water exchange was required in an LRT.
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Plate 2.20 Green water being filtered through a 50 μm2 mesh net into the water preparation tank.
Replacement water for the LRTs was mixed using a set of six combinations at two salinities, depending upon the requirements of each LRT. The target salinity when mixing replacement water for the LRTs was set at either 20 ‰ or 32 ‰. During the first few days of larviculture, the replacement water was mixed to a salinity of 20 ‰, to progressively increase the salinity of the culture water with successive daily water exchanges. Because larvae were hatched at a salinity of 10 ‰, a salinity of ~20 ‰ was attained by around day 7 of culture.
Once this point was reached for an individual LRT, all subsequent water replacements for that tank were mixed to 32 ‰. This raised the salinity to approximately 30 ‰ by day 26 to 30 of culture. This salinity was maintained until post-larvae were produced, after
74 Monal Lal MSc THESIS: CHAPTER TWO ______which point all water exchanges used treated freshwater to progressively reduce the salinity to 0 ‰. The six combinations for mixing replacement water were:
1. Green water + brown water 2. Green water + treated seawater 3. Brown water + treated freshwater 4. Green water + brown water + treated freshwater 5. Green water + brown water + treated seawater 6. Treated freshwater + treated seawater
When sufficient quantities of green and brown water cultures were available for harvest, each LRT was filled using a mixture of only green water and brown water (combination #1). This mixture was found to be critical for encouraging microalgal blooms and biofloc production and maintenance in the LRT. The combination was also used to restart microalgal blooms after a ‘crash’ and to increase biofloc volume.
When insufficient quantities of either brown water or green water were available for harvest, combinations #2 and #3 respectively were used. In these cases, treated freshwater or seawater was substituted for the microalgae. Combination #6 was also used in this situation, when epibiont infestation and/or disease indications were seen on the larvae or when reducing microalgal and biofloc volumes in the LRT.
Combinations #4 and #5 were primarily used for adjustment of the salinity and/or temperature of the mixed water, if these were not at their target levels by the time the required volume of water had been mixed.
LRTs were drained by siphoning water from near the bottom of the tank using 195 mm diameter plastic drainage funnels to which 30 mm diameter flexible food-grade polyethylene hoses were attached (Plate 2.21). The drainage funnels were fitted with nylon mesh of an appropriate size to retain larvae in the LRT whilst removing uneaten food particles and other unwanted organic matter. The mesh size was changed as the
75 Monal Lal MSc THESIS: CHAPTER TWO ______larvae grew larger in size. A mesh size of 100 μm2 was used from hatch until larvae had reached zoeal stage IV, 500 μm2 was used from stage IV to IX and 1 mm2 from stage IX to PL1. Usually two drainage funnels were used simultaneously to shorten the amount of time taken to perform a water exchange, especially when the 100 μm2 mesh was required to be used.
Plate 2.21 LRT being drained during a water exchange. Photo: Tomohiro Imamura
2.2.6.1.2 Biofloc management
A critical component of the Rua-Cell System is the formation and maintenance of biofloc in the LRT, which serves as live feed for the larvae and also has secondary benefits such as stabilization of water parameters including alkalinity, total hardness, pH and NH4+,
NO2, NO3 and NH3 concentrations, enhancing feed efficiency through microbial recycling and probiotic effects (Avnimelech, 2009).
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The primary source of biofloc in the LRTs was the addition of biofloc found to have formed in both the green water and brown water cultures. As culture progressed however, the majority of the biofloc particles present were those which formed naturally from coalescing microalgal cells encouraged to grow in the LRT.
Healthy microalgal blooms in the LRT maintained a fairly consistent volume of biofloc in the tanks. However when these blooms crashed, the decomposing microalgal cells collecting on the tank floor started to cause water quality problems and outbreaks of protozoan and epibiont infestations which affected larval health.
To counter this problem, each LRT floor was siphoned once or twice daily using a length of 110 cm PVC pipe with a diameter of 21 mm attached to a length of flexible polyethylene hose to remove dead larvae, uneaten food particles and biofloc that had sunk to the bottom. Aeration was ceased for 5 minutes prior to the tank floor being siphoned to allow for settlement of the particulate matter to be removed. Aeration was recommenced immediately after siphoning was completed.
When insufficient biofloc volumes were seen in the LRT, fresh biofloc was added to the tanks from both the green water and brown water cultures. The mixture of replacement water during water exchanges was also changed to encourage microalgal blooms in the LRT which would lead to biofloc production, viz. the use of green water and brown water only to exchange water for the affected tank. The maintenance of sufficient aeration volumes in the LRT was found to be important in keeping the biofloc suspended in the water column where it could be easily accessible to the larvae. Aeration volumes were initially set at approximately 15 – 30 mLs-1 for each airline, but increased to 100 mLs-1 and 300 mLs-1 when larvae had developed to the zoea V and VIII stages respectively to cater for this.
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2.2.6.2 Feeds and feeding
2.2.6.2.1 Prepared feeds
Larvae were fed a number of prepared feeds, which included three types of cooked custard feeds and Algamac 3050 flake (Aquafauna Bio-Marine).
2.2.6.2.1.1 Custard feeds
The prepared custard feeds were made as and when required with ingredients that were as fresh as possible. The refrigerated (~5 approximately 3 days, and therefore small amounts of these feeds were prepared to avoid spoilage and to ensure that larvae were offered fresh, high quality feeds.
Whole Boston Squid Loligo pealei were purchased from a wholesale seafood company (Lund’s Seafood Inc.). After removal of the pen and beak, entire individual squid were used for preparation of the squid custard feed. A key ingredient for preparation of the squid and shrimp custard feeds was a collection of freshwater and estuarine prawns known locally in the Fijian language as moci. The majority of these prawns are from the family Palaemonidae, with the dominant species being Palaemon concinnus, P. debilis, Macrobrachium grandimanus and M. equidens (Richards et al., 1994). These were purchased from the Nausori Municipal Market.
The ingredients for all of the prepared custard feeds are listed below in Table 2.1. To prepare each feed, all the ingredients requiring weighing were individually weighed, combined with the remaining ingredients and homogenized using a Philips HL1643 mixer-grinder. Following this, the mixture was steamed until cooked in a stainless steel bowl using a Sunbeam FP 5910 2400 W electric frying pan half-filled with water. For further details on custard feed preparation refer to Imamura et al., (2009).
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Table 2.1 Prepared custard feed ingredients modified from Imamura et al. (2009). a EPA 1000 mg capsules include 180 mg EPA and 120 mg DHA. b Refer to Appendix 2.2 for the composition of this ingredient. Egg Custard Squid Custard Shrimp Custard 1 egg yolk 25 g whole squid flesh 25 g whole prawns (approx. 20 g in weight) (including viscera) 20 g milk powder 25 g whole prawns 1 EPA capsule a (1000 mg) 15 g water 1 whole egg 1 Multi-vitamin capsule 1 EPA capsule a 1 egg yolk 10 mL of soyabean (1000 mg) (approx. 20 g in weight) cooking oil 1 Multi-vitamin capsule 1 EPA capsule a (1000 mg) 15 g Algamac 3050 flake
10 mL of soybean 1 Multi-vitamin capsule b cooking oil 10 mL of soybean cooking oil
1 lecithin capsule (1200 mg)
When the prepared custard feeds were offered to the M. lar larvae, they were first weighed individually according to the daily feed ration for each LRT, and then screened through a series of 15 cm diameter stainless steel feeding sieves into the water (Plate 2.22).
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Plate 2.22 Egg custard feed being screened through a 150 μm2 feeding mesh. Photo: Tomohiro Imamura
The feeding sieve mesh sizes were correlated to the size and development stage of the larvae in individual LRTs, to ensure that larvae would be able to capture and feed on particles of an appropriate size in relation to their body and mouth size. The feeding sieve mesh sizes used are detailed in Table 2.2
Table 2.2 Feeding sieve mesh sizes used for particular larval stages. Feeding sieve number Feeding sieve mesh size Used for larval stages (μm2) 1 150 zoea I to zoea III 2 400 zoea II to zoea V 3 750 zoea VI to zoea X 4 1000 zoea X to PL1+
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2.2.6.2.1.2 Algamac 3050 flake
This product is drum-dried Schizochytrium sp. algae in 1.5 mm coarse particle flake form. Algamac 3050 was used to supplement the prepared custard feeds as well as enrich Artemia na 7 '/ 8 9 ((:%2/ docosahexaenoic acid (DHA), which is known to be beneficial for crustacean larvae (Aquafauna, 2007).
The Algamac 3050 flake was weighed according to the daily feed ration for each LRT and either screened through the appropriately sized feeding mesh for zoeae I to IX larvae, or added directly to the water after larvae had developed past zoea X.
2.2.6.2.2 Artemia nauplii and meta-nauplii
Great Salt Lake (GSL) Brine Shrimp Artemia sp. cysts (INVE Aquaculture Inc.) were hatched and the resulting nauplii and meta-nauplii used as the primary live feed offered to the larvae of M. lar. The Artemia cysts were hatched in two 250 L dedicated cylindro- conical fibreglass hatch tanks. These tanks were operated alternately, with one tank being drained of hatched nauplii on a given day while at the same time fresh cysts were added to the other tank, which would become ready for harvest the following day.
Each hatch tank was vigorously aerated using five open-ended 5 mm diameter air lines at a rate of approximately 0.5 Ls-1 at each air line, which was individually weighed down with a cleaned stone weight. Both tanks were heated with Aqua One 300 W glass tube immersion heaters to maintain a temperature of 32 ± 1 used to cover the tank during the hatch process.
Treated seawater was used for hatching the nauplii, at a salinity of 30 ± 2 ‰, pH of 7.8 ± -1 0.2 and DO2 > 6.5 mgL . All seawater added to the hatch tanks was filtered through a 50 μm2 mesh net and chelated by adding EDTA at the rate of 0.005 gL-1.
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The required amount of cysts to be hatched for all operational LRTs for a given day was weighed into a 500 mL plastic beaker. The beaker was then filled with treated freshwater and the cysts left to hydrate for approximately 20 minutes. 6 mL of Chlorox bleach solution (containing at least 5 % available chlorine) was then added for disinfection and the beaker left for a further 5 to 10 minutes. The cysts were then washed under a running tap in a 100 μm2 mesh net until the odour of any residual chlorine was removed. The cysts were not decapsulated prior to addition to the hatch tank, and hatched at a density of up to 0.075 g of cysts per L in the absence of illumination for a period of up to 28 hours.
Nauplii were harvested by draining the tank into a 100 μm2 mesh net. Care was taken to exclude unhatched cysts and remnant cyst ‘shells’ (from hatched nauplii) from the net by draining the hatch tank slowly and also removing aeration from the tank for approximately 5 minutes prior to harvest. This allowed the hatched cyst shells to float and settle at the surface of the water where care could be taken to separate them from the nauplii being drained from the bottom of the tank.
After harvesting the nauplii, the hatch tank was thoroughly scrubbed using a clean sponge, washed with a bleach solution (containing at least 5 % available chlorine) and left to dry for at least 9 hours prior to being refilled.
After harvest the nauplii were transferred to an 80 L cylindro-conical FRP enrichment tank. This tank was painted black but had a clear region around the conical portion at the bottom tapered towards the drain valve. The enrichment tank was left unheated, and aerated using three 5 mm diameter air lines fitted with 4 cm corundum air diffusers at a rate of approximately 40 Ls-1 each. Air delivery was regulated by individual plastic screw valves installed on each air line. A circular fibreglass board was used to cover the tank during the enrichment process.
Nauplii were enriched with either freshwater microalgae or Algamac 3050 on alternate days for up to 6 hours post-harvest. Freshwater microalgae (Desmodesmus sp. and Chlorella sp.) were added by introducing green water filtered through a 50 μm2 mesh net
82 Monal Lal MSc THESIS: CHAPTER TWO ______to make up 25 % of the tank volume. The remainder of the tank volume consisted of treated seawater.
For enrichment using Algamac 3050, 0.2 g of Algamac 3050 flake particles per L of enrichment tank water were transferred to the mixing jar of a mixer-grinder with 1 L of treated freshwater. The mixer was then pulsed on its highest setting for approximately 5 minutes to create a suspension of individual Schizochytrium sp. cells between 6 to 8 μm in size. This suspension was then added to the nauplii enrichment tank.
Nauplii were harvested after enrichment using the same procedure as described earlier for harvesting from the hatch tanks. The nauplii were transferred to a clean 2 L plastic jug filled with treated seawater from where they were introduced to the LRTs. Varying amounts of nauplii were added to individual LRTs to maintain a density of 2.5 ind./mL.
Hatched Artemia when offered to the larvae after enrichment were up to 10 hours old, and were a mixture of instar I (~ 500 μm in size) nauplii and instar II (~ 600 μm in size) meta-nauplii. These were offered to all zoeal stages of M. lar larvae. From zoea X onwards, larvae were also offered larger Artemia meta-nauplii. The required numbers of enriched nauplii to be grown on to meta-nauplii were transferred to a 3 L plastic beaker filled with treated seawater. The beaker was aerated at approximately 30 mLs-1 using a 5 mm diameter air line. Air delivery was regulated by a plastic screw valve installed on the air line. The Artemia were fed green water and Algamac 3050 suspension, and cultured for approximately 36 hours before they were harvested and added to the LRTs, by which time they had developed into instar III and IV meta-nauplii and were between 800 to 900 μm in size.
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2.2.6.2.3 Feeding rations and schedules
Larvae were fed at 2-hourly intervals, from 0700 to 1900 daily. The custard feeds were offered in an alternating pattern as outlined in Figure 2.2, with Artemia being offered once daily in the afternoon. The Artemia was fed last as being a non-polluting feed whilst alive, it would sustain the larvae overnight without rapidly degrading the LRT water quality as the custard feeds would. When larvae had developed into the thirteenth zoeal stage, crushed formulated prawn pellet (32 % crude protein level, Crest Chicken Limited, Fiji) was offered to complement the custard feeds.
Feeding intervals Larval 0700 0900 1100 1300 1500 1700 1900 Feed EC SC ShC AM Art./mArt. CFPP Figure 2.2 Daily feed regime. EC = egg custard, SC = squid custard, ShC = shrimp custard, AM = Algamac 3050, Art. = Artemia nauplii, mArt. = Artemia meta-nauplii and CFPP = commercial formulated prawn pellet.
The LRTs were continuously illuminated, and larvae were observed to feed around the clock. Each LRT was stirred prior to each feed offering by running a 150 cm length of PVC pipe with a diameter of 40 mm along the floor of the tank. The end of the pipe in contact with the tank bottom was flattened into a paddle-like shape. This served to bring up particulate matter (mostly settled biofloc and uneaten feed particles) into the water column, and was done so that a visual estimate of uneaten feed remaining in the LRT could be made.
Larvae were fed ad lib., however, an effort was made to maintain adequate concentrations of feed in the LRT at all times so that the larvae did not starve. Feed rations at the fixed feeding intervals were largely determined by observation of the larvae. If the majority of the larvae were seen to be holding onto feed particles introduced earlier and there was
84 Monal Lal MSc THESIS: CHAPTER TWO ______residual feed in the LRT, then no additional feed was offered. Conversely, if there was no residual feed apparent and the larvae actively swam towards and grasped new feed introduced to the LRT, then the ration for that particular feed interval was added to the tank.
A general ratio followed when determining the feed ration for a particular feed interval was 1 g feed: 4000 individual larvae. This was doubled when larvae had developed past the fifth zoeal stage. Larvae were offered different feeds as they progressed through their development stages. A regime followed for feeding the larvae of M. lar is shown in Figure 2.3
Macrobrachium lar larval development stage Larval Zoea Zoea Zoea Zoea Zoea Zoea Zoea Feed I II III IV V VI VII Age 1-3 3-7 7-9 9-11 11-16 16-20 20-23 (days) EC Start SC Start ShC Start AM Start Art. Start
Larval Zoea Zoea Zoea Zoea Zoea Zoea PL1+ Feed VIII IX X XI XII XIII Age 23–26 26–31 31-39 39-45 45-48 48-77 77-110 (days) EC End SC End ShC End AM End Art. End mArt. Start End CFPP Start Figure 2.3 Larval feeding schedule. EC = egg custard, SC = squid custard, ShC = shrimp custard, AM = Algamac 3050, Art. = Artemia nauplii, mArt. = Artemia meta-nauplii and CFPP = commercial formulated prawn pellet.
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2.2.6.2.4 Biofloc and other live feeds
Larvae were likely to have consumed a number of other live feed items apart from Artemia nauplii and meta-nauplii. The majority of these live feed items comprised of biofloc, and biofloc-associated micro-organisms. A number of the biofloc-associated micro-organisms included various types of rotifers; the most abundant of which was a Colurella sp. (Fam.: Brachionidae) shown in Plate 2.22, along with various nematodes and protozoans.
Plate 2.22 Colurella sp. rotifers.
It proved to be fairly difficult to quantify biofloc volume in the LRT, however a general guide established was to maintain a concentration of between 1500 to 2500 pieces of biofloc per L. This proved to be an optimal density where larvae were easily able to locate and feed on it based on qualitative observations of larval feeding behaviour. A
86 Monal Lal MSc THESIS: CHAPTER TWO ______single ‘piece’ of biofloc was loosely defined as any aggregation of biofloc material up to 5 mm in size.
At times between scheduled feeding intervals if the larvae were observed to have consumed all feed from the previous offering, they were encouraged to feed on biofloc present in the LRT by stirring the tank floor with the PVC pipe paddle described in the previous section.
2.2.6.2.5 LRT water management
Due to the relatively large feed volume in circulation in the LRTs, special attention was required to be paid to water quality. Periodic checks were made on water quality at least twice daily, to ensure that optimal culture conditions were maintained. Particular attention was paid to water turbidity, biofloc volume and the amounts of uneaten feed in the LRT. When deteriorating water quality indicators were observed, water exchanges were performed in the affected LRTs.
The accumulation of unhatched Artemia cysts presented a particular problem, as they are known to harbour pathogenic bacteria and provide attachment surfaces for parasitic protozoans such as Epistylis spp., Zoothamnium spp. and Vorticella spp.. This problem was most obvious when larvae were developing from zoeal stages II to IV, as a 100 μm2 mesh was used to drain the LRTs during this period which retained any unhatched Artemia cysts in the tank. The problem was compounded by the fact that in order to maintain live microalgae and biofloc in the LRTs, large volume water exchanges were not performed if the larvae were in good health.
The issue was resolved in two ways. The first solution was used when larvae developed past zoea V and were large enough to allow a 500 μm2 mesh to be used. Here the cysts were able to be drained out of the LRT during routine water exchanges. The second and more immediate solution was to use a 300 μm2 mesh fitted onto two drainage funnels.
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The hoses attached to these funnels were directed into an 80 L plastic tub, from which a submersible pump returned water to the LRT through a 50 μm2 mesh net as shown in Plate 2.23. This was continued for approximately 15 minutes at a time as required. The procedure proved to be effective in removing the majority of unhatched Artemia cysts from the LRT, as well as excessive biofloc or uneaten feed.
Plate 2.23 Removal of Artemia cysts from an LRT. Photo: Tomohiro Imamura
2.2.6.3 Larval health management
The primary disease problems observed on M. lar larvae were epibiont protozoans such as Epistylis, Zoothamnium and Vorticella spp. along with melanised areas on various parts of the exoskeleton which were thought to be sites of infection by Vibrio-like bacteria.
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The method of treatment employed for all conditions was the same. For severe epibiont infestations or bacterial infection where a significant proportion of the larval population in an LRT was affected, 30 mL of 40 % formalin per 1000 L tank volume was added after the tank had been drained down to the 200 L mark. After 5 – 10 minutes, the LRT was re- filled with the appropriate mixture of treated seawater and treated freshwater (without any brown water or green water phytoplankton). When larvae were seen to improve, phytoplankton were reintroduced to the LRT. Treatment was continued until the majority of larvae sampled from the affected LRT were symptom free, which usually occurred between 2 to 4 days post-treatment.
For less severe infestations or infections, 20 mL of Treflan (C13H16F3N3O4) per 1000 L tank volume was used on the first day of treatment, which effected a concentration of approximately 0.05 ppm when the LRT was re-filled. The dosage was reduced to 10 mL per 1000 L for the second and subsequent days of treatment if this was required.
2.2.6.4 Larval observations
Observations on the health and activity of the larvae were regularly carried out between 0800 and 0900 every morning prior to water exchange in the LRTs. The criteria used in the observations are detailed overleaf in Figure 2.4 and Table 2.3 which contain a condition index evaluation system used for M. rosenbergii larvae.
Microscopy was also carried out on the larvae themselves to assess development stage (this is addressed in Chapter 4), on dead larvae to attempt to ascertain the cause of death and on “bottom matter” siphoned off the floor of the LRTs which contained a mixture of biofloc, uneaten feed, dead larvae, larval exuviae and other organic matter. Examination of bottom matter was carried out mainly to determine the levels of epibionts present. If heavy concentrations of epibionts were found in a particular LRT, then water exchange volumes were increased and/or Treflan treatment commenced until concentrations were seen to decrease.
89 Monal Lal MSc THESIS: CHAPTER TWO ______90
Figure 2.4 Visual criteria used for determining condition index in evaluating M. rosenbergii larval quality. Source: after Tayamen & Brown (1999) cited in Valenti et al. (2010) 90 Monal Lal MSc THESIS: CHAPTER TWO ______
Table 2.3 Criteria used for determining condition index in evaluating M. rosenbergii larval quality. Source: after Tayamen & Brown (1999) cited in Valenti et al. (2010) 91
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2.3 Results
2.3.1 Broodstock
2.3.1.1 Broodstock maintenance
M. lar broodstock were found to be fairly easy to maintain in captivity. It was found that relatively high stocking densities of up to 40 – 50 individuals per m2 in a single tank could be maintained for extended periods of time, provided that PVC pipe shelters along with ample feed were present. Shelters were necessary to avoid newly moulted individuals being cannibalised by other hard-shelled prawns.
The average body weight (ABW) for males and females was 34.0 and 17.5 g respectively. Average total length (TL) was 12.5 and 10.1 cm, and average carapace length was 5.5 and 4.2 cm for males and females respectively. Average cheliped length measured from the base of the coxa to the tip of the dactylus was 15.0 cm for males.
It was found that maintaining more than two large males in a tank with a floor area of 1 m2 resulted in fights for dominance and the death of all the smaller males until only one or two of the largest specimens remained. For this reason, no more than two to four males were collected for mating with spent females.
Ovigerous females whose larvae had hatched during the larval rearing trials when returned to the broodstock holding tanks, were found to come into berried condition within 2 – 3 weeks when a male was present. Because the clutch sizes produced by these matings and the resulting larvae were smaller than those from wild caught females (possibly due to broodstock nutrition among other factors), further investigation of captive maturation is warranted.
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2.3.1.2 Larval hatch
During early trials, it was found that some females released the majority of their larvae within a few hours of being placed in the hatch tank, while others retained larvae for up to 2 weeks before they became spent. This asynchronous hatching presented a problem for larval rearing as a single LRT would contain larvae of mixed ages and developmental stages. At any given point in time, up to three different developmental stages were observed in the LRTs, which meant that the feeding, aeration and water exchange regimes had to be altered to cater for all these developmental stages which have different requirements. This problem was countered somewhat by holding all ready to hatch broodstock in one LRT for up to a maximum of 3 days to build up the larval population, before removing all spent females and transferring the remainder which were still berried to another LRT to continue hatching of larvae. Any females which had become ready to hatch in the meantime in the broodstock holding tanks were also transferred to this ‘new’ LRT.
It was unclear as to why this occurred, and further investigation is required to identify the causative mechanisms. For this reason, at least 10 or 12 females were required for hatching in a single LRT in order to obtain sufficient numbers of larvae for rearing, as larval survival was found to be extremely low (see section 2.3.3.2 on larval rearing trials). When at least 10 females were transferred to a LRT for spawning, only 2 to 3 females underwent a complete hatch while the remainder partially spawned.
Increased mortality of female broodstock was observed when they were held in brackish water for more than 5 to 7 days prior to hatch. Mortality was virtually nil when females were held in freshwater until transfer to the hatch tanks 1 – 2 days prior to the expected time of hatch. Despite this, salinity tolerances for females may in fact be quite high, as two females were inadvertently held at 25 ‰ for approximately 12 days as the salinity of the LRT they spawned in was increased from 10 ‰ for rearing larvae. These females managed to escape being removed from the LRT after hatch had been completed, and appeared to suffer no ill effects from exposure to high salinity.
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2.3.1.3 Disease
Adult M. lar maintained in the laboratory were relatively disease free. The only disease problem encountered was areas of melanisation on the shells of affected individuals which appeared to be similar to the Brown Spot/Black Spot Disease which has been described for M. rosenbergii (see Plate 2.24).
Individuals were collected with melanised areas already present on their shells. Routine disinfection with formalin upon arrival at the laboratory was found to improve the condition. Recurrent incidences were found to occur in a few individuals when excessive detritus accumulated in the holding tank; however treatment with formalin as described in section 2.2.4 along with regular water exchanges served to minimize the number and severity of individuals affected.
Plate 2.24 Areas of melanisation on the shell of an M. lar specimen.
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2.3.2 Larval rearing
2.3.2.1 Larval observations
2.3.2.1.1 Growth
Growth in larval M. lar was found to be very similar to that of M. rosenbergii. The larvae were observed to undergo the processes of moulting and ecdysis at certain intervals in their development.
Moulting in the larvae occurred at any time of the day. Prior to undergoing a moult, larvae tended to settle at the bottom of the LRT until their old exoskeleton (exuvium) began to separate from the endocuticle through the process of apolysis. Following this, larvae stretched their abdomen forwards underneath the cephalothorax into an inverted “u” shape. This caused a split in the old exoskeleton along the rear margin of the carapace, and the larva then pulled its abdomen and tail region out of the exuvium. Once this was complete, it began swimming in the water column and pulled its cephalothorax complete with all appendages out of the exuvium. Usually the antennal scales are the last body parts to separate from the exuvium, and larvae were observed swimming in the water column with the exuvium attached to these points.
It was not possible to study the duration of the moult cycle for M. lar larvae, however it probably lasts for several hours from the onset of processes which begin the moult cycle until the newly secreted exoskeleton hardens in place. This was deduced from several larvae collected from a mass culture LRT. One zoea III larva collected with its carapace just beginning to separate along its rear margin was observed at 15 minute intervals under the microscope until it had shed its exuvium (see Plate 2.25), and begun actively swimming around. The observational period lasted 1 hour and 15 minutes.
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Plate 2.25 Recently moulted larva disentangling itself from its exuvium.
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2.3.2.1.2 Behaviour and feeding
M. lar larvae upon hatch swim tail-first and adopt an upside-down position with the telson uppermost. Swimming is achieved by rapidly beating the exopods of the maxillipeds and pereiopods, which are equipped with long, plumose natatory setae that propel the larva through the water. In the later larval stages from zoea X onwards, the pleopods also aid in swimming.
When ascending or descending through the water column the larvae move in a spiralling motion, extending or retracting the antennal scales and uropods for braking or to change direction. If startled, the larvae move rapidly away from the source of the disturbance by flexing the abdomen and using the telson and uropods to drive themselves backwards much like the adults do. When not actively feeding, the larvae tended to adopt a position in the middle of the water column close to the walls or above the floor of the LRT.
Zoea I larvae were found to be positively phototactic upon hatch, however this tendency decreased as they developed, and late-stage larvae were found to avoid sources of light altogether. An interesting observation was that M. lar larvae did not exhibit aggregating behaviour at the surface of the water column as larvae of M. rosenbergii do when aeration is suspended in the LRT.
The larvae of M. lar feed by swimming or floating around until within reach of a food item, which they capture by extending the endopods of the pereiopods and retracting them once the item has been grasped. The pereiopod endopods are equipped with setae which aid in maintaining a grip on the food item, which is then held and manipulated towards the mandibles for consumption.
In mid-stage larvae which have developed to zoea V and beyond, the fifth pair of pereiopods are larger than the other pairs and are held with the distal portions parallel to the carapace when food is captured. This forms a platform of sorts on which feed items are accumulated until they can be eaten. Plates 2.26 and 2.27 show a zoea VIII larva capturing a food item.
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Plate 2.26 Zoea VIII larva reaching out to capture a biofloc particle. Note the faecal trail underneath the telson and uropods.
Plate 2.27 The same larva in Plate 2.26 above shown manipulating the biofloc particle towards the mandibles.
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Larval M. lar are capable of capturing feed items significantly larger than themselves. Larvae were observed on a number of occasions holding onto pieces of custard feed up to three times larger than their carapace size. The larvae were not observed to display any cannibalistic tendencies as has been described for the larvae of M. rosenbergii, although unhatched eggs which remained floating in the water column were found to be eaten by zoea II larvae.
A point of difference noted between the feed preferences of M. lar and M. rosenbergii larvae was that the former showed a strong tendency to consume biofloc particles over Artemia nauplii when both were present in sufficient quantities. All observations of larval feeding behaviour revealed that larvae were seen to consume either the prepared custard feeds or biofloc particles. This issue requires further investigation, but may mean that M. lar larvae are more herbivorous in feed preferences than M. rosenbergii larvae.
Healthy larvae were seen to swim and feed continuously between resting periods and display full foregut, midgut and hindgut regions (Plate 2.28). Larvae sometimes trailed a strand of faeces from the anus equivalent to the length of the abdomen. This was used as an important indicator of larval health.
It was observed that the body colouration of larvae varied with the transparency of the LRT water. Larvae reared in water which had relatively high transparency due to low phytoplankton concentrations had contracted chromatophores and consequently transparent bodies, with the exception of the internal organs and cephalothorax structures. Larvae reared in water which had higher phytoplankton concentrations however, had greatly expanded chromatophores which lent their bodies a pale orange hue with red areas where the chromatophores were concentrated e.g. the zoea X larva shown in Plate 2.28. This is likely to be a camouflage response to avoid predation.
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Plate 2.28 Zoea X larva with full foregut, midgut and hindgut regions.
2.3.2.1.3 Disease
The larvae of M. lar were found to be susceptible to infestations by the protozoan epibionts Epistylis, Zoothamnium and Vorticella spp.. Infestations were found to be most prevalent during periods of high concentrations of organic matter in the LRTs when excessive levels of biofloc had accumulated.
A condition where melanised areas formed on various parts of the exoskeleton was also observed. This was attributed to sites of infection by Vibrio-like bacteria (Pillai et al., 2010), and affected body parts included those which are highly compressed or delicate in structure e.g. setae, spines, pleopod buds, antennae, antennules, antennal scales and pereiopod segments. In severe cases, the infection eroded away portions of larval
100 Monal Lal MSc THESIS: CHAPTER TWO ______appendages, as shown in Plate 2.29 below. Both conditions were able to be treated with applications of either Treflan or formalin to the LRT water.
Plate 2.29 Larval disease indications on a zoea X larva. a = Epistylis sp. epibionts, b = lateral margin of telson eroded from possible bacterial attack and c = bacterial (possibly Vibrio sp.) infection on surface of uropod endopod.
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2.3.2.1.4 Aberrant larvae/deformities
A discussion of “normal” larval morphology and development stages is provided in Chapter 4 of this thesis, and larval deformities or malformations observed in M. lar larvae are described in the following section.
The only incidence of a deformed/aberrant larva observed during the entire study period involved a zoea I larva which possessed a single eye, compressed carapace and bent telson and sixth abdominal somite upon hatching. This larva appeared to be active but swam with an irregular spiralling motion when not resting on the tank floor. A photograph of this larva is included below in Plate 2.30 a along with a photograph of a normal zoea I specimen shown in Plate 2.30 b for comparison.
Plate 2.30 a Deformed zoea I larva. Note the single eye and bent telson.
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Plate 2.30 b Normal zoea I larva.
2.3.2.2 Larval rearing trials
A total of 20 larval rearing trials were carried out during the entire study period at 3 different salinity regimes as determined by a series of salinity tolerance experiments which are described in Chapter 3 of this thesis. Larval performance during these trials is detailed for the 20 ‰, 25 ‰ and 30 ‰ regimes in Tables 2.4, 2.5 and 2.6 respectively. It must be noted that the data presented in the aforementioned tables are quite preliminary in nature.
The 20 ‰ regime trials were replicated in a total of 8 1000 L LRTs, while the 25 ‰ and 30 ‰ trials were replicated in 4 1000 L LRTs each. Only data from each of the most successful replicates (i.e. those with maximal larval survival) during each trial was chosen to be representative of each trial and is presented in Tables 2.4, 2.5 and 2.6. Trial number 20 was the only trial during which post-larvae (PL) were produced, with a total of 5 PL metamorphosing after 77, 78, 85, 101 and 110 days of culture respectively. A graph of larval survivorship for this trial is shown in Figure 2.7. 103 Monal Lal MSc THESIS: CHAPTER TWO ______
Table 2.4 20 ‰ regime trials. * = 100% mortality for this trial shortly after hatch. Trial Duration Total Time to 50 % Maximum Larval Number (Days) number of mortality of salinity development larvae larvae attained stage hatched (Day of culture) (‰) attained 1 32 4,800 ~11 20.9 zoea V 2 22 7,800 ~5 19.8 zoea V 3 6 8,830 ~5 14.3 zoea II 4 8 7,440 ~4 21.2 zoea III 5 25 8,250 ~10 20.5 zoea V 6 35 4,000 ~8 21.2 zoea VII 7 5 8,162 * 19.9 zoea II 8 11 6,840 ~9 20.1 zoea IV 9 6 2,800 * 18.3 zoea II 10 19 2,090 ~13 21.4 zoea V 11 9 22,532 ~7 21.6 zoea IV 12 7 12,800 ~2 21.3 zoea II
Table 2.5 25 ‰ regime trials. * = 100% mortality for this trial. Trial Duration Total Time to 50 % Maximum Larval Number (Days) number of mortality of salinity development larvae larvae attained stage hatched (Day of culture) (‰) attained 13 6 7,516 * 24.2 zoea II 14 9 9,600 ~7 24.5 zoea III 15 25 8,866 ~12 26.0 zoea VIII 16 41 3,500 ~21 26.7 zoea XI
Table 2.6 30 ‰ regime trials. Trial Duration Total Time to 50 % Maximum Larval Number (Days) number of mortality of salinity development larvae larvae attained stage hatched (Day of culture) (‰) attained 17 39 9,396 ~5 30.0 zoea IX 18 11 7,150 ~5 28.5 zoea III 19 12 17,050 ~7 30.5 zoea V 20 110 (65 + 45) 6,000 ~12 31.4 PL1+
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Patterns of growth observed in M. lar larvae were regular until the fifth zoeal stage was reached. Figures 2.5 and 2.6 show the day of first appearance and intermoult durations respectively for all larval developmental stages observed during the 30 ‰ rearing trial which successfully produced PL.
Development through zoeal stages I to IV appears to occur fairly regularly with an average intermoult duration of approximately 4 days (Figure 2.6). From zoeal stages V to VIII, intermoult duration changes to approximately 8 days, but remains regular. A further change is observed from zoeal stages IX to XI, where an average intermoult duration of approximately 12.6 days occurs. From this point onwards, larval development is highly irregular with intermoult durations of 21 and 63 days for zoea XII and XIII respectively.
Metamorphosis into PL by zoea XIII larvae was also prolonged, taking a total of 34 days from the time of metamorphosis of the first till last PL.
85 80 77 75 70 65 60 55 50 48 45 45 39 40 35 31 30 26 25 23 20 20 16 15 11 9 10 7 Day of first appearance of larval stage larval appearance of Day first of 3 5 1 0 Z 1Z 2Z 3Z 4Z 5Z 6Z 7Z 8Z 9Z 10Z 11Z 12Z 13PL1 Larval Development Stage
Figure 2.5 Graph of 30 ‰ trial number 20 showing the day of first appearance of larval developmental stage.
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70 65 63 60 55 50 45 40 35 34 30 25 21
Larval stage duration stage Larval 20 14 13 15 11 10 9 888 5 5 3 3 4 0 Z 1 Z 2 Z 3 Z 4 Z 5 Z 6 Z 7 Z 8 Z 9 Z 10 Z 11 Z 12 Z 13 PL1
Larval Development Stage
Figure 2.6 Graph of 30 ‰ trial number 20 showing larval stage intermoult durations.
Larval mortality proved to be very high throughout all trials but exceptionally so during the first few days of culture, with 50 % of the total larval population in the LRTs dying before day 14 of culture. The one exception was trial number 16 in the 25 ‰ regime, which reached day 21 before this occurred. A similar trend was observed during trial number 20 as shown in Figure 2.7. The overall survival rate from hatch till metamorphosis was 0.08 %.
The most advanced larval stages reached during the 20 ‰ and 25 ‰ regime trials were zoea VII and zoea XI respectively. Although larval development progressed farther under the 25 ‰ regime, it is interesting to note that larval survival was prolonged during some 20 ‰ trials and similar to the most successful 25 ‰ regime trials whereby larvae survived for more than 20 days.
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Population Expon. (Population) 6400 6000 6000 5600 ) 5200 4800 4400 4000 3600 3500 3200 2800 2400
Larval Population 2000 2550 1540 1600 1200 1200 (Average number of individuals 800 1050 420 400 50 500 10 8 5 5 0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 Day of Culture Figure 2.7 Graph of larval survivorship during 30 ‰ regime trial number 20. The increase in larval population over the period from days 1 to 6 is due to continued input of larvae from spawning broodstock. Broodstock were removed on day 6.
2.3.2.3 Larval rearing salinity and temperature
Particular attention was paid to culture medium salinity and temperature during the larval rearing trials. Trends for these two parameters during trial number 20 which successfully produced PL are shown in Figures 2.8 a and b overleaf and described below.
Through gradual salinity changes which were made starting from 11.2 ‰ at hatch, salinities above 28 ‰ were achieved by day 13 and remained above this level for the duration of the trial until PL were produced. Culture medium temperatures remained fairly stable between 28 ± 0.8 ºC for the duration of the trial.
Due to very poor larval survival during trial number 20 with only ten larvae remaining alive by day 65, it became impractical to rear the surviving larvae in the 1000 L LRT. This tank was consequently drained and the survivors transferred to a 60 L cylindro- conical fibreglass LRT and the trial continued.
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After the first PL1 was observed on day 77, a reduction in salinity from 30 ‰ to 24.3 ‰ was carried out with the addition of treated freshwater over days 80 and 81 with the view of encouraging metamorphosis in the remaining larvae. From this point on, further gradual reductions in salinity were carried out until salinities of 1.4 ‰ and 0 ‰ were reached by day 95 and 96 respectively.
The first PL1 produced was removed and transferred to a separate 60 L cylindro-conical fibreglass LRT. The salinity and temperature in this LRT were maintained at 28.8 ‰ and 28 ± 0.5 respectively for a period of 24 hours, before salinity was reduced in 5 ‰ steps each day by exchanging 20 – 25 % of the LRT volume until 0 ‰ was reached.
This procedure was also carried out for the next three PL1 collected from the LRT. For the last PL1 collected, an attempt was made to gauge freshwater tolerance of M. lar PL shortly after metamorphosis. The PL1 was transferred to another 60 L cylindro-conical fibreglass LRT and the salinity of the culture medium reduced from 28.3 ‰ to 0 ‰ over 24 hours. This was achieved by replacing between 30 and 50 % of the water in the LRT at 6 hourly intervals with treated freshwater. The PL1 did not appear to show any ill- effects from this rapid salinity reduction and moulted into the PL2 instar 6 days later.
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Salinity (‰) Temperature ( 33 28.4 32 31 30 28.3 29 28 27 26 28.2 25 24 23 28.1 22 21 20 28.0 19 18 17
109 27.9 16 15
Salinity (‰) 14 27.8 13 ( Temperature 12 11 10 27.7 9 8 7 27.6 6 5 4 3 27.5 2 1 0 27.4 1 3 5 7 9 11131517192123252729313335373941434547495153555759616365 Day of Culture Figure 2.8 a Graph of 30 ‰ trial number 20 salinity and temperature trends during days 1 to 65. Monal Lal MSc THESIS: CHAPTER TWO ______
Salinity (‰) Temperature ( 33 29 32 31 30 28.8 29 28 27 28.6 26 25 24 28.4 23 22 21 28.2
110 20 19 18 28 17 16 15 27.8
Salinity (‰) 14 13 12 27.6 ( Temperature 11 10 9 27.4 8 7 6 27.2 5 4 3 27 2 1 0 26.8 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 Day of Culture Figure 2.8 b Graph of 30 ‰ trial number 20 salinity and temperature trends during days 66 to 96. Monal Lal MSc THESIS: CHAPTER TWO ______
2.4 Discussion
2.4.1 Broodstock maintenance
2.4.1.1 Stocking density for holding broodstock
It is apparent that M. lar broodstock may be held at relatively high densities in captivity when provided with artificial substrates. The densities used during this study of 40 – 50 individuals per m2 are at the higher end of the spectrum of densities recommended for M. rosenbergii broodstock. Daniels et al. (2010) recommend 1 individual per 20 – 60 L for M. rosenbergii, which is the equivalent of 16 – 50 individuals per m2 when considering a 1000 L tank with a floor area of 1 m2, while Nandlal and Pickering (2006b) recommend holding 2 individuals per m2.
M. lar displays gregarious behaviour in the wild (Pickering, unpubl.), and this aspect of its biology implies that fewer resources in terms of numbers of tanks etc. may be required for maintaining a given number of broodstock if larviculture is found to be commercially feasible.
There is scope for further research into broodstock management and maintenance of M. lar, as comparatively little is known about issues such as sexual maturation and fecundity in captivity, appropriate stocking densities and sex ratios, social hierarchical interactions and optimal broodstock nutrition among others for this species.
2.4.1.2 Larval hatch
It appears to be important to maintain hatch tanks for M. lar under very dimly lit or totally dark conditions; because in the wild, larval hatch is primarily confined to evenings. Kubota (1972) reported that peak catches of M. lar larvae in a Hawaiian stream occurred between 1900 and 2300. Nandlal (2010) reported similar findings and that females were often disturbed when lights over tanks were switched on at night, although he mentions that in some cases larvae hatched during daylight hours.
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The problems with asynchronous and prolonged hatching by ovigerous females encountered during this study require further investigation. One aspect of this is to determine how much control female M. lar are able to exert on the period over which their larvae hatch, and the causative mechanisms if any exist.
Atkinson (1973) reports that females may delay hatching by decreasing or stopping the rate of beating of the pleopods if unsatisfactory conditions prevail at the time the larvae are ready to hatch. These conditions include those of temperature (above 26 unsuitable illumination i.e. constant dark or constant light. If these conditions persist, the female may only release 200 to 300 larvae each day or under extreme circumstances eat the eggs.
If this observation is correct, then the problem of prolonged hatch may be attributed to a larval dispersal and survival adaptation of this species whereby larvae are hatched in a staggered fashion to ensure maximum survival. This would be important in the event that if a larval cohort hatches all at once, they may encounter unfavourable environmental conditions or be heavily preyed upon as they drift out to sea.
Another possibility is that unfavourable conditions were provided in the hatch tanks which were unsuitable for the larvae themselves, and it was this which prolonged the period during which hatch occurred. This will require investigation of the physical parameters involved which need to be within their optimal ranges for large numbers of larvae to hatch.
One such parameter may be the pH of the hatch tank water. Law et al. (2002) investigated the effect of M. rosenbergii egg hatchability at various pH levels and found that at pH levels of 6.5 and 7.5, hatching rates drastically dropped to 5.00 ± 3.50 % and 13.33 ± 2.98 % respectively from 92.22 ± 1.72 % at a pH of 7.0. All other pH levels above or below this range resulted in hatch rates of zero. Monitoring of the hatch tank water showed that pH did not vary considerably from a level of 7.8; nonetheless it was not possible to control pH during this study. Another factor may have been salinity, as females were held at 10 ‰ during hatch to facilitate increasing culture medium salinity
112 Monal Lal MSc THESIS: CHAPTER TWO ______quickly. Yen and Bart (2008) and Soundarapandian et al. (2009) demonstrated that female M. rosenbergii broodstock reproduced earlier and produced more offspring when held at lower salinities. Conversely, larval hatch of M. rosenbergii has been observed to be prolonged at 0 ‰, and much more synchronous at 12 ‰ (Singh, 2011).
Atkinson (1973) describes a method for stripping the eggs of ovigerous females and maintaining the egg mass in a flask of freshwater to counter this problem, however attempts to do the same during this study failed with very few larvae hatching and the majority of the embryos contained within the stripped eggs dying.
Other factors affecting time and duration of hatch in crustaceans that have been identified include salinity (Valenti et al., 2010; Willführ-Nast, et al., 1993; Ching & Velez Jr., 1985; Ngoc-Ho, 1976), temperature (Manush et al., 2006; Brillon et al., 2005; Mossolin & Bueno, 2002; G. G. Smith et al., 2002; Gomez Diaz & Kasahara, 1987), agitation of the egg mass by the female (Katre & Pandian, 1972) and photoperiod (Katre & Pandian, 1972).
The numbers of larvae hatched were relatively low, with most trials being run with an average of less than 10,000 larvae at their commencement. The low total larval output by the females may have been due to their size, as it has been established that there is a clear relationship between female size and the number of eggs she is able to brood (Daniels et al., 2010; Anger, 2001; Anger & Moreira, 1998; Shakuntala, 1977); although this is doubtful. Ovigerous females collected during the study had an average carapace length of 4.2 cm, which would imply the number of eggs they were able to carry would be at the higher end of the range of 924 – 38,800 eggs that Kubota (1972) reports for the species.
113 Monal Lal MSc THESIS: CHAPTER TWO ______
2.4.1.3 Disease
Although the “black spot” disease concern manifested by areas of melanisation on the shells of some animals did not present a problem during this study, it may be an issue for further work involving maintaining adult M. lar in captivity for extended periods of time.
Kubota (1972) mentions that 17.4 % of all animals sampled in the wild during his study in a Hawaiian stream displayed symptoms, and that maintaining prawns at high densities under high temperatures and in water of poor quality may exacerbate the condition.
He also mentions that the incidence of the disease is higher in larger and older specimens, possibly being related to the inability of these individuals to clean themselves regularly. Juvenile specimens did not appear to be affected as much, as they moulted more frequently than older specimens.
While over the short term, maintaining broodstock under conditions of appropriate hygiene should ensure that they remain disease free, further investigation of this condition is warranted over the long term to identify causative mechanisms and methods of prevention and treatment.
114 Monal Lal MSc THESIS: CHAPTER TWO ______
2.4.2 Larval rearing
2.4.2.1 Larval observations
Although the larvae of M. lar were found to be similar to the larvae of other Macrobrachium spp. including M. rosenbergii in many respects, there were also areas where there were important differences. These areas of difference include different patterns of larval behaviour, growth and feed preferences. The latter two are discussed in more detail in the next section 2.4.2.2 on the larval rearing trials, while the patterns of larval behaviour are discussed here.
The larvae of M. lar displayed a more benthic habit in the LRTs (even with the presence of aeration) than that which has been observed for M. rosenbergii, where healthy larvae are active and in the absence of aeration remain near the water surface (Valenti et al., 2010). This agrees somewhat with observations by Atkinson (1973), who mentions that larvae occupied the upper portion of the water column. Reasons which may account for this include predator avoidance and the use of sub-surface currents for larval transport out of coastal waters during pelagic larval dispersal.
This behavioural trait may be important in future larviculture efforts with this species, especially for providing feed for the larvae where they are able to most easily access it. Development of a semi-buoyant feed which maintains a position in the water column just below the surface is one area that can be investigated.
Cannibalism where live individuals were consumed was also not observed during this study, whereas this has been documented for M. rosenbergii larvae (Valenti et al., 2010). This differs with the observations of Nandlal (2010) who reports that M. lar larvae did display cannibalistic tendencies and attacked and consumed weak or dead larvae. It is possible that differences in nutrition between the two studies may be an explanation.
115 Monal Lal MSc THESIS: CHAPTER TWO ______
2.4.2.2 Larval rearing trials
2.4.2.2.1 Survival
The overall survival rate from hatch till metamorphosis during the trial which successfully produced PL was very low, at 0.08 %. Unfortunately, due to time, budgetary and resource constraints, further trials were not able to be carried out to determine whether this rate could be improved upon.
Larval survival rates are in all likelihood very low in the wild, on the order of < 0.1 % (Jennings et al., 2006; Bagenal, 1967); and depend on a variety of factors including temperature, salinity, food availability and development/settlement cues (Anger, 2001; Willführ-Nast et al., 1993).
For crustacean species that are being investigated for their culture potential and for which optimal larviculture techniques are still being developed, larval survival rates in early trials are for the most part not much better than those inferred for wild larvae. As an example, initial larviculture research on the Mud Crab Scylla sp. in Indonesia produced survival rates till metamorphosis of 0.07 – 0.19 % and 0.5 – 3.2 % (Cholik, 1999).
Larval survival rates that have been reported for other Macrobrachium spp. have also been comparatively low during initial attempts at larviculture, but have improved with continued refinement of culture techniques. Arguably the best example of this is the larviculture of M. rosenbergii. When post-larvae for this species were first produced in the laboratory, the larval survival rate till metamorphosis was reported to be 16 – 17 % (Ling, 1962, 1961). Today, survival rates are between 40 – 50 % in flow-through hatchery systems, 60 – 80 % in Thai backyard hatcheries and between 60, 75 and 80 % in experimental and commercial recirculation systems; with development durations of 29 to 35 days (Valenti et al., 2010). It can thus be expected that there will be room for improvement in M. lar larval survival as a result of further research.
116 Monal Lal MSc THESIS: CHAPTER TWO ______
Survival rates reported for larviculture of other Macrobrachium spp. are variable. Survival till metamorphosis was 12 % for M. vollenhovenii (Willführ-Nast et al., 1993), 21 % and 2.5 % for M. acanthurus and M. carcinus respectively (Dobkin et al., 1974), 9 % for M. acanthurus (Choudhury, 1971a), > 90 % for M. amazonicum (Anger et al., 2009), 20 % for M. americanum (Holtschmit & Pfeiler, 1984) and ~ 59 % for M. nipponense (MacLean & Brown, 1991).
It is difficult to compare these survival rates with those of M. lar, as none of the species listed above have larvae which require fully marine conditions for development. Of those species which have a requirement for brackish water of approximately 20 ‰ or higher are M. vollenhovenii (16 – 24 ‰), M. acanthurus (< 20 ‰) and M. americanum (20 – 30 ‰ for early larval stages only) (Willführ-Nast et al., 1993; Holtschmit & Pfeiler, 1984; Dobkin et al., 1974 and Choudhury, 1971a).
Another consideration may be that due to the prolonged dispersal phase that M. lar passes through during larval development, survival for extended periods of time in the larval form may be genetically “hard wired”. Despite this, optimising culture methods can be expected to greatly shorten development time. An example of this can be seen in the current study, where the first PL1 was produced on day 77 of culture whereas Atkinson (1977, 1973) during his study reached the eleventh zoeal stage on day 89 before all larvae died.
2.4.2.2.2 Growth
A more complete discussion of the growth pattern for M. lar larvae observed during this study is provided in Chapter 4 of this thesis, however a brief consideration is presented here.
The most obvious trend displayed in Figures 2.5 and 2.6 is that of increasing intermoult period from averages of 4 – 8 and then 12.6 days during the onset of zoeal stages I, V and IX respectively. It is well known that Macrobrachium spp. exhibit “developmental
117 Monal Lal MSc THESIS: CHAPTER TWO ______plasticity” in terms of the number of instars, morphological development stages and developmental pathways they pass through prior to metamorphosis (Anger, 2001).
It is thought that this is a response to a number of factors which include prevailing unfavourable environmental conditions, inappropriate nutrition and the presence or absence of settlement cues (Anger, 2001).
It is likely that the M. lar larvae in this study underwent mark-time moulting in response to either unfavourable environmental conditions or inappropriate nutrition or both; and further comprehensive investigation is required to definitively determine this. This issue is discussed in greater detail in Chapter 4. Despite this, 5 larvae managed to complete their development and metamorphose to PL.
2.4.2.2.3 Feeds and feeding
The observation that the larvae of M. lar may have differing feed preferences to those of other Macrobrachium species which have been investigated thus far requires further investigation. The larvae of most Macrobrachium spp. are omnivorous, with carnivorous tendencies. This has been shown to be especially true for M. rosenbergii during the first few larval stages, after which they become more omnivorous from stage VII onwards (Dhont et al., 2010).
Reasons stated for this include the fact that larvae remain quite primitive during early development, with partially developed systems for digestion, sight and chemoreception. The gut remains fairly poorly developed until larval stages V and VI are reached, and hence they have a low digestive capacity. It is because of this that early stage larvae are reliant on highly digestible live feeds i.e. zooplankton which may provide exogenous prey enzymes to begin the proper processes of digestion (Dhont et al., 2010).
The primary feed used for most of these other species including M. rosenbergii (Uno & Kwon, 1969; Ling, 1962, 1961), M. vollenhovenii (Willführ-Nast et al., 1993), M. carcinus (Choudhury, 1971b, 1971c), M. novaehollandiae (Greenwood et al., 1976), M.
118 Monal Lal MSc THESIS: CHAPTER TWO ______americanum (Holtschmit & Pfeiler, 1984; Monaco, 1975), M. equidens (Ngoc-Ho, 1976) and M. acanthurus (Choudhury, 1971a, 1970) among others is the nauplii of Artemia spp..
If the larvae of M. lar are proven to indeed show a preference for feed containing material of plant rather than animal origin, this may imply lower feed-associated costs during hatchery production as plant-based sources are generally cheaper to obtain. Artemia currently play an essential role as a live feed in freshwater prawn hatcheries and are indispensable. To date, only partial substitution of Artemia nauplii with other live and inert feeds is possible in larval diets (Dhont et al., 2010).
Previous studies that involved rearing the larvae of M. lar have all used Artemia nauplii as the staple feed with varying results, however all failed to reach the post-larval stage (Nandlal, 2010; Atkinson, 1977, 1973; Kubota, 1972). Supplementary feeds used have included ox liver particles (Nandlal, 2010) and Melon Fly Bactrocera (Dacus) cucurbitae larvae along with a prepared feed incorporating high gluten wheat flour (20 %), corn flour (17.3 %), shrimp meal (20 %), iodised salt (0.4 %) and “Vitamix” premix (1 %) (Atkinson, 1977, 1973).
The success of this study in producing PL may be partly attributed to the provision of a suitable larval diet. The components of the diet which may have played larger roles in adequately meeting the nutritional requirements of some of the larvae may be the prepared custard feeds and biofloc during the earlier larval stages. It is likely that the custard feeds played a greater role in ensuring continued larval development during the later stages when they were easier to digest and hence provided better nourishment, while biofloc was particularly important during the first few larval stages.
Biofloc technology (BFT) is a relatively recent development in aquaculture and provides a number of advantages to the culture system which incorporates it. These include the control of nitrogenous wastes, greater feed efficiency (through microbial recycling of uneaten feed), the provision of a naturally occurring live feed source (the bioflocs themselves) and possible probiotic effects of certain biofloc components (Avnimelech, 2009). Avnimelech (2009) mentions that suspended biofloc has been demonstrated to be
119 Monal Lal MSc THESIS: CHAPTER TWO ______eaten and contribute significantly to the protein requirements of a number of species which have been reared in BFT systems including Tilapia, certain Carp and the marine shrimp Litopenaeus vannamei and Penaeus monodon.
Unfortunately, biofloc volumes were not quantified during this study; however this may be carried out in future investigations to determine parameters such as optimal biofloc volumes for rearing the larvae of M. lar and other Macrobrachium spp..
2.4.2.2.4 Salinity
The success of this study in producing PL may also be attributed in part to providing the larvae with an adequate rearing environment, particularly in terms of salinity. Although a more detailed discussion of the salinity requirements of M. lar is provided in Chapter 3 of this thesis, a brief consideration of the larval salinity requirements as inferred from the results of the mass culture trials at the 20, 25 and 30 ‰ regimes is given here.
Prolonged survival of M. lar larvae in the 20 ‰ regime trials may indicate that they are able to survive under less than optimal conditions of salinity for short periods of time, e.g. in estuarine areas with fluctuating salinities in anticipation of currents which would enable transport farther out to sea where conditions of salinity are higher and more stable, and consequently better suited for survival and growth. This implies that larvae are quite euryhaline during early development, as it has now been established that salinities higher than 30 ‰ are critical for larval development to progress past stages VII and VIII.
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2.4.2.2.5 Culture system
There has been considerable debate over whether greenwater or clearwater-type culture systems are better suited for rearing the larvae of Macrobrachium spp. It appears that both systems have their merits, although clearwater systems have been proven to be easier to manage (Valenti et al., 2010).
During this study, the propagation of biofloc in the LRTs as a result of the phytoplankton maintained in culture was likely to have provided adequate nutrition to some of the larvae; and was responsible at least in part, for enabling them to complete development and metamorphose into PL.
A number of researchers have investigated the potential role that microalgae play in the successful larviculture of Macrobrachium spp. These include Lober and Zeng (2009), who found higher survival and shorter development duration in an Australian strain (lineage II) of M. rosenbergii reared at higher versus lower microalgal concentrations, reduced ammonia levels when algae were present in the LRT with M. rosenbergii larvae (Cohen et al., 1976) and enhanced survival and metamorphosis rates when M. rosenbergii larvae were cultured with seven different species of unicellular algae (Manzi & Maddox, 1977; Manzi et al., 1977).
Although the larvae of Macrobrachium spp. are known to be visual, particulate feeders (Atkinson, 1977, 1973), and do not feed directly on algal cells except via accidental ingestion in negligible amounts (Cohen et al., 1976); the benefits of enrichment of Artemia nauplii on microalgae which are then consumed by the larvae have been well documented (Dhont et al., 2010; Valenti et al., 2010).
Cohen et al. (1976) mention that the presence of algae in the culture medium facilitate the growth of Macrobrachium larvae only indirectly by removing toxic material viz. ammonia from the culture medium. This is somewhat inaccurate when considering the incorporation of microalgal cells into biofloc particles, as they would offer similar benefits as enriched Artemia nauplii when consumed by the larvae. Further mention is
121 Monal Lal MSc THESIS: CHAPTER TWO ______made that in systems where algae are incorporated, the balance of the ecological system operating in the LRT is more complicated, as more trophic levels exist and less control can be exercised over the whole system.
An interesting finding was made by Cheah and Ang (1979) who reared M. rosenbergii larvae using no intensive hatchery techniques in a greenwater system operated completely without water exchange. The LRTs were topped up with greenwater to counter losses due to evaporation, and larvae were reared under two salinity regimes of 6 – 8 ‰ and 12 – 14 ‰. Their results yielded survival rates to PL of 39.6 % and 36.9 % for the two regimes respectively, with no significant difference between the two.
Regarding future M. lar larviculture research, the greenwater technique described in this study is a starting point to investigate the reliability of producing PL in the laboratory. Once greater knowledge of the nutritional and other requirements of the larvae become established, experimentation with clearwater systems may be possible.
2.5 Conclusion
As a result of these investigations carried out into developing a larviculture technique, it became possible to successfully rear the larvae of M. lar from hatch until metamorphosis into PL. The knowledge base on the larval requirements for this species has been expanded, and a benchmark for future larviculture research involving M. lar has been established.
The production of PL during this study places the School of Marine Studies at the University of the South Pacific as possibly the first institution in the world to report completion of the larval phase of development of M. lar in captivity.
A number of important observations have been made regarding the maintenance of M. lar broodstock in the laboratory. Overall, it was found that broodstock were relatively easy to maintain in captivity. The subjects of prolonged and partial larval hatches will require further investigation, along with issues such as sexual maturation and fecundity in
122 Monal Lal MSc THESIS: CHAPTER TWO ______captivity, appropriate stocking densities and sex ratios, social hierarchical interactions and optimal broodstock nutrition.
Overall larval survival rate till metamorphosis in the single trial which produced PL was very low, and further research into optimising culture methods is essential in order to improve this. Larvae were also observed to undergo mark-time moulting, especially during the later zoeal stages as metamorphosis approached, which lengthened overall culture duration. This is generally attributed to inappropriate culture conditions, especially in terms of larval nutrition.
If the hypothesis that M. lar larvae require a significant component of plant-based nourishment in their diet can be proven, there are positive implications for hatchery operating costs, because there may be less reliance on animal based feeds such as Artemia nauplii. The importance of biofloc in the diet of the larvae warrants further investigation, as greater efficiencies in feed utilization may be achieved with the implementation of BFT in the larviculture system.
123 Monal Lal MSc THESIS: CHAPTER THREE ______
CHAPTER THREE
DETERMINATION OF SALINITY AND TEMPERATURE OPTIMA FOR LARVAL REARING OF Macrobrachium lar
3.1 Introduction
The physical parameters of the rearing environment provided for the larvae of any type of aquatic organism in culture are of critical importance for successful efforts to rear them. These physical parameters include salinity, temperature, pH, dissolved oxygen concentration, light, hydrostatic pressure, turbulence and the presence of pollutants and suspended matter among others (Zheng et al., 2008; Anger, 2001; Maciolek, 1972).
Two important parameters which need to be within certain ranges to yield optimal growth and survival of any species being investigated for culture potential, are those of salinity and temperature. Once the optima for these parameters have been established, further work can be carried out in order to determine the appropriate ranges for other environmental parameters, as techniques for culture are refined and developed (Zheng et al., 2008; Anger, 2001; Maciolek, 1972).
Salinity is a key factor which has been observed to influence the rates of survival and development of decapod crustacean larvae (Kinne, 1971 in Anger, 2001). The salinity tolerances of aquatic species are closely associated with the ability to maintain their internal media largely independent of external conditions, and fluctuations may cause rapid changes in ion concentrations of the blood and other tissues (Anger, 2001). Most crustacean larvae actively regulate internal ion concentrations by osmo-ionic regulatory processes, which have functional limits that depend on the salinity of the external environment (Brown et al., 2010; Anger, 2001).
The temperature of the environment of decapod crustacean larvae is another important extrinsic factor in the regulation of growth, development and metabolism, as like many other aquatic organisms they are poikilothermic in nature (Zheng et al., 2008). It has been established that larval moulting frequency, the final size and biomass of individual larval
124 Monal Lal MSc THESIS: CHAPTER THREE ______stages, larval activity and swimming speed (thermokinesis) and larval respiration rate all vary as a function of temperature (Anger, 2001).
Experimental work with the larvae of various species of decapod crustaceans suggests that growth is maximum near a particular temperature where the physiological performance of a given larval stage or species is optimal, and decreases at both higher and lower temperatures (Matsuda & Yamakawa, 1997; Sulkin & McKeen, 1996, 1994; Minagawa, 1990; Laughlin & French, 1989; MacKenzie, 1988; Anger, 1987; Dawirs et al., 1986; Kunisch & Anger, 1984; Rothlisberg, 1979 and Regnault, 1969 all in Anger, 2001).
3.1.1 Previous investigations of the salinity and temperature requirements of Macrobrachium species.
The salinity and temperature requirements of a number of crustacean species that are in various stages of assessment for their culture potential, or already commercially cultivated, have been investigated from a number of perspectives.
The majority of studies on the larval development of various Macrobrachium spp. have evaluated the direct effects of salinity and temperature (either separately or in combination), on the rates of larval survival and development e.g. Wong (1987); Holtschmit & Pfeiler (1984); Lee & Fielder (1981); Subramanian et al. (1980); Dugger & Dobkin (1975); Dobkin et al. (1974); Dobkin (1971) and Williamson (1971). These investigations typically describe the earliest attempts at rearing larvae of the species concerned in the laboratory, and aim to establish the conditions of salinity and temperature required for larviculture along with descriptions of larval developmental morphology.
A number of studies have examined the improvement of larval freshwater tolerance through selective breeding (Wong & McAndrew, 1990), the effects of salinity and temperature on embryonic development and larval viability (Smith et al., 2009; Manush et al., 2006; Brillon et al., 2005; Ching & Velez Jr., 1985) and investigations of post-
125 Monal Lal MSc THESIS: CHAPTER THREE ______larval cold tolerance, salinity tolerance and osmoregulation (Yen & Bart, 2008; Chen & Chen, 2003; Silverthorn & Reese, 1978; Sandifer et al., 1975).
Other studies investigated variations of salinity and temperature and their effects on larval induced thermo-tolerance and stress resistance (Rahman et al., 2004), ammonia and pH toxicity (Chen & Chen, 2003; Armstrong et al., 1978) and immune responses (Cheng et al., 2003; Cheng & Chen, 2000, 1998).
The Giant River Prawn M. rosenbergii is by far the most researched and highly commercialized of the species of Macrobrachium that have been described worldwide (Nandlal, 2005), owing to its fast growth rates, large size, attractive meat quality and omnivorous feeding habit which make it adaptable to both small and large-scale farming operations (Nandlal & Pickering, 2006b, 2006a). Despite these positive attributes for culture, previous experiments by fisheries biologists to rear larvae of this species had been unsuccessful before it was discovered by FAO expert Dr. Shao-Wen Ling in 1961 that the larvae required brackish conditions for survival beyond 5 days (New, 2010).
Today, the culture of M. rosenbergii is a global billion dollar industry, and a number of investigations have since been carried out on various aspects of its hatchery and grow-out phases of culture; including several studies on salinity and temperature optima for larviculture.
Ling (1961) initially reported that the optimal salinities for larval rearing of M. rosenbergii are 12 – 14 ‰, while Uno and Kwon (1969) reported approximately 10.5 ‰ (stated as 30 % seawater) with optimal temperatures of 28 ± 0.5< Takano (1987a) who stated ranges of 8 – 10 ‰ at 26 – 31 ! ! Daniels et al. (2000) in Cheng et al. (2003) found that the thermal tolerance range for this species is 18 – 34 (-–32 < ! however a comparison with data for other species of Palaemonid prawn summarised in Table 3.1 overleaf shows that the species of Macrobrachium that have been investigated thus far may be divided into four groups.
126 Monal Lal MSc THESIS: CHAPTER THREE ______
Table 3.1 Larval salinity and temperature optima investigations of other species of Palaemonid prawn. Optimal Acclimation protocol Optimal Species salinity range (‰) temperature Source range ( Macrobrachium 32.9 – 26 – 29.5 Ngoc-Ho (1976) equidens M. grandimanus 17.5 – 35 Not mentioned, larvae 27 Shokita (1985) (J. W. Randall, 1840) hatched at 10 ‰ M. intermedium ~35; no PL produced Larvae hatched at ~35 ‰ 18 Williamson (1971) (Stimpson, 1860) M. acanthurus 23.5 – 35 Hatch at ~17.5 ‰, direct 26 – 30 Dobkin (1971) transfer to test salinities M. acanthurus 20 Acclimated volumetrically Not Choudhury (1971a)
127 over 4 – 6 hours controlled, varied from 23–27 M. acanthurus ~21 Slow volumetric Not Choudhury (1970) (60% seawater) acclimation controlled M. acanthurus 16 – 18 Not mentioned 30 Dobkin et al. (1974) M. vollenhovenii 16 – 24 Gradual acclimation 28.1 ± 0.12 Willführ-Nast et al. (1993) Palaemonetes 15 – 30 – 25 Knowlton (1970); also in vulgaris (Say, 1818) Lee & Fielder (1981) P. vulgaris 10 – 30 – 20 – 25 Sandifer (1973) in (Say, 1818) Lee & Fielder (1981) M. americanum 20 – 30 for early larval No specific mention 28 ± 0.1 Holtschmit & Pfeiler stages, from IV to VI (1984) 15–20 M. americanum 15 ± 0.1 2 hours to reach 15 ± 0.1 ‰ 29.5 ± 0.5 Monaco (1975)
127 Monal Lal MSc THESIS: CHAPTER THREE ______
M. novaehollandiae 23 Larvae hatched at 23 ‰ 13 ± 5 , Greenwood et al. (1976) (De Man, 1908) max. = 28 M. olfersii ~21 ; no PL produced Larvae hatched at ~21 ‰ 30 Dugger & Dobkin (1975) (60% seawater) M. carcinus 14 – 16 Slow acclimation Not Choudhury (1971c) controlled, varied from 24–28 M. carcinus 14 – 17.5 Acclimated volumetrically Not Choudhury (1971b) over 6 hours mentioned M. formosense 13 – 16 – 24 – 28 In Shokita (1985) Bate, 1868 M. idae 5 – 15 Direct transfer in 5 ‰ steps 25 ± 2.0 Subramanian et al. (1980) from freshwater 128 M. australiense Up to 15 No acclimation 30 Lee & Fielder (1981) M. australiense 5.25 – 7 Larvae hatched at culture 21 – 28 Fielder (1970) (15 – 20 % seawater) salinity M. rosenbergii 12 – 12.3 Slow acclimation 28 ± 0.5 Uno & Kwon (1969) M. rude 10 – 12 – – Haque (1980) in (Heller, 1862) Khan et al. (1984) M. birmanicus 1 * 10–12 Direct transfer 30–32 Khanet al. (1984) (Schenkel, 1902) M. amazonicum 10 Not mentioned 29 Anger et al. (2009) M. nipponense 10 No specific mention, Not Wong (1987) appears to be direct transfer mentioned. M. niloticum 0 Not mentioned Not Williamson (1971) (P. Roux, 1833) mentioned M. lamarrei 0 – – Amin (1979) in Khan et al. (1984)
M. dayanum 2 0 – – Khanam (1981) in Khan et (Henderson, 1893) al. (1984) 128 Monal Lal MSc THESIS: CHAPTER THREE ______
M. lamarrei lamarrei 0–Not In Shokita (1985) (H. Milne-Edwards, mentioned 1837) M. kistnense 0 – 28 – 29 In Shokita (1985) (Tiwari, 1952) M. hendersodayanum 0–Not In Shokita (1985) (Tiwari, 1952) mentioned M. shokitai 0 – 28 – 29.5 Shokita (1973) in Shokita Fujino and Baba, 1973 (1985) M. asperulum 0 – 27.8 – 28.2 In Shokita (1985) (Von Martens, 1868) 129 * Invalid taxon. Now known as Macrobrachium malcolmsonii malcolmsonii (H. Milne-Edwards, 1844). 1 Stated as M. birmanicus in the text. Invalid synonym. 2 Stated as M. dayanus in the text. Invalid synonym.
129 Monal Lal MSc THESIS: CHAPTER THREE ______
These groups comprise of species which: i) are able to complete their larval development entirely in freshwater (0 ‰). ii) require salinities up to 10 ‰ for successful development. iii) require salinities between 10 and 20 ‰ for successful development and iv) require salinities greater than 20 ‰ for successful development.
The temperature optima data for the majority of the species listed are largely similar, ranging between 20 to 30<#! distribution.
Kubota (1972) reports that berried female M. lar make downstream breeding migrations to brackish waters to release newly hatched larvae which drift with the current (Short, 2004), until they encounter full-strength seawater where they remain in the plankton to complete their development. He also states that hatching can also take place in freshwater, although larvae must reach brackish water within 3 to 4 days if they are to survive (Nandlal, 2010; Short, 2004; Atkinson, 1977, 1973; Kubota, 1972).
From what is known about the life history characteristics of M. lar, it is likely its larval salinity requirements will fall into the fourth group of species, and its temperature requirements are likely to be similar to those of other species which have a tropical distribution.
3.1.2 Previous work on investigating culture conditions for the larvae of M. lar
A number of researchers have previously worked on the larval rearing of M. lar; viz. Kubota (1972), Atkinson (1977, 1973), M. S. Muranaka in Hanson & Goodwin (1977), Takano (1987b), Nandlal (2010), Sethi and Roy in Kutty & Valenti (2010) and Sethi et al. (2011), with varying levels of progress.
130 Monal Lal MSc THESIS: CHAPTER THREE ______
Of the seven investigations reported in the literature, details of salinity and temperature studies have only been described for three studies (Nandlal, 2010; Atkinson, 1977, 1973 and Hanson & Goodwin, 1977).
The earliest study by Kubota (1972) reported rearing of larvae over 21 days to the fifth zoeal stage before total larval mortality eventuated. However, details on the salinity and temperature conditions maintained are not mentioned, with the author stating that methods used for rearing M. rosenbergii were used as a guide.
A subsequent attempt by Atkinson (1977, 1973) resulted in further progress. Larvae were reared under two conditions of salinity (15 and 35 ‰) and three conditions of temperature (21 – 22.5 ($$ – 27.2 (.6 – 30.5 Results indicated that a salinity of 35 ‰ and a temperature of 23 – 26.5 of 89 days to the eleventh zoeal stage before total mortality was observed.
Some work by Michael S. Muranaka very briefly reported in Hanson & Goodwin (1977), states that “M. lar was successfully brought through to juvenile”, however further information on larval development is not reported. There is also scarce information on salinity and temperature requirements, apart from a finding that “M. lar rearing is more difficult than that of M. rosenbergii, and that larvae must remain in 35 ‰ seawater, after which salinity is lowered. The required salinity range is not yet firmly established, but may be as high as 25 ‰”. They also report that PL “must be kept in salinities at least this high”.
The most recent study by Nandlal (2010) reports rearing larvae to the seventh zoeal stage over 45 – 50 days at a salinity of 33 – 34 ‰ and temperature of 28 ± 0.5 , before all larvae experienced mortality.
Overall, the current state of knowledge on the larval development of M. lar is fragmentary, largely due to the difficulties encountered by previous researchers in rearing the larvae through to metamorphosis. There is a distinct lack of further information resulting from the work of M. S. Muranaka who had reportedly succesfully managed to
131 Monal Lal MSc THESIS: CHAPTER THREE ______produce PL, while all others have experienced total larval mortality before metamorphosis.
The research carried out for this chapter was aimed at determining the salinity and temperature ranges optimal for the survival and growth of M. lar larvae under laboratory conditions, so that efforts to rear larvae till metamorphosis may be successful and problem areas in larviculture technique with respect to salinity and temperature identified.
The experiments carried out on salinity tolerance and temperature optima were operated in tandem with the mass culture experiments described in Chapter 2 of this thesis, to allow for alteration and refinement of the mass culture technique used according to new information gathered from the results of the salinity and temperature experiments as they came to hand. This allowed for further progress to be made with larval rearing as the larvae developed through the more advanced zoeal stages, and ultimately metamorphosed into PL.
3.1.3 Research objectives
Research carried out for this chapter was aimed at answering the question: what are the optimal conditions of salinity and temperature required for successful culture of M. lar larvae? In order to answer this question, the specific objectives of the experiments designed were to: i) (a) determine the salinity ranges for optimal survival and growth of M. lar larvae at specific morphological stages of development. (b) develop a suitable salinity acclimation protocol which would maintain optimal larval survival and growth as they are exposed to increasing salinities up to the final target culture salinity of 30 ‰, and ii) (a) determine a temperature range for optimal survival and growth of M. lar larvae during culture.
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3.2 Methodology
3.2.1 Methodology development
3.2.1.1 Salinity tolerance study
As larvae of the Monkey River Prawn are known to hatch in either fresh or slightly brackish water and drift downstream as they disperse out to sea (Kubota, 1972), it was theorised that they are expected to require increasing levels of salinity as they develop.
Based on this, it was thought that the range of salinities conducive to optimal growth and survival may be different between larval stages; with these ranges encompassing progressively higher salinity values up to 35 ‰ until the PL1 stage is reached.
Consequently, the primary focus of this study was to determine the optimal salinity tolerance ranges for growth and survival of M. lar larvae at several stages of development. Larvae were sourced from mass cultures maintained in parallel with the experimental setup, from which larvae of the desired developmental stage could be collected for experimentation.
Previous studies have employed a number of methods for determining the salinity tolerance ranges of larvae and juveniles of a number of Macrobrachium and marine finfish species. Usually a number of test salinities are selected, and the larvae are either acclimated gradually (Nandlal, 2010; Zheng et al., 2008; Estudillo et al., 2000; Wong, 1987; Sandifer et al., 1975; Dugger & Dobkin, 1975; Choudhury 1971c, 1971a, 1971b) or transferred directly (Estudillo et al., 2000; Holtschmit & Pfeiler, 1984; Lee & Fielder, 1981; Subramanian et al., 1980; Atkinson, 1977, 1973; Dugger & Dobkin, 1975) into the test salinity treatments.
In investigations where larvae were acclimated to the test salinities, acclimation rates and procedures varied, according to the salinity increase increments e.g. 1 and 2.5 ‰ per day (Sandifer et al., 1975), or over various time intervals e.g. 4 – 6 hours (Choudhury, 1971a,
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1971c), 6 hours (Choudhury, 1971b) and between 18 – 120 hours (Nandlal, 2010) until target salinities were reached. Further details have been summarised earlier in Table 3.1.
For this study, a two-phase approach was taken to determine salinity range optima for larvae of the Monkey River Prawn by way of short-duration salinity tolerance tests. The first phase of the investigation was termed the ‘coarse-resolution series of tolerance tests’, whereby larvae of different developmental stages were acclimated to four different test salinity regimes viz. 0, 10, 20 and 30 ‰, to determine the proximate salinities which produced optimal survival and growth. During acclimation to the test salinities, data on larval survival was collected to ensure that larval mortality during the tolerance tests could not be attributable for the most part to the acclimation process itself. If however, mortalities did occur, then they could be recorded. Unfortunately, seawater of 35 ‰ was unavailable at the Seawater Laboratory, due to the proximity of the seawater intake to the shoreline and the consequent influence of freshwater influx; so the effect of full-strength seawater on the growth and survival rates of M. lar larvae could not be examined.
The second phase of the investigation was termed the ‘fine-resolution series of tolerance tests’, and was based upon the results of the coarse-resolution phase of testing. These tests exposed larvae of the same developmental stages as those in the previous phase to a range of test salinities in 5 ‰ increments bordering the best test salinity from the coarse- resolution phase. Therefore, for example; if zoea III larvae were found to survive and grow best at 20 ‰ during the coarse-resolution phase of testing, then a second batch of zoea III larvae were exposed to 10, 15, 20, 25 and 30 ‰ test solutions for the fine- resolution testing phase.
This was done so that the coarse-resolution tests could be repeated to verify the earlier results and to mitigate any temporal variation effects between the two sets of experiments. The protocol also allowed for the determination of a narrower range of salinity values which could have produced optimal survival and growth.
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3.2.1.2 Temperature tolerance study
The temperature tolerance investigation developed for this chapter largely followed the methods of earlier researchers, whereby larvae were gradually acclimated from the temperature of the LRT they were hatched and cultured in to a series of test temperature treatments; see Smith et al. (2009), Zheng et al. (2008), Manush et al. (2006), Brillon et al. (2005) and Silverthorn & Reese (1978). Test temperature values that were likely to be optimal for growth and survival based on studies with other tropical Macrobrachium species were selected for this investigation.
3.2.2 Larval mass cultures
A series of mass cultures of M. lar larvae were maintained throughout the duration of the salinity and temperature investigations to ensure a constant supply of larvae for experimentation. The findings of the salinity tolerance investigations were used to modify the mass culture procedures, particularly with regard to the regimes used for increasing salinity and maintenance of the temperature range within the culture medium.
Up to four circular 1000 L flat-bottomed polyethylene LRTs were maintained at any one time during this period according to the procedures outlined in Chapter 2 of this thesis. Larvae of the required stage were collected from the LRTs as and when they were needed. As heavy mortalities (see Chapter 2) were encountered in all of the mass culture attempts, only larvae up to zoeal stage VII were available in sufficient quantities to allow for meaningful experimentation.
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3.2.3 Salinity tolerance experiments on M. lar larvae
3.2.3.1 Collection and acclimation of larvae
Larvae at zoeal stages I, III, V and VII of development were used in the coarse-resolution and fine-resolution salinity tolerance experiments. Odd-numbered larval stages were used so that development of larvae in treatments of optimal salinities could be measured and recorded as they moulted to the subsequent stage. These larvae were then acclimated to the test salinity solutions they would be exposed to for the duration of the tolerance testing.
Each larval stage was tested separately in sequence, when sufficient numbers of individuals of each stage were available to be used in the experiments. A total of 240 and 300 individual larvae of each developmental stage were required for the coarse-resolution and fine-resolution tolerance tests respectively. To account for mortalities due to handling and other factors prior to the start of the experiments, 50 extra larvae of each developmental stage were collected from the mass culture LRTs, bringing the total number of larvae collected to 290 and 350 for the coarse-resolution and fine-resolution tests respectively.
All larvae were collected from the LRT using a larval counting bowl. Larvae were then transferred into a petri dish filled with water from their LRT using a wide bore (3 mm diameter) pipette for examination under a binocular dissecting microscope. Larvae were examined to determine their stage of development and to check for the presence of any unhealthy or abnormal/deformed larvae which were discarded. Healthy larvae at the required stage of development were then transferred to the test salinity acclimation tanks.
3.2.3.2 Acclimation set up
A series of test salinity solutions were prepared using treated freshwater and treated seawater (see Chapter 2 for details). For the coarse-resolution phase of testing, 0, 10, 20 and 30 ‰ solutions were prepared. For the fine-resolution phase of testing, a range of
136 Monal Lal MSc THESIS: CHAPTER THREE ______solutions including 0, 5, 10, 15, 20, 25 and 30 ‰ were prepared depending on the zoeal stage being tested and the outcome of the coarse-resolution phase tests.
Each solution was prepared by addition of the required amounts of treated seawater and/or freshwater to a clean 20 L plastic bucket through a 50 μm2 mesh net. The solution was vigorously aerated at the rate of 0.5 Ls-1 using an open-ended air line to ensure thorough mixing. The salinity of the solution was checked during the mixing process with a YSI 85 salinity, temperature and conductivity meter to ensure that the target salinity was achieved to ± 0.5 ‰.
The mixed solutions were then maintained in individual, cleaned 50 L plastic jerry cans (Plate 3.1) and supplied with aeration at the rate of approximately 40 – 50 mLs-1. Air delivery was regulated by individual plastic screw valves installed on each air line. Each solution was maintained for no more than 3 days after which it was discarded and a fresh mixture prepared.
Plate 3.1 50 L jerry cans used for storage of the mixed test salinity solutions.
A drip system was used to acclimate larvae to their test salinities. Individual 1 L drip bags (previously containing Hartmann’s solution) were cleaned with treated freshwater and re- filled with the respective test salinity solutions.
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Calibrated adjustable burettes were attached to the necks of the drip bags and their 3 mm delivery lines run into the acclimation tanks. One drip bag was used per acclimation tank. A single 5 mm diameter air line attached to a 40 mm corundum air diffuser provided aeration inside each drip bag at a rate of approximately 15 – 30 mLs-1. Air delivery was regulated by individual plastic screw valves installed on each air line. The test salinity solutions were aerated to ensure they remained thoroughly mixed and well oxygenated as they were added to the acclimation tanks.
The aim of the drip system was to gradually replace all of the LRT water that larvae were transferred to the acclimation tank in at a uniform rate across all treatments over a period of 24 hours. Preliminary trial work with the system discovered that a delivery rate of 0.7 mLmin-1 was adequate for this purpose, to ensure that all larvae were transferred to their test solutions at approximately the same time. This was necessary so that no bias was afforded to those larvae that needed to make a large ‘jump’ in salinity e.g. from 10 ‰ to 30 ‰ compared to those that were being transferred across smaller increments e.g. 10 ‰ to 15 ‰.
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Plate 3.2 Drip bag and burette units.
A total of four or five 3 L rectangular polyethylene acclimation tanks were set up corresponding to the drip bags for the coarse or fine-resolution phase tests respectively. These tanks were placed on a table in the laboratory and filled with a litre of mass culture LRT water (Plate 3.3).
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Plate 3.3 Salinity tolerance test acclimation tanks.
At the 1 L mark, a hole was drilled to allow for the placement of a 5 mm diameter plastic screw valve to which a 1 m length of air line was attached (Plate 3.4). This air line was run off the table onto the floor of the laboratory towards a drain to allow the tank to empty via gravity when filled to more than 1 L capacity. A freeboard of 7 cm (2 L capacity) remained above the drainage level to accommodate extra water added to the tanks by the drip system.
Plate 3.4 Salinity tolerance test acclimation tanks showing drain valves.
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The plastic screw valve was calibrated to release water from the acclimation tank at a rate to match the incoming rate of water from the drip bags. The end of the drip bag delivery line was placed at the opposite end of the tank from the drain valve and secured in place with a plastic laundry clip. Inside the acclimation tank, a 5 cm length of 5 mm diameter air line was fitted to the end of the plastic screw valve to the end of which a piece of 100 μm2 mesh net was attached using a plastic cable tie (Plate 3.5). This was to retain larvae in the acclimation tank.
Plate 3.5 Salinity tolerance test acclimation tanks showing the placement of the drain valves, retaining screens, air diffusers and drip bag delivery lines.
Each acclimation tank was aerated at the rate of 4.5 mLs-1 using a 20 mm diameter spherical corundum airstone. Air delivery was regulated by individual plastic screw valves installed on each air line.
All acclimation tanks were monitored at intervals of 2 hours to ensure that the drip bag and drainage systems were working correctly, and that the larvae remained viable. Any
141 Monal Lal MSc THESIS: CHAPTER THREE ______dead or moribund individuals observed were removed using a wide bore (3 mm diameter) pipette. The numbers of all dead and moribund individuals were recorded before they were discarded. Changes in salinity and temperature in each acclimation tank were also recorded during this period. Each tank was covered with its lid when not being examined.
Throughout the acclimation period, monitored water parameters remained as follows: -1 4+ temperature of 28 ± 0.5 -./6( 1 2 > 6.5 mgL and average NH and NH3 concentrations no higher than 1.5 and 0.1 ppm respectively.
Upon completion of the acclimation period, larvae were gently individually transferred to the salinity tolerance testing set up using a wide bore (3 mm diameter) pipette. In cases where larval mortality during an acclimation run resulted in insufficient numbers of larvae being available for experimentation, all larvae were discarded and a fresh batch of larvae acclimated.
3.2.3.3 Coarse-resolution tolerance tests
Larvae at zoeal stages I, III, V and VII were exposed to a range of 0, 10, 20 and 30 ‰ test salinity solutions in 1 L glass jars (Plate 3.6) for a test period of 5 days. Larvae at each stage of development were tested separately in successive trials as sufficient numbers of larvae became available from the mass cultures, starting with zoea I. 20 larvae of the required stage were transferred from the acclimation tanks into a single test jar. Test jars were set up in triplicate for any given test salinity, and therefore a total of 12 jars were used for the four test salinities at each of the larval developmental stages.
Each test jar was filled to a volume of 800 mL with the required test salinity solution prepared earlier as described under section 3.2.3.2, and aerated at the rate of 4.5 mLs-1 using individual 5 mm diameter air lines terminating in a plastic pipette tip. The pipette tip allowed the jar to be aerated without causing excessive turbulence while maintaining a weak current in the jar to keep food particles in suspension.
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Plate 3.6 1 L tolerance test jar.
All the test jars were placed in a water bath system to maintain an even temperature. Three polystyrene boxes 55 cm in length, 40 cm in width and 17 cm in depth with a thickness of 25 mm filled with seawater were used as the containers for the water bath. A total of 12 test jars could be accommodated inside one polystyrene container.
Each container was heated to maintain 28 ± 0.5 !4 1(663 immersion heaters. Three evenly spaced 5 mm diameter air lines attached to 40 mm corundum air diffusers provided aeration inside each container at the rate of 50 mLs-1 to circulate the seawater in the water bath (see Plates 3.7 and 3.8). Throughout the testing period, monitored water parameters remained as follows: pH of 7.8 ± 0.2, DO2 > 6.5 -1 4+ mgL and average NH and NH3 concentrations no higher than 1.5 and 0.1 ppm respectively.
Test salinity treatments were assigned randomly to the jars positioned in the water bath system.
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Plate 3.7 Salinity tolerance test water bath system set up.
Plate 3.8 Salinity tolerance test water bath system in operation.
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3.2.3.4 Fine-resolution tolerance tests
The experimental set up for the fine-resolution tolerance tests was identical to that described earlier for the coarse-resolution phase of tests, with the only difference being that larvae at zoeal stages I, III, V and VII were exposed to finer increments of the test salinity solutions in the 1 L test jars, viz. 0, 5, 10, 15, 20, 25, and 30 ‰.
3.2.3.5 Experiment maintenance
The entire experimental set up was illuminated for 24 hours each day to duplicate the conditions under which larvae were raised in the mass culture systems they were sourced from. Two fluorescent light tube fittings containing a single 4 feet Osram 36 W ‘warm white’ tube and a single 4 feet Eurolux 36 W ‘cool white’ tube each were suspended over the experimental set up using 4 mm nylon ropes approximately 75 cm above the water bath system. This provided a total light output of approximately 6700 lux at the surface of the water in the test jars.
Larvae were offered feed four times a day at four-hourly intervals starting at 0800. The first three feed offerings were a mixture of egg, squid and shrimp custards depending on the stage of development of the larvae being tested. Biofloc and Algamac (Aquafauna Biomarine Inc.) particles were also incorporated into these feed offerings. The last feed offering was solely Artemia nauplii to nourish the larvae overnight. Larvae were fed ad lib..
Water was exchanged in the test jars at the rate of 30 %day-1 for the duration of the tolerance tests. Up to 50 or 80 %day-1 was exchanged if the quality of water was found to have deteriorated significantly since the previous exchange. Uneaten feed particles (including Artemia nauplii and meta-nauplii), excessive biofloc and dead or moribund larvae were removed at this time as well using a 5 mm diameter length of air line as a siphon hose.
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3.2.3.6 Larval examination and data collection
Larvae were examined twice daily to determine the number of surviving individuals and their stage of development. Dead and moribund larvae were removed from the test jars and examined under the microscope. Larvae were considered to be dead when a heart beat could not be discerned and the pulsing activity of the hind-gut had ceased. Moribund larvae were characterised by their lack of swimming and feeding activity and opaque- white body colour which preceded death. Healthy larvae were seen to actively swim and feed and have transparent body tissues.
The numbers of surviving larvae and their stages of development at the end of each day were recorded. The survival data was used to produce survivorship performance curves for each zoeal stage tested, while the data on developmental stages of the larvae was employed to sketch graphs comparing increases (or otherwise) of larval zoeal development stage between treatment salinities, for both coarse and fine-resolution phases of investigation.
3.2.3.7 Data analyses
Data organization and entry was carried out using Microsoft Excel for Microsoft Office 2003 suite and statistical analyses were carried out using GenStat® Version 12.1 (VSN International Ltd.) software, on the Microsoft Windows XP OS platform.
A One-Way Analysis of Variance (ANOVA) was carried out on percentage survival data for the fine resolution test salinities.
Due to the restrictions imposed by the high mortality of M. lar larvae in culture and the resulting small sample sizes that this investigation was forced to use, data were inspected visually to ensure there were no large deviations from normality.
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The data on larval growth at fine resolution test salinities were excluded from further analysis as they did not easily lend themselves to statistical tests due to the small sample sizes involved.
3.2.4 Temperature tolerance tests
3.2.4.1 Collection and acclimation of larvae
Larvae at the first zoeal stage of development were used in the temperature tolerance experiments. All larvae were collected from the LRT using a larval counting bowl 18 – 24 hours post-hatch and transferred into a petri dish filled with water from their LRT, using a wide bore (3 mm diameter) pipette for examination under a binocular dissecting microscope.
Larvae were examined to determine their stage of development and to check for the presence of any unhealthy or abnormal/deformed larvae which were discarded. Healthy larvae at the required stage of development were then acclimated to the test LRTs where they would be exposed to the test temperature treatments.
The test LRTs were prepared to match the water parameters of the mass culture LRT that larvae were to be sourced from. Particular care was taken to ensure that salinity and temperature in particular varied by no more than 0.5 ‰ and 0.2 ! mass culture LRT values. Once larvae had been stocked in the test LRTs, the glass tube immersion heaters each LRT was equipped with were turned on to gradually acclimate larvae to the test temperatures required. Other water parameters remained as follows: pH -1 4+ of 7.8 ± 0.2, DO2 > 6.5 mgL and average NH and NH3 concentrations no higher than 1.5 and 0.1 ppm respectively.
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3.2.4.2 Temperature tolerance tests
Approximately 1400 larvae were stocked in individual 60 L cylindro-conical fibreglass LRTs and exposed to three test temperature ranges; viz. 26 ± 0.5 (./6$ /6/ 0.5 < !=< thrice.
Larvae were not counted individually to minimize stress due to handling, and for this reason estimated counts were used. Care was taken to ensure that relatively uniform numbers of larvae were stocked in each LRT.
Plate 3.9 Temperature experiment LRTs.
Each LRT was heated to maintain its assigned temperature range with an Aqua One 200 W glass tube immersion heater (Plate 3.10). All water exchanges were carried out as described for the mass culture LRTs in Chapter 2 of this thesis. Aeration was provided by means of two 5 mm diameter air lines at a rate of approximately 80 – 100 mLs-1 at each air line, which were individually weighed down with cleaned stone weights.
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Plate 3.10 LRT equipped with Aqua One 200 W immersion heater.
The entire experimental set up (Plate 3.9) was illuminated for 24 hours each day to duplicate the conditions under which larvae were raised in the mass culture systems they were sourced from. Two fluorescent light tube fittings containing a single 4 feet Osram 36 W ‘warm white’ tube and a single 4 feet Eurolux 36 W ‘cool white’ tube each were suspended over the set up using 4 mm nylon ropes at a distance of approximately 75 cm. This provided a total light output of approximately 6700 lux at the surface of the water in the test LRTs.
Larvae were offered feed four times a day at four-hourly intervals starting at 0800. The first three feed offerings were a mixture of egg, squid and shrimp custards following the protocol described in Chapter 2. Biofloc and Algamac particles were also incorporated into these feed offerings. The last feed offering was solely Artemia nauplii to nourish the larvae overnight. Larvae were fed ad lib..
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3.2.4.3 Larval examination and data collection
Larvae were examined once daily to determine the number of surviving individuals and their stage of development. Dead and moribund larvae removed from the LRTs were examined under the microscope. Larvae were considered to be dead when a heart beat could not be discerned and the pulsing activity of the hind-gut had ceased.
When signs of disease such as epibiont infestation or bacterial lesions were observed on larvae in a particular LRT, the affected tank was treated as described in Chapter 2.
3.2.4.3.1 Larval survival
Larval survival was determined by estimating the number of surviving larvae in each test LRT every second day. Larvae were first evenly distributed in the LRT by gently mixing the water using a larval counting bowl. Five separate counts were made by removing 1 L of water from the LRT and accurately counting all larvae present in the counting bowl. The LRTs were stirred between counts and any dead or moribund individuals removed for microscopy.
3.2.4.3.2 Larval growth
Larval growth was evaluated by calculating the Larval Staging Index (LSI) according to Mallasen & Valenti (2006).
Where: ni = number of larvae in each stage
B S n Si = larval stage N = total number of larvae examined LSI i i N i = 1 – 5; representing each larval stage
In order to determine the LSI for a particular LRT, three samples of larvae were randomly selected using a larval counting bowl. For each sample, the number of larvae at a particular stage of development was recorded. This data was then used to calculate the LSI using the formula stated earlier.
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3.2.4.4 Data analyses
Statistical analyses were carried out using GenStat® Version 12.1 software. A Repeated Measures Analysis of Variance (RM-ANOVA) was carried out on each of the data sets for larval percentage survival and growth (measured by LSI) at the three test temperatures. Data were inspected visually to ensure there were no large deviations from normality; however the RM-ANOVA test chosen was robust enough to account for slight deviations from the assumptions of a normally distributed set of data (GenStat, n.d.).
The data on larval survival was transformed using an arcsine-square root transformation (Sokal & Rohlf, 1973) prior to running the RM-ANOVA. Data on larval growth did not require transformation. A posteriori analyses were carried out for both the growth and survival data by One-Way ANOVA among the treatments within each day of culture to determine the time period at which a significant separation in the trends was seen. Least significant differences (LSDs) were also used to examine the separation of the means.
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3.3 Results
3.3.1 Salinity tolerance tests
Data on larval survival gathered during the acclimation periods for both the coarse and fine-resolution salinity tests is not reported here as survival remained at 100 % throughout all acclimation runs with the exception of one, which experienced severe mortality and from which all larvae were discarded.
3.3.1.1 Larval survival during coarse-resolution tolerance tests
Larval survivorship during the coarse-resolution phase of testing is displayed in Figures 3.1, 3.2, 3.3 and 3.4 for zoeal stages I, III, V and VII respectively. Larval survival at the end of the tests was highest (36 %) at 10 ‰ for zoea I, both 20 ‰ and 30 ‰ (50 % and 63 % survival respectively) for zoea III and 30 ‰ for zoea V (83 % survival) and zoea VII (96 % survival) respectively. All larvae maintained in freshwater did not survive past the fourth day of the experiment.
A distinct preference for salinities trending towards that of full-strength seawater is apparent as the larvae moult through zoeal stages III to V, with fully marine conditions being optimal for survival and growth beyond this developmental stage.
Mean survival between the 10 ‰ and 20 ‰ treatments for zoea I were largely similar (Figure 3.5), as was the case for the 20 ‰ and 30 ‰ treatments for zoea III and V larvae which were the only treatments containing surviving larvae at the end of the tolerance test. Exposing zoea I larvae to 30 ‰ resulted in poor survival. For zoea VII larvae, survival was a little better in the 30 ‰ than the 20 ‰ treatment. This data was used to select the range of salinities to be evaluated for the fine-resolution phase of testing.
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100
90
80
70
60 0 ‰ Survival (%) 50 10 ‰
40 20 ‰ 30 ‰ 30
20
10 30 ‰ 20 ‰ 0 10 ‰ Salinity 0 1 2 0 ‰ 3 4 Day 5
Figure 3.1 Survivorship performance of zoea I M. lar larvae during coarse-resolution salinity testing.
100
90
80
70
60 0 ‰ Survival (%) 50 10 ‰ 40 20 ‰ 30 ‰ 30
20
10 30 ‰ 20 ‰ 0 10 ‰ Salinity 0 1 2 0 ‰ 3 4 Day 5
Figure 3.2 Survivorship performance of zoea III M. lar larvae during coarse- resolution salinity testing.
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100
90
80
70
60 0 ‰ Survival (%) 50 10 ‰
40 20 ‰ 30 ‰ 30
20
10 30 ‰ 20 ‰ 0 10 ‰ Salinity 0 1 2 0 ‰ 3 4 Day 5
Figure 3.3 Survivorship performance of zoea V M. lar larvae during coarse- resolution salinity testing.
100
90
80
70
60 0 ‰ Survival (%) 50 10 ‰ 40 20 ‰ 30 ‰ 30
20
10 30 ‰ 20 ‰ 0 10 ‰ Salinity 0 1 2 0 ‰ 3 4 Day 5
Figure 3.4 Survivorship performance of zoea VII M. lar larvae during coarse- resolution salinity testing.
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0‰ 10‰ 20‰ 30‰ c 100
90 b
80
70 a a a a
60 a a 50
40 Survival (%) 30 a
20 b 10
0 I III V VII Larval Stage Figure 3.5 Effect of coarse resolution test salinities on survival of M. lar larvae (Mean ± S.E., n = 4). The superscripted letters indicate significant differences within each stage (p < 0.05).
3.3.1.2 Larval growth during coarse-resolution tolerance tests
Larval growth as measured by increase in larval stage number during the coarse- resolution phase of testing is displayed in Figures 3.6, 3.7, 3.8 and 3.9 for zoeal stages I, III, V and VII respectively. The decrease in larval stage number for the 0 ‰ and 10 ‰ treatments in Figure 3.8 for stage V was due to the slow mortality rate of individuals which did not moult into subsequent development stages. This decreased the average stage number which displayed a negative trend as these individuals eventually died. Larval development at the end of the tests was fastest at 10 ‰ for zoea I where the subsequent larval stage was reached on day 3 of the experiment, while other treatments contained larvae which lagged in development. 20 ‰ appears to be suitable for zoea III, as it was the only treatment to contain surviving larvae by the end of the test. The 20 ‰ and 30 ‰ treatments were optimal for zoea V, with similar performance between treatments, and 30 ‰ for zoea VII. These findings agree with the coarse-resolution test salinity survival data, indicating a strong preference for fully marine conditions as the larvae of M. lar develop through their first few zoeal stages.
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2
1.8
1.6
1.4
1.2 0 ‰ Larval Stage Increase 1 10 ‰ 0.8 20 ‰ 30 ‰ 0.6
0.4
0.2 30 ‰ 20 ‰ 0 10 ‰ Salinity 0 1 2 0 ‰ 3 4 Day 5
Figure 3.6 Growth performance of zoea I M. lar larvae during coarse-resolution salinity testing.
3.3
3.25
3.2
3.15
3.1 0 ‰ Larval Stage Increase 10 ‰ 3.05 20 ‰ 3 30 ‰
2.95
2.9 30 ‰ 20 ‰ 2.85 10 ‰ Salinity 0 1 2 0 ‰ 3 4 Day 5
Figure 3.7 Growth performance of zoea III M. lar larvae during coarse-resolution salinity testing.
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6
5
4
0 ‰ Larval Stage Increase 3 10 ‰ 20 ‰ 2 30 ‰
1 30 ‰ 20 ‰ 0 10 ‰ Salinity 0 1 0 ‰ 2 3 4 Day 5
Figure 3.8 Growth performance of zoea V M. lar larvae during coarse-resolution salinity testing.
8.4
8.2
8
7.8
7.6
7.4 0 ‰ Larval Stage Increase 10 ‰ 7.2 20 ‰ 7 30 ‰ 6.8
6.6
6.4 30 ‰ 20 ‰ 6.2 10 ‰ Salinity 0 1 0 ‰ 2 3 4 Day 5
Figure 3.9 Growth performance of zoea VII M. lar larvae during coarse-resolution salinity testing.
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3.3.1.3 Larval survival during fine-resolution tolerance tests
Larval survivorship during the fine-resolution phase of testing is displayed in Figures 3.10, 3.11, 3.12 and 3.13 for zoeal stages I, III, V and VII respectively, with results of the One-Way ANOVA on the effect of larval survival shown in Table 3.2. Larval survival at the end of the experiments was highest at 15 ‰ for zoea I at 40 %. Survival for zoea III larvae was more varied, with the highest value obtained at 20 ‰ (90 %); however, treatments maintained at 25 ‰ and 30 ‰ showed appreciable results as well (83 % and 86 % respectively). Zoea V larvae survived best at 30 ‰ (90 %), but the 25 ‰ treatment produced a comparable result of 80 %. A similar result was observed with zoea VII larvae in the 20 ‰, 25 ‰ and 30 ‰ treatments, which experienced 83 %, 96 % and 93 % final survival respectively.
100
90
80
70 5 ‰ 60 10 ‰ Survival (%) 50 15 ‰ 40 20 ‰ 30 25 ‰ 20 25 ‰ 10 20 ‰ 15 ‰ 0 Salinity 10 ‰ 0 1 2 5 ‰ 3 4 Day 5
Figure 3.10 Survivorship performance of zoea I M. lar larvae during fine-resolution salinity testing.
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100
90
80
70 5 ‰ 60 10 ‰
Survival (%) 50 15 ‰
40 20 ‰
30 25 ‰ 30 ‰ 20 30 ‰ 25 ‰ 10 20 ‰ 15 ‰ Salinity 0 10 ‰ 0 5 ‰ 1 2 3 4 Day 5
Figure 3.11 Survivorship performance of zoea III M. lar larvae during fine-resolution salinity testing.
100
90
80
70 10 ‰ 60 15 ‰ Survival (%) 50 20 ‰ 40 25 ‰ 30 30 ‰ 20 30 ‰ 10 25 ‰ 20 ‰ 0 Salinity 15 ‰ 0 1 2 10 ‰ 3 4 Day 5
Figure 3.12 Survivorship performance of zoea V M. lar larvae during fine-resolution salinity testing.
159 Monal Lal MSc THESIS: CHAPTER THREE ______
100
90
80
70 10 ‰ 60 15 ‰ Survival (%) 50 20 ‰ 40 25 ‰ 30 30 ‰ 20 30 ‰ 10 25 ‰ 20 ‰ 0 Salinity 15 ‰ 0 1 2 10 ‰ 3 4 Day 5
Figure 3.13 Survivorship performance of zoea VII M. lar larvae during fine-resolution salinity testing.
Table 3.2 Summary of One-Way ANOVA results on the effect of larval survival at fine-resolution test salinities. % survival at F value P value LSD larval stage I 2.60 0.100 35.77 III 14.33 <.001 33.55 V 58.92 <.001 16.94 VII 18.18 <.001 22.03
Differences in mean survival data between the 15 ‰ and 20 ‰ treatments for zoea I larvae (Figure 3.14) showed large variation (LSD = 35.77) and were not significant (One-
Way ANOVA: F4,10 = 2.6 and p > 0.05; as summarised in Table 3.2). These were the only remaining treatments containing surviving larvae at the end of the experiment. Indications are that zoea I and II larvae are able to tolerate salinities up 20 ‰, however overall survival was better under conditions ranging from 10 ‰ to 15 ‰.
Once larvae moult into the third and fourth zoeal stages, they appear to require a salinity of at least 20 ‰, but are able to tolerate salinities as high as 30 ‰. Larvae exposed to
160 Monal Lal MSc THESIS: CHAPTER THREE ______treatment salinities below 20 ‰ recorded final percentage survival values of less than 20 %. A similar trend was observed with zoea V and VI larvae, with largely similar final survival rates in the 25 ‰ and 30 ‰ treatments but poor survival below 25 ‰.
With zoea VII and VIII larvae, slightly different salinity preferences were apparent. Survival was optimal at 25‰ and 30 ‰, however appreciable survival was also noted in the 15 ‰ and 20 ‰ treatments.
Differences in the data for zoea III (One-Way ANOVA: F4,10 = 14.3 and p < 0.001), zoea
V (One-Way ANOVA: F4,10 = 58.9 and p < 0.001) and zoea VII (One-Way ANOVA:
F4,10 = 18.2 and p < 0.001) were found to be highly significant (refer to Table 3.2).
A reduction of variation in the data between treatments as the larvae develop demonstrated by decreasing LSD values of 33.6, 16.9 and 22.0 for zoeal stages III, V and VII respectively (Table 3.2), may indicate that the larvae of M. lar become progressively more euryhaline as they develop until they moult into zoea VIII.
10‰ 15‰ 20‰ 25‰ 30‰
b b b b b c c 100 bc
90 b 80
70
60
50
40 Survival (%) a 30 a a a a
20
10
0 I III V VII Larval Stage Figure 3.14 Effect of fine-resolution test salinities on survival of M. lar larvae (Mean ± S.E., n = 5). The superscripted letters indicate significant differences within each stage (p < 0.05).
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3.3.1.4 Larval growth during fine-resolution tolerance tests
Larval growth as measured by increase in larval stage number during the coarse- resolution phase of testing is displayed in Figures 3.15, 3.16, 3.17 and 3.18 for zoeal stages I, III, V and VII respectively.
Larval development at the end of the experiments was fastest at 15 ‰ for zoea I where the subsequent larval stage was reached on day 2 for both treatments, while other treatments lagged in development. Development for zoea III larvae in the 20 ‰ treatment was accelerated, with all larvae reaching zoea IV by day 3, and all 25 ‰ and 30 ‰ treatment larvae also moulting to this stage a day later.
Moulting frequency was most rapid in the 30 ‰ treatment for zoea V larvae, followed by larvae in the 25 ‰ treatment. A very similar result was observed for zoea VII larvae, with all larvae in the 30 ‰ treatment moulting to stage VIII on day 3 of the experiment, closely followed by 25 ‰ treatment larvae which had moulted by day 4.
Comparing mean larval development increases over the experimental period (Figure 3.19), zoea I larval development increased at the same rate between the 15 ‰ and 20 ‰ treatments. The same trend was observed between the 25 ‰ and 30 ‰ treatments for zoea III larvae, however the 30 ‰ treatments containing zoea V and VII larvae developed much faster (an averaged larval stage increase of approximately 1.6), when compared to the other treatments. This data displays similar trends to those evident in the survivorship performance of the larvae, indicating that those larvae which survived best at the “optimal” test salinities also developed the most during the test period.
162 Monal Lal MSc THESIS: CHAPTER THREE ______
2
1.8
1.6
1.4 5 ‰ 1.2 10 ‰ Larval Stage Increase 1 15 ‰ 0.8 20 ‰ 0.6 25 ‰ 0.4 25 ‰ 0.2 20 ‰ 15 ‰ Salinity 0 10 ‰ 0 1 2 5 ‰ 3 4 Day 5
Figure 3.15 Growth performance of zoea I M. lar larvae during fine-resolution salinity testing.
4.5
4
3.5
3 5 ‰ 10 ‰ 2.5 Larval Stage Increase 15 ‰ 2 20 ‰ 1.5 25 ‰
1 30 ‰ 25 ‰ 0.5 15 ‰ Salinity 0
0 1 5 ‰ 2 3 4 5 Day
Figure 3.16 Growth performance of zoea III M. lar larvae during fine-resolution salinity testing.
163 Monal Lal MSc THESIS: CHAPTER THREE ______
7
6
5 10 ‰ 4 15 ‰ Larval Stage Increase 3 20 ‰ 25 ‰ 2 30 ‰
1 30 ‰ 25 ‰ 20 ‰ Salinity 0 15 ‰ 0 10 ‰ 1 2 3 4 Day 5
Figure 3.17 Growth performance of zoea V M. lar larvae during fine-resolution salinity testing.
9
8
7
6 10 ‰ 5 15 ‰ Larval Stage Increase 4 20 ‰
3 25 ‰ 30 ‰ 2 30 ‰ 1 25 ‰ 20 ‰ 0 Salinity 15 ‰ 0 1 2 10 ‰ 3 4 Day 5
Figure 3.18 Growth performance of zoea VII M. lar larvae during fine-resolution salinity testing.
164 Monal Lal MSc THESIS: CHAPTER THREE ______
10‰ 15‰ 20‰ 25‰ 30‰
2
(Stage) 1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
Averaged Larval Development Increase Increase Development Larval Averaged 0 I III V VII Larval Stage Figure 3.19 Effect of fine resolution test salinities on growth of M. lar larvae (Mean ± S.E., n = 5).
3.3.1.5 Salinity acclimation protocol for larvae
Based on the optimal rates of larval survival and development in the results of the salinity tolerance tests, a preliminary salinity acclimation protocol was developed for use in further M. lar larviculture work. This protocol is detailed in Table 3.3.
Table 3.3 Salinity acclimation protocol for larviculture of M. lar. Developmental stage Salinity range zoea I – II 10 – 15 ‰ zoea III – IV 15 – 25 ‰ zoea V – VI 25 – 30 ‰ zoea VII – XIII 30 ‰ + up to 35 ‰ PL1 35 ‰ acclimated down to 0 ‰
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3.3.2 Temperature tolerance tests
Larval survivorship and growth measurements along with statistical analysis of the data gathered during the temperature tolerance tests are reported here.
3.3.2.1 Larval survival at treatment temperatures
Larval survivorship measured during the temperature tolerance tests is displayed in Figure 3.20. Larvae exposed to the 30 > survival (18 %) at the end of the experiment compared to the other two treatments, which were 10.7 % and 3.1 % for the 28 (% !
The survival trend between the three treatments remained fairly uniform from the start of the experiment until day 6, when the 28 /6 (% treatment. It is apparent that the survival rate for M. lar larvae improves with an increase in temperature.
26 28 30
100 90 80 70 60 50 40 Survival (%) 30 20 10 0 0246810
Day of Culture Figure 3.20 Effect of treatment temperatures on larval survival (Mean ± S.E., n = 3).
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The differences in the data as a result of the effect of treatment were found to be significant (RM-ANOVA: F2,6 = 5.5 and p = 0.04; see Table 3.4), as was the effect of interaction between treatments and time (Repeated Measures ANOVA: F10,30 = 3.7 and p = 0.04), confirming that the 28 /6 than the 26
Table 3.4 Results of Repeated Measures ANOVA for the effect of temperature on survival of M. lar larvae.
Source of variation d.f. s.s. m.s. v.r. F pr. Subject stratum Temperature 2 0.085082 0.042541 5.47 0.044 Residual 6 0.046632 0.007772 1.88
Subject.Time stratum d.f. correction factor 0.3962 Time 5 9.644053 1.928811 465.89 <.001 Time.Temperature 10 0.152293 0.015229 3.68 0.036 Residual 30 0.124202 0.004140 Total 53 10.052262 (d.f. are multiplied by the correction factors before calculating F probabilities)
A posteriori analysis by One-Way ANOVA and LSDs (Table 3.5) to determine the points during the experiment at which larval survival rates differed, showed that separation of the 28 /6 (% atment on day 6 (One-Way ANOVA:
F2,6 = 13.8 and p = 0.006) and separation of the 30 (. eatment on day 10 (One-Way ANOVA: F2,6 = 6.7 and p = 0.03) were significant.
Table 3.5 Summary of One-Way ANOVA results on the effect of larval survival against treatment temperatures. Day of Culture F value P value LSD 0--- 2 0.57 0.591 0.1745 4 1.07 0.401 0.1336 6 13.83 0.006 0.0892 8 4.53 0.063 0.1626 10 6.71 0.030 0.1759
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3.3.2.2 Larval growth at treatment temperatures
Larval growth as measured by LSI during the temperature tolerance tests is displayed in Figure 3.21. The 30 4 ? stages much faster than did the other treatments. Observations made on larval staging in order to calculate LSI found that in the majority of samples, larvae collected from the 30 ? treatments. By the end of the experiment, averaged LSI was 3.36, 4.16 and 4.85 for the 26 (. /6 !
26 28 30
6
5
4
3
2
Larval Staging Index (LSI) 1
0 0246810
Day of Culture Figure 3.21 Effect of treatment temperatures on larval growth (Mean ± S.E., n = 3).
The differences in the data as a result of the effect of treatment were found to be significant (RM-ANOVA: F2,6 = 13.9 and p < 0.01; see Table 3.6), as was the effect of interaction between the treatments and time (RM-ANOVA: F10,30 = 7.2 and p = 0.002); inferring that the 30 (% (. treatments.
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Table 3.6 Results of Repeated Measures ANOVA for the effect of temperature on growth of M. lar larvae.
Source of variation d.f. s.s. m.s. v.r. F pr. Subject stratum Temperature 2 5.20348 2.60174 13.85 0.006 Residual 6 1.12702 0.18784 6.40
Subject.Time stratum d.f. correction factor 0.4646 Time 5 67.69571 13.53914 461.36 <.001 Time.Temperature 10 2.10730 0.21073 7.18 0.002 Residual 30 0.88038 0.02935 Total 53 77.01388 (d.f. are multiplied by the correction factors before calculating F probabilities)
A posteriori analysis by One-Way ANOVA (Table 3.7) to determine the points during the experiment at which larval growth rates differed, showed that separation of all the treatment trends from each other on day 2 (One-Way ANOVA: F2,6 = 10.8 and p = 0.01;
LSD = 0.31), day 8 (One-Way ANOVA: F2,6 = 21.35 and p = 0.002; LSD = 0.40) and day 10 (One-Way ANOVA: F2,6 = 52.0 and p < 0.001; LSD = 0.36) of the experiment were significant.
Table 3.7 Summary of One-Way ANOVA results on the effect of larval growth against treatment temperatures. Day of Culture F value P value LSD 0--- 2 10.80 0.010 0.3122 4 3.38 0.104 0.624 6 3.75 0.088 0.746 8 21.35 0.002 0.4048 10 52.04 <.001 0.3570
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3.4 Discussion
3.4.1 Salinity tolerance experiments
The salinity tolerance experiments indicate that survival and growth of M. lar larvae are higher under conditions of salinity which are either entirely fresh or slightly brackish at hatch and gradually increase up to full-strength seawater by the time the mid-way point in larval development is reached. Larvae of M. lar are unable to survive in freshwater beyond a period of 4 days, confirming that this species has a truly oceanic larval dispersal phase (Nandlal, 2010; Mather et al., 2006; Mather, 2002; Atkinson, 1977, 1973; Kubota, 1972 and Maciolek, 1972), which helps to account for it being by far the most widespread species of the genus Macrobrachium (Short, 2004).
A comparison of this life history trait with those of other Macrobrachium spp. shows that there are relatively few species which have larvae that require salinities for successful development which approach fully marine conditions. These species include M. equidens which requires approximately 33 ‰ (Ngoc-Ho, 1976 in Shokita, 1985), M. grandimanus which requires 17.5 – 35 ‰ (Shokita, 1985), M. intermedium whose lifecycle has not been completed in captivity yet but with the first 8 zoeal stages being reared at 35 ‰ (Williamson, 1971), M. acanthurus which was reared between 23.5 and 35 ‰ by Dobkin (1971) M. vollenhovenii which has a requirement for between 16 and 24 ‰ (Willführ- Nast et al., 1993) and M. americanum which requires between 20 and 30 ‰ for its early larval development after which salinity is reduced to between 15 and 20 ‰ (Holtschmit & Pfeiler, 1984). Holthuis (1980) states that M. latidactylus and M. latimanus may also have a marine larval development phase.
An interesting observation by the carcinologist L. B. Holthuis is that out of all these species, M. intermedium is “the only known species of the genus that spends its entire life in the sea” (Holthuis, 1980; Williamson, 1971), while the rest occupy brackish-water or entirely freshwater habitats as adults (Short, 1998, 2004, n.d.; Holthuis, 1980).
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This correlates with observations made by other researchers (de Grave et al., 2008; Alekhnovich & Kulesh, 2001; Jalihal et al., 1993; Shokita, 1985), which distinguish three types of larval developmental patterns in freshwater prawns of the genus Macrobrachium on the basis of criteria such as the number and size of eggs, larval development duration, environmental parameters for larval development and the number of zoeal stages; viz. prolonged/normal, semi-abbreviated/partially abbreviated and abbreviated/completely abbreviated development as discussed earlier in Chapter 2.
The two measures of larval survival and larval growth used here to determine salinity optima displayed the same pattern; i.e. the salinities observed to be optimal for survival were also optimal for growth.
One of the limitations of the salinity tolerance study was the difficulty in maintaining larvae in small culture volumes for extended periods of time. Very heavy mortality was observed when attempts were made to rear larvae in the 1 L glass jars used for the tolerance tests beyond a period of 10 days. Larvae reared in mass culture in 1000 L LRTs however, did not appear to encounter this problem. A similar finding was reported by Nandlal (2010). One reason for this may be that larvae reared in mass culture could have had access to a live food source e.g. biofloc, of which larvae reared in the glass jars were deprived, although an effort was made to mitigate this circumstance by adding biofloc particles to the jars when the larvae were fed.
Another limitation of the salinity investigations was the unavailability of sufficient numbers of larvae for experimentation beyond stage VII. For a complete investigation of the larval salinity requirements of M. lar, it would have been ideal to be able to test zoea larvae from stages IX, XI and XIII as well as PL, to determine their salinity tolerance ranges. It has been inferred from the results of the current study that the larvae of M. lar require a constant salinity for successful development beyond stages VII and VIII until they metamorphose into PL, because this is the case for other species of Macrobrachium e.g. M. rosenbergii (Nandlal & Pickering, 2006b; Takano, 1987a; Uno & Kwon, 1969; Ling, 1961). Indications to support this are reports by Holthuis on the Siboga Expedition in Short (2004) that two specimens of juvenile M. lar were collected from a depth of 55
171 Monal Lal MSc THESIS: CHAPTER THREE ______m in the Bay of Bima, Sumbawa, Indonesia on a reef with a mud and fine coral sand substrate along with observations by Kubota (1972) in Hawaii of newly-settled juveniles in a freshwater stream 100 yards from the ocean. This implies that larval development is completed under entirely marine conditions, and juveniles migrate towards land and travel upstream into the high elevation streams which are the adult habitat.
It was unfortunate that seawater of 35 ‰ was unavailable at the Seawater Laboratory, so the effect of full-strength seawater on the growth and survival rates of M. lar larvae could not be determined. This is an area that future M. lar larviculture research could address to determine if an improvement in larval culture performance may be seen.
3.4.2 Temperature tolerance tests
Results of the temperature tolerance investigations revealed that a temperature range of 30 ± 0.5 M. lar larvae. This is similar to the requirements of M. rosenbergii, with reported ranges of 28 ± 0.5 @ A 2 won, 1969), 26 – 31 (Takano, 1987a), 28.9 ($6/6$<B C in Atkinson, 1973) and 27 – 32 et al., 2000 in Cheng et al., 2003).
The most comprehensive published investigation to date on the temperature requirements of M. lar larvae has been carried out by Atkinson (1973), where ranges of 21 – 22.5 25.5 – 27.2 (.6– 30.5 23 – 26.5 (4 and 30 He also reports that as M. lar is an insular species, it is probably less tolerant of higher temperatures than M. rosenbergii, which is an estuarine, continental species. This differs from the findings of the current study, which found the optimal range to be on the higher end of Atkinson’s reported range.
Larviculture research by Nandlal (2010) was carried out at 27 – 30 likely tolerance range of 18 – 28 M. lar, as they disperse via ocean currents. If the lower thermal tolerance limits for this species are indeed as low as 18 this implies a prolonged pelagic development time as larval respiration and moulting
172 Monal Lal MSc THESIS: CHAPTER THREE ______frequency rates vary as a function of temperature (Anger, 2001). Atkinson (1973) mentions that the larvae of M. lar may undergo an extensive development period, probably as long as at least three months based on these larval life history characteristics. This was reflected in the results of the current study, where the first and last PL produced metamorphosed after 77 and 110 days of culture respectively (see Chapter 2), at a temperature of 28 ± 0.5
An ideal extension to the temperature tolerance investigations carried out here would be to investigate the upper and lower temperature limits which permit larval survival and growth. Unfortunately, this was beyond the scope of this study as time and budgetary constraints restricted the focus of the study to determining an optimal temperature for successful mass culture.
3.4.3 Further work
Improvement of mass larviculture techniques to improve larval survival past zoea VII and VIII will facilitate further culture condition experimentation with M. lar larvae. Repetition of the investigations carried out in this chapter to both verify the results reported here and to test a narrower range of test salinites will aid in fine-tuning the approaches taken for larviculture of M. lar.
3.5 Conclusion
As a result of the current investigation, baseline data on the salinity and temperature requirements of M. lar larvae have been collected and will be useful in further larviculture research work with this species. A salinity acclimation protocol has also been developed that will be useful in developing a mass culture technique for hatchery production of PL.
It has now been established that the larvae of M. lar are able to hatch in either freshwater or brackish-water of approximately 10 ‰, but require gradually increasing salinities post- hatch reaching 30 – 35 ‰ by stage V – VI, which probably needs to be maintained until
173 Monal Lal MSc THESIS: CHAPTER THREE ______metamorphosis into PL. Stage I larvae kept in freshwater die within 4 days unless transferred to brackish-water.
A temperature of 30 ± 0.5 > growth rates, and is similar to the requirements of other Macrobrachium spp. which have a tropical distribution e.g. M. rosenbergii.
174 Monal Lal MSc THESIS: CHAPTER FOUR ______
CHAPTER FOUR
LARVAL DEVELOPMENT STAGES OF THE MONKEY RIVER PRAWN Macrobrachium lar
4.1 Introduction
This chapter describes the morphological development of the larvae of M. lar reared from hatch until metamorphosis into post-larva. The procedures used for rearing the larvae are described in Chapter 2 of this thesis.
4.1.1 Larval development in Macrobrachium prawns
4.1.1.1 Patterns of larval development
Patterns of larval development in Macrobrachium spp. have been observed to fall into three major categories; viz. typical or prolonged/normal, semi– or partially-abbreviated and abbreviated or completely abbreviated; on the basis of criteria such as the number and size of eggs, larval development duration, environmental parameters required for larval development and the number of zoeal stages (de Grave et al., 2008; Alekhnovich & Kulesh, 2001; Jalihal et al., 1993; Shokita, 1985;). These have been described in greater detail earlier in Chapter 2.
Among the species which exhibit the typical or prolonged/normal type of development, there are comparatively few which, like M. lar, require salinities for successful development that approach fully marine conditions.
These species include M. equidens which requires approximately 33 ‰ (Ngoc-Ho, 1976 in Shokita, 1985), M. grandimanus which requires 17.5 – 35 ‰ (Shokita, 1985), M. intermedium whose lifecycle has not been closed in captivity yet but with the first 8 zoeal stages being reared at 35 ‰ (Williamson, 1971) and M. acanthurus which was reared between 23.5 and 35 ‰ by Dobkin (1971).
175 Monal Lal MSc THESIS: CHAPTER FOUR ______
Other species include M. vollenhovenii which has a requirement for between 16 and 24 ‰ (Willführ-Nast et al., 1993) and M. americanum which requires between 20 and 30 ‰ for its early larval development after which salinity is reduced to between 15 and 20 ‰ (Holtschmit & Pfeiler, 1984).
Holthuis (1980) states that M. latidactylus and M. latimanus may also have a marine larval development phase. A summary of developmental characteristics and rearing condition requirements of Macrobrachium spp. whose larviculture has been investigated is provided in Table 4.1 overleaf.
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Table 4.1 Developmental characteristics and rearing conditions of Macrobrachium spp. Abbreviations: S = small, M = medium and L = large. In cases where several studies have been reported on the same species, only the study describing the shortest development time has been listed. Source: Shokita (1985) and Willführ-Nast et al. (1993). Species Egg Number of Development Optimal Source type zoeal stages duration (day) and salinity range temperature ( (‰) M. lar S 13 77 – 110 at 28 ± 0.8 30 + This study M. sp. (not specified) S 12 11 at 26 – 29.2 32.1 – 32.9 Ngoc-Ho (1976) M. equidens S 10 36 – 53 at 26 – 29.5 32.9 Ngoc-Ho (1976)
M. grandimanus S 9 27 – 30 at 27 17.5 – 35 Shokita (1985) M. intermedium S 10; no PL ? ~35 Williamson (1971)
177 produced M. acanthurus S 10 32 – 42 at ? ~21 Choudhury (1970) (60% seawater) M. vollenhovenii S 8 – 9 63 at 28.1 ± 0.12 16 – 24 Willführ-Nast et al. (1993) M. americanum S 11 53 at 29 ± 0.5 15 ± 0.1 Monaco (1975) M. novaehollandiae S 10 41 – 58 at 13 – 28 23 Greenwood et al. (1976) M. olfersii S Over 12 ? ~21 – 35; no Dugger & Dobkin (1975) PL produced M. carcinus S 12 55 – 56 at 24 – 28 14 – 16 Choudhury (1971c) M. formosense S 9 22 at 24 – 28 13 – 16 In Shokita (1985) M. australiense M 3 6 at 21 – 28 0 Fielder (1970) M. rosenbergii S 11 28 ± 0.5 12 – 12.3 Uno & Kwon (1969) M. rudis ? 6 ? at 25 – 32 10 – 12 Haque (1980) in Khan et al. (1984) M. birmanicus ? 5; no PL ? at 10 – 12 Khan et al. (1984) produced 20 – 25 until Z V
177 Monal Lal MSc THESIS: CHAPTER FOUR ______
M. amazonicum S 8 – 9 23 – 26 at 24 ± 0.25 10 In Shokita (1985) M. nipponense S 9 15 – 20 at 27.8 – 8.26 – 9.32 In Shokita (1985) 28.2 M. niloticum S 8 – 10 ? 0 Williamson (1971) M. lamarrei M 3 ? 0 In Shokita (1985) M. dayanum ? 7 ? at 27 – 40 0 Khanam (1981) in Khan et al. (1984) M. lamarrei lamarrei M 3 5 – 6 at ? 0 In Shokita (1985) M. kistnense M 3 4 – 5 at 28 – 29 0 In Shokita (1985) M. hendersodayanum L 0 3 – 4 0 In Shokita (1985) M. shokitai L 1 20 hours at 28 – 29.5 0 Shokita (1973) in Shokita (1985) M. asperulum L 1 2 days at 27.8 – 28.2 0 In Shokita (1985) 178
178 Monal Lal MSc THESIS: CHAPTER FOUR ______
4.1.1.2 Growth processes
The larval development of crustaceans is usually described as a sequence of morphologically distinct stages, and transition through these stages occurs through the processes of moulting and ecdysis (Brown et al., 2010; Anger, 2001; Hickman Jr. et al., 2001).
The processes of moulting (formation of a new cuticle) and ecdysis (shedding of the old exocuticle and solidification of the new cuticle) are under hormonal (endocrine) control, and dependant on a number of factors including salinity, temperature, photoperiod and nutrition (Brown et al., 2010; Anger, 2001; Hickman Jr. et al., 2001). While some of these factors e.g. temperature affect primarily the frequency with which the animal moults, others e.g. nutrition appear to exert an influence on the increase in size at ecdysis (Anger, 2001).
The organs responsible for regulation of the moult cycle are the major hormone- producing organs; viz. the Y-organs, the X-organ-sinus-gland complex and the mandibular organs (Brown et al., 2010; Anger, 2001; Gore, 1985).
The moult cycle as originally described by Drach in 1939 with subsequent modifications by others is composed of five principal moult-stages (see Brown et al., 2010; Anger, 2001 and Gore, 1985). These are described below after Anger (2001) and Hayd et al. (2008) in a simplified form with the omission of details of the various sub-stages. A diagram depicting the structure of the crustacean cuticle is included in Figure 4.1 for reference.
179 Monal Lal MSc THESIS: CHAPTER FOUR ______
Figure 4.1 The generalised structure of the crustacean cuticle. Source: Stevenson (1985) in Brown et al. (2010).
Early postmoult (Stage A); modified after Hayd et al. (2008) and Anger (2001)
This moult-stage occurs fairly rapidly, lasting only a few minutes in larval crustaceans and less than an hour in juveniles; and is marked by a rapid increase in body size, the evagination of setae, spines and appendages along with stretching of the thin and limp cuticle. This occurs during and shortly after ecdysis (Stage E), and is achieved by the uptake of considerable amounts of water.
During early postmoult, the animal may perform pumping movements and drink water but otherwise remain inactive. In species which produce pelagic larvae, individuals tend to sink in the water column and cease to feed as the newly secreted cuticle and associated structures are still soft, and preclude the capture and consumption of food items.
180 Monal Lal MSc THESIS: CHAPTER FOUR ______
Late postmoult (Stage B); modified after Hayd et al. (2008) and Anger (2001)
The body of the animal reaches its final dimensions during this stage, the cuticle is strengthened and the epidermal tissues become denser. Calcification of the cuticle now begins and along with tissue development, these processes continue into the intermoult stage. Marine decapods take up calcium from their environment, whereas terrestrial and freshwater taxa mobilise carbonate deposits laid down in the hepatopancreas.
Intermoult (Stage C); modified after Hayd et al. (2008) and Anger (2001)
The endocuticle becomes fully developed during this stage, with total cuticle thickness reaching its maximum for the current moult event. The epidermis however, continues to grow, while further changes in the cuticle prior to ecdysis (Stage E) come to an end. The intermoult period is the longest moult-stage in the entire moult cycle and maximum growth in terms of biomass also occurs during this interval.
Premoult (Stage D); modified after Hayd et al. (2008) and Anger (2001)
The animal starts to prepare for ecdysis during this stage. This is marked by a withdrawal of the epidermis from the cuticle (the process of apolysis), and epidermal structures begin to invaginate. Apolysis is brought about by proteolytic and chitinolytic enzymes, and has been observed to occur first in those structures which are programmed to undergo marked morphogenetic changes. In late premoult, existing setae become fully separated from the old cuticle and any new structures which will appear after the moult event are formed. The new cuticle makes its first appearance here, and the new structures created during late premoult are now fixed and easily seen by their cuticular outline under microscopic examination.
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Ecdysis (Stage E); modified after Hayd et al. (2008) and Anger (2001)
This moult-stage is typically brief and lasts less than a minute in most larval decapods. Immediately prior to ecdysis, the animal takes up water and followed by this the old cuticle ruptures between the carapace and the pleon (abdomen); allowing the animal to remove itself from the old exoskeleton (exuvium). The exuvium is eventually separated from the body by movements of the animal to shed it.
Apart from bringing about the changes in the integument of decapod crustaceans which have been described earlier, the moult cycle also effects changes in the anatomy, biochemistry and physiology of other organ systems, including the hepatopancreas and haemolymph. Other cyclical patterns related to the moult cycle occur in the feeding, growth, chemical composition, autotomy and regeneration, respiration and excretion of the animal (Hayd et al., 2008; Anger, 2001).
There exist three known critical points during the moult cycle of decapod crustaceans; viz. the Point of No Return (PNR), the Reserve Saturation Point (RSP) and the Exuviation Threshold (Anger, 2001; Gore, 1985); the positions of which vary within the moult-cycle according to the various factors (environmental and otherwise) that influence the processes of moulting and ecdysis mentioned at the beginning of this section. One such factor of particular importance is that of nutrition, as a continuous lack of food (starvation) has been demonstrated to completely inhibit moulting (Anger, 2001).
Descriptions of these three critical points are given overleaf and shown in Figure 4.2.
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Figure 4.2 Critical points during the moult cycle of a decapod crustacean larva, showing the Reserve Saturation Point (RSP) and Point of No Return (PNR). Source: Gore (1985).
Reserve Saturation Point (RSP)
The RSP, also called the Point of Reserve Saturation (PRS), is defined by Anger (2001) as “the point in a moulting cycle where well-fed larvae have gained sufficient organic matter or energy (they have “saturated” their reserves) to develop successfully through premoult and ecdysis, independent of the presence or absence of food.” The larva may then complete a moult without needing to feed again.
Point of No Return (PNR)
If a larva is starved for a period from the time of hatch, it loses its ability to recover from nutritional stress. The point at which this occurs is called the “Point of No Return”, and even if starved larvae which have passed their PNR are subsequently fed, they may be able to survive for an extended period of time but will inevitably die without moulting to the next stage (Anger, 2001; Gore, 1985).
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Exuviation threshold
This is the critical point at which larvae may complete ecdysis and survive, or die while doing so once they progress past the PNR. When previously starved larvae are fed, some individuals are able to develop through some of the premoult stages and reach the Exuviation Threshold. These larvae however moult unsuccessfully and die in the transition between late premoult and ecdysis (Anger, 2001; Gore, 1985).
4.1.2 Important terms and concepts used in describing the morphological development of Macrobrachium larvae
4.1.2.1 Morphological terms
A number of morphological terms used to describe the biology of crustacean larvae have been used in the literature (often interchangeably), so the precise terms which will be used in the results section of this chapter to describe the morphological development of M. lar larvae will be defined here. The definitions below follow Anger (2001). A glossary of anatomical terms used to describe the morphology of crustacean larvae has also been included as Appendix 1.1.
Phase
This term denotes a sequence of morphologically equivalent developmental stages, e.g. all naupliar stages combined (e.g. for penaeid shrimp larvae) or all zoeal stages combined (e.g. for Macrobrachium larvae).
Instar (Moulting cycle stage/numerical stage)
An instar is defined as the duration of development over a single intermoult period for a larva; and the number of instars occurring in a larval development sequence is equivalent to the total number of moults passed through. The appearance of a new instar may be associated with a difference in the size and weight of the larva but not necessarily a
184 Monal Lal MSc THESIS: CHAPTER FOUR ______morphological change. Under ideal conditions, different instars may represent different stages. Successive instars within a given phase are denoted with Roman numerals, e.g. zoea I, zoea II etc..
Stage (Morphological stage)
Morphologically distinguishable instars are in general considered and named as different developmental or morphological stages. Roman numerals are assigned to denote successive stages within a given phase, e.g. zoea I, zoea II etc.. Note that the numbering system for stages is the same as that for instars.
Metamorphosis
This is the process by which a sudden and dramatic change in the morphology of two subsequent stages occurs, and is usually accompanied by changes in behaviour, feeding, ecology and physiology.
4.1.2.2 Zoea and prezoea larvae
All decapod crustaceans hatch from the egg as a zoea or prezoea larva. Zoea larvae are distinct from other larval types e.g. the nauplius and mysis forms found in penaeids and other taxa and the megalopa larvae of crabs.
They are characterised by features which include functional thoracopods (appendages attached to the cephalothorax) and paired compound eyes. Their antennae and antennules perform functions associated with the mechanical and chemical perception of prey and their mandibles are designed for biting food. The maxillules and maxillae are also functional and used to aid in feeding (Anger, 2001).
In the thoracic appendages, at least two pairs of maxillipeds are functional and the exopods fulfil general swimming functions while the endopods are used for holding food
185 Monal Lal MSc THESIS: CHAPTER FOUR ______items. Pleonal (abdominal) appendages are consistently absent or only rudimentary, and where they do appear they take the form of non-functional buds (Anger, 2001).
Some decapod taxa hatch from the egg as a prezoea stage, which normally lasts only for a few minutes before it moults into a zoea or equivalent stage. Prezoeae have no functional appendages or other larval organs and either do not swim or move by means of abrupt abdominal movements. Prezoeae have been reported for several decapod taxa (Anger, 2001), including Macrobrachium novaehollandiae (Gore, 1985; Greenwood et al., 1976).
4.1.2.3 Post-larva
This stage of development usually marks the beginning of the juvenile phase of development, and in some cases has also been referred to as the decapodid stage (Anger, 2001). For the purposes of this study however, the term post-larva will be used. The final moult during the larval phase of development may result in a dramatic change in morphology and behaviour of the animal, although in some decapod taxa these changes are more gradual and less obvious.
The post-larval phase is characterised by the pleopods becoming fully functional and being solely responsible for swimming, the pereiopods losing their natatory exopods and now being used for walking only while the same occurs with the maxillipeds which are used only for feeding. In some cases, vestiges of the natatory exopods may remain but are lost during subsequent moults. The biramous pleopods located in five pairs under the abdomen are often coupled together in pairs by the appendices internae, which are specialised hook-like structures located on the inner margins of the pleopod endopods. This is so that they are able to beat in a coordinated way to provide manoeuvring control during swimming (Anger, 2001). Some decapod taxa may develop through more than one post-larval stage (Anger, 2001; Gore, 1985). Post-larvae may also be assigned a number in subscript to denote the instar number. Hence the first PL instar moulted into from the last zoeal stage is PL1, and the following instar is PL2 etc..
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4.1.2.4 Variability in morphological development
Typical developmental traits of shrimps and prawns belonging to the infra-order Caridea include either gradual or dramatic morphological changes as the animal completes larval development. In the latter case, the number of instars and their morphology can vary intraspecifically (Anger, 2001).
In some cases, certain species pass through a variable and often unusually high number of larval stages. An example of this is Macrobrachium rosenbergii where routine hatchery operations generally recognise 11 zoeal stages (Uno & Kwon, 1969), whereas a laboratory-based study distinguished 17 distinct zoeal stages for the same species (Gomez Diaz & Kasahara, 1987). Even individuals of the same species hatched from a single female or belonging to a single cohort may display variability in developmental rates and/or pathways (Anger, 2001).
Decapod crustaceans exhibit a number of phenomena associated with variability in their developmental patterns. These are fully described in Gore (1985) and Anger (2001), and a few relevant to this study are described below:
Mark time moulting
This situation has been well documented in a number of decapod crustacean larviculture studies and occurs when a particular zoeal stage enters a sequence of moults during which very little change in morphology takes place, although each instar in this sequence may show a slight increase in size and/or weight. The larva may continue in this pattern for an indeterminate period of time and either eventually die, or resume an apparently normal moulting mode and complete its development (Gore, 1985).
Mark time moulting has been reported frequently in the phyllosoma larvae of scyllarid and panulirid lobsters as well as a number of Macrobrachium spp. which undergo the typical or prolonged/normal type of development (Gore, 1985). It may be a strategy to
187 Monal Lal MSc THESIS: CHAPTER FOUR ______delay metamorphosis until specific environmental cues indicate that suitable habitat for settlement and colonisation is available.
Terminally additive staging
The phenomenon of terminally additive staging is very similar to mark time moulting, with the exception that any additional instars the larva passes through are added after and not within the series of moults that occur during regular, normal development. The additional instars display only minor changes in morphology, but may be distinguished by visible differences in size. This extends the total larval development period indefinitely (Gore, 1985).
Terminally additive staging along with mark time moulting have both been documented in M. rosenbergii larvae (Ismael & New, 2004).
Skipped staging
Skipped staging occurs when one or more instars normally seen during the regular development sequence are bypassed as the larva develops towards metamorphosis. It is more common in those taxa (e.g. caridean shrimps and prawns) which normally undergo greatly extended larval development patterns (Gore, 1985), such as the typical or prolonged/normal type of development of Macrobrachium spp.
4.1.2.5 Larval dispersal, settlement and colonisation strategies
As M. lar is by far the most widespread species of the genus and has a broad Indo-West Pacific distribution (Short, 2004; Suzuki, 2001), a consideration of the larval dispersal, settlement and colonisation strategies known to be exhibited by some decapod crustaceans is important to understand the pattern of larval development shown by this species.
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There are two principal strategies which occur in the life cycles of estuarine species of decapod crustaceans. These are the export and retention strategies.
The export strategy involves dispersal of the larval stages to adjacent coastal or offshore marine areas out of the vicinity of the adult habitat, where conditions for larval development are more favourable. The retention strategy on the other hand is demonstrated by adaptation of all life cycle stages to estuarine conditions, which allows retention of larvae within the parental environment (Anger, 2001).
The life history of M. lar is an example of the former strategy, which has allowed the species to colonise habitats that are geographically widely separated, such as many of the major volcanic islands of the Pacific region (Nandlal, 2010; Short, 2004; Atkinson, 1977, 1973). A conceptual model of the export strategy is explained diagrammatically in Figure 4.3.
Figure 4.3 Conceptual model of the export strategy of larval dispersal as given by the example of a generalised grapsid crab. Source: Anger (2001).
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Anger (2001) mentions that as a consequence of the export strategy; larvae of species which utilize this pattern of dispersal do not require the evolution of special adaptations against osmotic, thermal or intense pelagic predation pressures, and hence resemble the larvae of fully marine species in many of their traits.
4.1.3 Description of larval development of Macrobrachium lar
The descriptions of the morphological development of M. lar larvae provided in this chapter have intentionally been kept simple, for the purpose of easily identifying developmental stages.
Typical descriptions of crustacean larval development from an academic standpoint are often very detailed and complete. Rather than adopting this approach, morphological descriptions provided here are from a more practical perspective; particularly with regard to larval staging, which has been undertaken by concentrating on a few, major and easily detectable features rather than minor morphological changes.
The same approach has been used in the construction of the guide to identifying the larval stages of M. lar included in the results section; the primary intention of which is to provide a means of simply and rapidly identifying live specimens for any future larviculture work which may be carried out.
4.1.4 Research objectives
Research carried out for this chapter was aimed at answering the question: is it possible to describe all larval developmental stages from hatch to metamorphosis, and elucidate the precise number of larval stages? In order to answer the larval development component of this question, the following specific objectives were designed:
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i) To simply describe the embryonic development stages of M. lar with the view of determining time till hatch of ovigerous females maintained in captivity. ii) To describe larvae of M. lar through all larval developmental stages until metamorphosis into post-larvae. iii) To produce a simple larval development guide for identifying the developmental stages of M. lar larvae.
4.2 Methodology
4.2.1 Embryonic development study
To observe the developmental stages of the eggs, 20 randomly selected females from each of the egg mass development categories described earlier were subjected to egg mass biopsies. These females were maintained separately from those animals which were to be used for hatching larvae in dedicated floating PVC pipe cages at 28 ± 0.5 as described in Chapter 2 of this thesis.
Females were removed from the cages by raising the frames in the water column and gently netting individuals using a nylon dip net with a 10 mm mesh size. Care was taken to minimize the amount of time the eggs were exposed to air. Once in the net each female was removed by hand and gently turned over to expose the ventral surface of the abdomen. Care was taken to ensure that the females did not struggle in the net to minimize the amount of handling stress and to avoid the loss of eggs when the egg mass scraped against the mesh of the net.
A pair of stainless steel fine-tipped dissecting forceps was disinfected by immersion in an
80 % ethyl ethanol (C2H5OH) solution and then used to remove a small portion of the egg mass. Where necessary, a disinfected stainless steel retractor was used to bend the pleopods backwards to expose portions of the egg mass for removal. The sites on the egg mass chosen for biopsy were randomly selected. The female was immediately returned to
191 Monal Lal MSc THESIS: CHAPTER FOUR ______the floating PVC pipe cage she was removed from after the biopsy sample had been collected.
The biopsy sample was wet-mounted on a glass cavity slide using freshwater with a coverslip and observed at low power (×10 objective) under a binocular compound light microscope. The egg development features were observed and recorded along with measurements of the long and short axes of a sample of 100 eggs of each developmental stage. A calibrated eyepiece graticule was used for all microscopic measurements. Using these measurements, the volumes of the eggs at various stages of development was also calculated, after Collart and Rabelo (1996).
C lh2 S V D T E 6 U
Where: l = egg length (short axis) in mm
V = Egg volume in mm3 h = egg height (long axis) in mm
The eggs were also photographed under the microscope using a digital camera with a tilting lens attachment for photomicroscopy. Observations of the features of general embryonic development visible during microscopy were recorded at the intervals during which biopsy samples were collected, in a similar fashion to that described for Macrobrachium olfersii by Müller et al. (2003) as shown in Figure 4.4. Complete descriptions of embryogenesis were not carried out, as the scope of this study was restricted to determining defining characteristics of egg development to assess time till hatch.
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Figure 4.4 Embryonic development of Macrobrachium olfersii.c–g: ventral view, h–j: lateral view. ab = abdomen, an = antennulae, at = antennae, ba = blastoporal area, bl = blastomeres, cf = cleavage furrows, cp = caudal papilla, ey = eye, gd = germinal disk, mn = mandible, ol = optic lobe, pa = postnaupliar appendages, st = stomodaeum, te = telson, yk = yolk, ym = yolk mass. Source:Mülleret al. (2003).
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4.2.2 Larval development study
4.2.2.1 Rearing of larvae
Larvae were reared in a mass culture experiment which began on the 15th of October 2009 and ended on the 18th of December 2009 after 65 days of culture. The general methodology and procedures used are described in Chapter 2.
A total of four circular 1000 L flat-bottomed polyethylene tanks were used to start this experiment, however only a single tank contained surviving larvae by day 65 of culture. On the 65th day of culture, 10 surviving larvae were transferred from that 1000 L mass culture polyethylene LRT into a 60 L cylindro-conical FRP LRT where they were cultured for a further 45 days.
4.2.2.2 Larval microscopy
All microscopy was carried out using a binocular compound microscope fitted with a calibrated eyepiece graticule. All photomicroscopy was carried out using a digital camera with a tilting lens attachment mounted on one of the microscope eyepieces.
Larvae were routinely examined once daily at approximately 0900 throughout the larviculture run. When disease outbreaks or other problems were encountered, microscopy was carried out more frequently, at least two or three times daily.
Larvae to be examined under the microscope were removed from the LRTs using a larval counting bowl and gently transferred onto a cavity slide using a wide bore (3 mm diameter) pipette with a few drops of tank water from the counting bowl. No coverslip was used, as this was found to squash the specimens. All observations and photographs taken were of live specimens and specimens were either preserved immediately afterwards in a solution of 80 % ethyl ethanol (C2H5OH) for larval staging work, or returned to the LRT.
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4.2.2.3 Determining larval developmental stages
A select number of morphological features present on the larvae were examined and changes in these features as they developed were recorded and used for characterising the larval developmental stages for M. lar.
These morphological features included carapace armature including the rostrum, supra- orbital spines and pterygostomian spines; developments of the tail fan (telson and uropods); pereiopods (walking legs) and pleopods (swimmerets); and the antennules and antennae. Larvae were also measured to determine their total and carapace lengths.
Larval measurements taken are shown below in Figure 4.5 and the features examined are shown in the drawings displayed in Figures 4.6, 4.7 and 4.8. A total of 10 larvae of the same apparent morphological developmental stage and age were sampled and used in making determinations of larval stages. All of these larvae were thoroughly examined to ensure that the morphological features being recorded were consistent among the individuals sampled.
Once determinations of stage had been made and found to be fairly consistent according to all observations recorded, specimens representative of each larval developmental stage were lodged at the Marine Reference Collection of the School of Marine Studies, Faculty of Science, Technology and Environment, University of the South Pacific, Suva, Fiji Islands under Catalogue Number 5940.
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Figure 4.5 Larval body measurements. CL = carapace length, AL = abdominal length and TL = total length.
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Figure 4.6 External characteristics of an M. lar larva. Only appendages present on the left side are shown here. Antenn. = antennule, Antenn. flag = antennular flagellae, Ant. flag. = antennal flagellum, Scaph. = scaphocerite (antennal scale), S. o. s. = supra-orbital spine, Ab. 1 – 6 = abdominal somites 1 through 6, Tel. = telson, Uro. end. = uropod endopodite, Uro. exo. = uropod exopodite, Pl. 1 – 5 = pleopods 1 through 5 and Per. 1 – 5 = pereiopods (walking legs) 1 through 5. Diagram is not drawn to scale.
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Figure 4.7 Rostrum and carapace armature on an M. lar larva. D.c. = dorsal carina of the rostrum, v.c. = ventral carina of the rostrum, S. o. s. = supra-orbital spine and Pteryg. sp. = pterygostomian spines. Diagram is not drawn to scale.
Figure 4.8 Pleopod morphology details. The arrow indicates the appendix interna. Diagram is not drawn to scale.
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4.2.2.4 Specimen drawings
All larval specimens were drawn in lateral view (except for zoea I which includes a dorsal view) by free hand sketches using a 2B pencil. These drawings were used to produce simple line diagrams showing the body outlines of the larvae. Internal structures including organs and musculature along with external chromatophore patterns were not drawn. Photographs taken of live specimens were used to aid in construction of the drawings. Only the appendages on the left hand side of the larva were drawn. All diagrams were then outlined using a 0.6 mm BIC Exact-tip pen in black ink before being scanned at a resolution of 600 dpi and processed using Adobe Photoshop version 7.0 software.
4.2.2.5 Specimen photograph processing
Adobe Photoshop version 7.0 software was also used for processing photographs taken of larval specimens. This included the addition of scale bars in microscopy photographs and adjusting contrasts and colour balances to produce the finished images. It would have been ideal to fix and mount a specimen permanently, and then produce a composite image using multiple photographs at differing depths of focus. Unfortunately this was not possible as live specimens were used which had to be returned to the LRT shortly after photography was complete.
For zoeal stages VII to PL1, individual larvae had grown too large to fit within the field of view of the microscope used, even at the weakest magnification levels in the lowest power objective. It therefore became necessary to take several photographs showing different portions of a single specimen. These images were then merged using Adobe Photoshop version 7.0 to create a composite image of the whole larva.
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4.2.3 Larval development staging guide
A guide to simply and rapidly determine the developmental stage of M. lar larvae was developed, based on larval features which were relatively simple and easily recognisable on live specimens.
Diagrams showing these features were drawn and included along with whole-body larval diagrams in the guide.
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4.3 Results
4.3.1 Embryonic development of M. lar larvae
The external characteristics of the eggs of M. lar underwent a number of visible changes as the embryos developed, and these are described below according to sampling intervals. Photographs of eggs at these sampling intervals are displayed in the colour plates section on pages 202 to 204.
14 days – Plates 4.1 a and b
The eggs of M. lar are an orange-amber colour at this stage. Cleavage has already occurred, however no embryonic tissues are visible and the germinal disc is not present. The bulk of the egg volume is occupied by the yolk mass.
18 days – Plates 4.2 a and b
At this time the embryonised nauplius has started to form and begins to grow along the long axis of the egg. This is shown by the clear area extending along one side of the egg adjacent to the yolk mass.
21 days – Plates 4.3 a and b
The heart can be seen beating at the base of the clear area and eyespots begin to form, being long and thin at first. The eyespots are easily recognised by being darkly pigmented, contrasting with the amber colour of the yolk mass.
23 days – Plates 4.4 a and b
Eyespots have increased in size and the yolk mass has become reduced in size. The eyes have become more oval in shape and individual ommatidia (facets) of the compound eye can be seen. Post-naupliar appendages can also be discerned.
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25 days – Plates 4.5 a and b
By day 25, the eyes have further increased in size, occupying a significant volume of the anterior portion of the embryo as post-naupliar development enters its final stages. The yolk mass is visibly reduced when compared to the previous interval.
27 days – Plates 4.6 a and b
The abdominal somites now become apparent and the embryo begins to move inside the egg. Mandibles, antennae and antennules are also visible. The embryo can be seen to be bent into a “u” shape, with the abdomen extending underneath the head region. The telson now becomes visible and is folded over the eyes. The eyes are now the most prominent feature inside the egg. The yolk mass has been incorporated inside the larval hepatopancreas.
29 days – Plate 4.7 a and b
This interval defines the pre-hatching embryo. The yolk mass is highly reduced and occupies the foregut and midgut regions of the digestive tract. The larval tissues are the most prominent feature inside the egg. Close to hatching, the chorion detaches from the surface of the embryo allowing it to make stretching and rolling movements in preparation for hatch. The chorion will be shed during eclosion as the larva emerges.
The eggs of M. lar are an elongated ellipsoid shape, and increase in volume as the embryo develops. Egg measurements taken at the sampling intervals are given in Table 4.2.
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Table 4.2 Egg development of M. lar at 28 ± 0.5 . Approximate Average long axis Average short axis Average egg age (LA) of egg (SA) of egg volume (Days) (mm ± S.E.) (mm ± S.E.) (mm3 ± S.E.) 14 0.574 ± 0.002 0.490 ± 0.004 0.072 ± 0.013 18 0.607 ± 0.003 0.513 ±0.002 0.084 ± 0.007 21 0.626 ± 0.005 0.531 ± 0.001 0.093 ± 0.008 23 0.698 ± 0.008 0.541 ± 0.001 0.107 ± 0.011 25 0.701 ± 0.004 0.539 ± 0.001 0.107 ± 0.006 27 0.725 ± 0.004 0.548 ± 0.002 0.114 ± 0.007 29 0.760 ± 0.004 0.578 ± 0.005 0.133 ± 0.013
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COLOUR PLATES
Egg Development (“b” plates are larger views of “a”)
ab Plate 4.1 Eggs of M. lar after approximately 14 days of development at 28
ab Plate 4.2 Eggs of M. lar after approximately 18 days of development at 28 Early embryonic tissues are seen adjacent to the yolk mass.
ab Plate 4.3 Eggs of M. lar after approximately 21 days of development at 28 Eyespots are now visible and the heart can be seen beating.
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ab Plate 4.4 Eggs of M. lar after approximately 23 days of development at 28 Eyespots enlarge at this stage.
ab Plate 4.5 Eggs of M. lar after approximately 25 days of development at 28 The appendages of the larva can now be discerned through the outer membrane of the egg.
ab Plate 4.6 Eggs of M. lar after approximately 27 days of development at 28 The yolk mass has been incorporated into the body of the larva and the embryo can be seen moving inside the egg.
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ab Plate 4.7 Eggs of M. lar hatching after 29 days of development at 28 D A shows the larva stretching the outer membrane of the egg during hatch and B shows the newly hatched larva.
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Zoea I
Plate 4.8 M. lar first zoea larva (zoea I).
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Plate 4.9 Rostrum of the first zoea larva.
Plate 4.10 First zoea hepatopancreas showing lipid globules.
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Plate 4.11 First zoea non-articulating sixth abdominal somite join with telson.
Plate 4.12 First zoea telson.
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Zoea II
Plate 4.13 M. lar second zoea larva lateral view (zoea II).
Plate 4.14 M. lar second zoea dorsal view.
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Plate 4.15 Rudimentary uropod development visible. Ex = exopods.
Plate 4.16 Partial articulation between the telson and sixth abdominal somite. The arrow indicates where the join is forming.
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Plate 4.17 Supra-orbital spine is now present. s. o. s. = Supra-orbital spine.
Plate 4.18 Pterygostomian spine is now present. p. s. = Pterygostomian spine.
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Plate 4.19 Eyes are now stalked.
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Zoea III
Plate 4.20 M. lar third zoea larva dorsal view (zoea III).
Plate 4.21 M. lar third zoea larva lateral view.
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Plate 4.22 Uropods now emergent and rudimentary endopods are seen developing inside the telson.
Plate 4.23 Three segments present in the antennal flagellum.
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Plate 4.24 a First tooth appears on the dorsal carina of the rostrum.
Plate 4.24 b Closer view of the first rostral tooth.
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Zoea IV
Plate 4.25 M. lar fourth zoea larva dorsal view (zoea IV).
Plate 4.26 M. lar fourth zoea larva lateral view.
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Plate 4.27 Second tooth appears on the dorsal carina of the rostrum.
Plate 4.28 Buds of the fifth pereiopods (arrows) are apparent.
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Plate 4.29 Chromatophores on appendages of the fourth zoea larva.
Plate 4.30 Uropod endopods are now emergent.
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Zoea V
Plate 4.31 M. lar fifth zoea larva dorsal view (zoea V).
Plate 4.32 M. lar fifth zoea larva lateral view.
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Plate 4.33 Four segments present in the antennal flagellum.
Plate 4.34 a The fifth pereiopod (walking leg) has now developed (arrow).
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Plate 4.34 b Enlarged view of the fifth pereiopod.
Plate 4.35 The telson is now almost completely rectangular.
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Zoea VI
Plate 4.36 M. lar sixth zoea larva lateral view (zoea VI).
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Plate 4.37 One tooth still present along the dorsal carina of the rostrum, however 2 setae have emerged in front of the first tooth.
Plate 4.38 Pleopod buds emergent. Buds for the third, fourth and fifth pairs are seen here.
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Zoea VII
Plate 4.39 M. lar seventh zoea larva lateral view. Composite image (zoea VII). 225 Monal Lal MSc THESIS: CHAPTER FOUR ______
Plate 4.40 a Pleopod buds have elongated. A bud for the second pair of pleopods is now emergent for this larva.
Plate 4.40 b Closer view of pleopods. Pleopods which have emerged earlier (usually the third and fourth pairs) now start becoming biramous.
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Plate 4.41 Protrusion develops in front of the first dorsal rostral tooth where the next tooth will emerge.
Plate 4.42 Eight segments present in the antennal flagellum. The usual range for this stage is six to eight segments.
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Zoea VIII
Plate 4.43 M. lar eighth zoea larva lateral view. Composite Image (zoea VIII).
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Plate 4.44 Second tooth appears on the dorsal carina of the rostrum. Total tooth count is three for this stage.
Plate 4.45 The antennular flagellum starts to develop and now has two segments (arrows).
229 Monal Lal MSc THESIS: CHAPTER FOUR ______
Plate 4.46 Eight segments present in the antennal flagellum.
Plate 4.47 Pleopod pairs three to five are now biramous and developing setae. The first pleopod pair is now emerging as a bud while the second pair is an elongated uniramous bud.
230 Monal Lal MSc THESIS: CHAPTER FOUR ______
Zoea IX
Plate 4.48 M. lar ninth zoea larva lateral view. Composite image (zoea IX). 231 Monal Lal MSc THESIS: CHAPTER FOUR ______
Plate 4.49 Third tooth appears on the dorsal carina of the rostrum (arrow indicates location of the first tooth hidden behind the eye). Total tooth count is four for this stage.
Plate 4.50 Three segments present in the antennular flagellum.
232 Monal Lal MSc THESIS: CHAPTER FOUR ______
Plate 4.51 Nine segments present in the antennal flagellum.
Plate 4.52 All pleopods are now biramous and possess setae.
233 Monal Lal MSc THESIS: CHAPTER FOUR ______
Plate 4.53 Buds of the appendices internae seen developing on the third and fourth pairs of pleopods. The arrows point to these protuberances on the endopods of the pleopods.
234 Monal Lal MSc THESIS: CHAPTER FOUR ______
Zoea X
Plate 4.54 M. lar tenth zoea larva lateral view. Composite image (zoea X).
235 Monal Lal MSc THESIS: CHAPTER FOUR ______
Plate 4.55 Fourth tooth appears on the dorsal carina of the rostrum. Total tooth count is five for this stage.
Plate 4.56 Four segments present in the antennular flagellum.
236 Monal Lal MSc THESIS: CHAPTER FOUR ______
Plate 4.57 Ten segments present in the antennal flagellum.
Plate 4.58 Chelae appear at the ends of the second pair of pereiopods.
237 Monal Lal MSc THESIS: CHAPTER FOUR ______
Zoea XI
Plate 4.59 M. lar eleventh zoea larva lateral view. Composite image (zoea XI). 238 Monal Lal MSc THESIS: CHAPTER FOUR ______
Plate 4.60 Fifth tooth appears on the dorsal carina of the rostrum. The larva shown had six teeth altogether, although the range for this stage can include up to seven teeth.
Plate 4.61 Sixteen segments present in the antennal flagellum.
239 Monal Lal MSc THESIS: CHAPTER FOUR ______
Plate 4.62 Chelae on second pair of pereiopods now more prominent (arrow).
Plate 4.63 Appendices internae now fully formed (arrows).
240 Monal Lal MSc THESIS: CHAPTER FOUR ______
Zoea XII
Plate 4.64 M. lar twelfth zoea larva lateral view. Composite image (zoea XII). 241 Monal Lal MSc THESIS: CHAPTER FOUR ______
Plate 4.65 Seventh tooth appears on the dorsal carina of the rostrum. Total tooth count is eight for this stage.
Plate 4.66 Nine segments present in the antennular flagellum.
242 Monal Lal MSc THESIS: CHAPTER FOUR ______
Plate 4.67 Nineteen segments present in the antennal flagellum.
Plate 4.68 Chelae on second pair of pereiopods more developed than previously and possess setae.
243 Monal Lal MSc THESIS: CHAPTER FOUR ______
Plate 4.69 Eight setae present on rear margin of fifth pair of pleopods.
244 Monal Lal MSc THESIS: CHAPTER FOUR ______
Zoea XIII
Plate 4.70 M. lar thirteenth zoea larva lateral view. Composite image (zoea XIII).
245 Monal Lal MSc THESIS: CHAPTER FOUR ______
Plate 4.71 Eighth tooth appears on the dorsal carina of the rostrum. Total tooth count is nine for this stage.
Plate 4.72 Thirteen segments present in the antennular flagellum.
246 Monal Lal MSc THESIS: CHAPTER FOUR ______
Plate 4.73 Twenty-nine segments present in the antennal flagellum.
Plate 4.74 Chelae now very prominent and used by the larva in feeding.
247 Monal Lal MSc THESIS: CHAPTER FOUR ______
Plate 4.75 Eleven setae present on rear margin of fifth pair of pleopods.
248 Monal Lal MSc THESIS: CHAPTER FOUR ______
Post-larva; PL1
Plate 4.76 M. lar first Post-Larva. Composite image.
249 Monal Lal MSc THESIS: CHAPTER FOUR ______
Plate 4.77 Dorsal carina rostral tooth count remains unchanged at eight.
Plate 4.78 First tooth appears on the ventral carina of the rostrum.
250 Monal Lal MSc THESIS: CHAPTER FOUR ______
Plate 4.79 Telson now triangular.
Plate 4.80 Second chelipeds significantly larger than other pereiopods.
251 Monal Lal MSc THESIS: CHAPTER FOUR ______
Plate 4.81 Natatory exopods (arrows) now highly reduced in comparison to the endopods (pereiopods). This was seen in only two individual post- larvae.
252 Monal Lal MSc THESIS: CHAPTER FOUR ______
4.3.2 Observations on the larval development of M. lar
The larvae of M. lar developed through 13 zoeal stages before metamorphosing to PL1 in th this study. The first post-larva (PL1) was observed on the 30 of December 2009, after 77 days of culture. A total of five post-larvae were produced, after 77, 78, 85, 101 and 110 days of culture respectively. Overall percentage survival to PL1 was 0.08%, and 0.27 % to zoea XII/XIII. Chapter 2 of this thesis describes the procedures used for larval rearing in detail.
Patterns of growth observed in M. lar larvae were regular until the fifth zoeal stage was reached. Figure 4.9 shows the intermoult durations for all larval developmental stages observed, and Table 4.3 details the age of first appearance and size ranges for these stages.
There are a number of critical points noted during development which have been indicated on Figure 4.9. Development through zoeal stages I to IV appears to occur fairly regularly with an average intermoult duration of approximately 4 days. From zoeal stages V to VIII, intermoult duration changes to approximately 8 days, but remains regular. A further change is observed from zoeal stages IX to XI, where an average intermoult duration of approximately 12.6 days occurs.
From this point onwards, larval development is highly irregular with intermoult durations of 21 and 63 days for zoea XII and XIII respectively. Metamorphosis into PL1 by zoea XIII larvae was also prolonged, taking a total of 34 days from the time of metamorphosis of the first till last PL1.
253 Monal Lal MSc THESIS: CHAPTER FOUR ______
Table 4.3 Age of first appearance and size ranges of M. lar larvae. Stage Age Carapace length Total length (Day of first (mm) (mm) appearance) Zoea I 1 0.25 ± 0.20 0.8 ± 0.21 Zoea II 3 0.35 ± 0.10 1.1 ± 0.32 Zoea III 7 0.40 ± 0.10 1.2 ± 0.25 Zoea IV 9 0.45 ± 0.15 1.3 ± 0.38 Zoea V 11 0.50 ± 0.20 1.5 ± 0.28 Zoea VI 16 0.70 ± 0.15 2.7 ± 0.36 Zoea VII 20 0.65 ± 0.25 2.9 ± 0.42 Zoea VIII 23 0.80 ± 0.22 3.2 ± 0.31 Zoea IX 26 1.18 ± 0.12 4.15 ± 0.29 Zoea X 31 1.20 ± 0.15 4.20 ± 0.32 Zoea XI 39 1.20 ± 0.18 4.40 ± 0.28 Zoea XII 45 1.65 ± 0.26 5.0 ± 0.37 Zoea XIII 48 1.80 ± 0.32 5.4 ± 0.41 PL1 77 2.25 ± 0.38 6.2 ± 0.63
70 65 63 60 55 50 45 40 12334 35 30
25 21
Larval stage duration 20 14 13 15 11 9 10 888 5 4 5 3 3 0 Z 1 Z 2 Z 3 Z 4 Z 5 Z 6 Z 7 Z 8 Z 9 Z 10 Z 11 Z 12 Z 13 PL1
Larval Development Stage
Figure 4.9 Graph of 30 ‰ trial number 20 showing larval stage intermoult durations. Critical points in development are marked with arrows.
254 Monal Lal MSc THESIS: CHAPTER FOUR ______
4.3.3 Descriptions of larval development stages
Zoea I
Figure 4.10 Lateral and dorsal views of M. lar zoea I.
A diagram showing the lateral and dorsal views of zoea I is displayed in Figure 4.10 above, with a photograph of a specimen shown in Plate 4.8.
Zoea I larvae of M. lar possess a very short, straight rostrum which is not toothed (Plate 4.9). The telson (Plate 4.12) does not possess uropods and is roughly heart-shaped and does not articulate, forming a solid join with the sixth abdominal somite (Plate 4.11).
255 Monal Lal MSc THESIS: CHAPTER FOUR ______
This stage does not possess fully formed walking legs (pereiopods), with the first two pairs being present as buds only. The first three maxillipeds are present.
The eyes are sessile and located on the anterior half of the cephalothorax. The body is highly transparent, and lipid globules which are the remnants of the egg yolk mass can be seen in the foregut and midgut regions (Plate 4.10).
Most zoea I larvae were not observed to feed immediately after hatch, however some individuals were seen to seize and feed on egg custard and/or biofloc particles.
256 Monal Lal MSc THESIS: CHAPTER FOUR ______
Zoea II
Figure 4.11 Lateral view of M. lar zoea II.
A diagram showing a lateral view of zoea II is displayed in Figure 4.11 above, with lateral and dorsal view photographs of specimens shown in Plates 4.13 and 4.14 respectively.
The rostrum remains largely unchanged from zoea I in zoea II larvae, however the carapace now has additional armature with the presence of a pair of supra-orbital spines (Plate 4.17) and a pair of pterygostomian spines (Plate 4.18).
The most noticeable feature about this stage is the presence of stalked eyes (Plate 4.19). A join starts forming between the sixth abdominal somite and telson (Plate 4.16), allowing particle articulation between these two body parts. Within the telson, rudimentary uropod exopods may be seen forming which will appear in the next stage (Plate 4.15).
The antennal flagellum at this stage is present but not segmented. The first two pereiopods have developed and appear similar to the third maxilliped.
257 Monal Lal MSc THESIS: CHAPTER FOUR ______
Zoea III
Figure 4.12 Lateral view of M. lar zoea III.
A diagram showing a lateral view of zoea III is displayed in Figure 4.12 above, with lateral and dorsal view photographs of specimens shown in Plates 4.21 and 4.20 respectively.
The first tooth on the rostrum appears during this stage, located immediately behind the eyes on the dorsal carina of the rostrum (Plates 4.24 a and b). It becomes apparent that the pterygostomian spine has two points.
The antennal flagellum becomes divided, and now contains three segments (Plate 4.23). The uropod exopods emerge, and rudimentary uropod endopods can be seen developing inside the telson (Plate 4.22).
All maxillipeds and pereiopods are better developed in this stage, with the fourth pereiopod appearing as a biramous bud.
258 Monal Lal MSc THESIS: CHAPTER FOUR ______
Zoea IV
Figure 4.13 Lateral view of M. lar zoea IV.
A diagram showing a lateral view of zoea IV is displayed in Figure 4.13 above, with lateral and dorsal view photographs of specimens shown in Plates 4.26 and 4.25 respectively.
The second tooth on the rostrum appears during this stage, appearing in front of the first tooth (Plate 4.27). The fifth pereiopod appears as a uniramous bud, while the fourth pereiopod is no longer a bud and possesses all segments.
The uropod endopods now emerge, making the tail fan complete (Plate 4.30). This stage is often noticeably pigmented, with chromatophores being distributed over various parts of the body (Plate 4.29).
259 Monal Lal MSc THESIS: CHAPTER FOUR ______
Zoea V
Figure 4.14 Lateral view of M. lar zoea V.
A diagram showing a lateral view of zoea V is displayed in Figure 4.14 above, with lateral and dorsal view photographs of specimens shown in Plates 4.32 and 4.31 respectively.
This stage possesses four segments in the antennal flagellum (Plate 4.33) and one segment in the antennular flagellum. A fully developed fifth pereiopod (Plates 4.34 a and b) also emerges. All pereiopods are present now.
The telson also changes shape at this stage, becoming almost rectangular (Plate 4.35) from the triangular heart shape of the first zoeal stage. This transition occurs gradually over the preceding stages.
260 Monal Lal MSc THESIS: CHAPTER FOUR ______
Zoea VI
Figure 4.15 Lateral view of M. lar zoea VI.
A diagram showing a lateral view of zoea VI is displayed in Figure 4.15 above, with a lateral view photograph of a specimen shown in Plate 4.36.
The rostrum in this stage remains unchanged, with the exception of the appearance of two or more setae in front of the second tooth (Plate 4.37). This stage possesses five segments in the antennal flagellum and still one segment in the antennular flagellum.
The main feature of this stage is the appearance of pleopod buds, usually for only the third and fourth and occasionally the fifth pairs of pleopods.
261 Monal Lal MSc THESIS: CHAPTER FOUR ______
Zoea VII
Figure 4.16 Lateral view of M. lar zoea VII.
A diagram showing a lateral view of zoea VII is displayed in Figure 4.16 above, with a lateral view photograph of a specimen shown in Plate 4.39. This stage is often the first point at which mark-time moulting may be encountered, and hence variability in morphological development was seen on a number of occasions.
The rostral tooth count for this stage can vary between two and three teeth on the dorsal carina (Plates 4.40 a and b). Upon reaching stage III, it was observed that some individuals had their first rostral tooth emerging almost parallel to the supra-orbital spine, thus the tooth was located well behind the eye (this has been termed the post-orbital tooth). In other individuals, their first rostral tooth emerged almost immediately behind or parallel to the eye. It was seen that in individuals which had their first rostral tooth well behind the eye, they possessed a total of three rostral teeth by the time they moulted to stage VI, whereas the others had only two. Those individuals which had only two teeth occasionally developed a protrusion in front of the second dorsal tooth where the next tooth would emerge (Plate 4.41).
This stage possesses six to eight segments in the antennal flagellum (Plate 4.42) and still one segment in the antennular flagellum. The pleopod buds have also become more developed. As buds for the third and fourth pleopods were the first to emerge in the preceding stage, they have elongated; and buds for the second and fifth pleopods have 262 Monal Lal MSc THESIS: CHAPTER FOUR ______emerged. It was observed that in individuals which already possessed a fifth pleopod bud, it elongated as well, along with the third and fourth buds.
Zoea VIII
Figure 4.17 Lateral view of M. lar zoea VIII.
A diagram showing a lateral view of zoea VIII is displayed in Figure 4.17 above, with a lateral view photograph of a specimen shown in Plate 4.43.
The rostral tooth count for this stage is usually three teeth along the dorsal carina, however some individuals may still possess two teeth, as described for zoea VII (Plate 4.44). This stage possesses eight segments in the antennal flagellum (Plate 4.46), and now two segments in the antennular flagellum (Plate 4.45).
Pleopod development progresses further during this stage. All pleopods which had elongated during the previous stage, are all biramous now and possess natatory setae. In most individuals, the second pleopod bud elongates, and the first pair of pleopods emerges as a simple bud.
263 Monal Lal MSc THESIS: CHAPTER FOUR ______
Zoea IX
Figure 4.18 Lateral view of M. lar zoea IX.
A diagram showing a lateral view of zoea IX is displayed in Figure 4.18 above, with a lateral view photograph of a specimen shown in Plate 4.48.
The rostral tooth count for this stage is three or four teeth along the dorsal carina (Plate 4.49). Individuals which possess only three teeth do not have a post-orbital rostral tooth. This stage possesses nine segments in the antennal flagellum (Plate 4.51) and three segments in the antennular flagellum (Plate 4.50).
All pleopods now become biramous and possess setae, making their development almost complete (Plate 4.52). Now that the endopods of the third and fourth pleopods are fully formed, buds of the appendices internae begin to appear along their inner margins (Plate 4.53).
264 Monal Lal MSc THESIS: CHAPTER FOUR ______
Zoea X
Figure 4.19 Lateral view of M. lar zoea X.
A diagram showing a lateral view of zoea X is displayed in Figure 4.19 above, with a lateral view photograph of a specimen shown in Plate 4.54.
The rostral tooth count for this stage is four or five teeth along the dorsal carina (Plate 4.55). Individuals which possess only three teeth do not have a post-orbital rostral tooth. This stage possesses ten segments in the antennal flagellum (Plate 4.57), and four segments in the antennular flagellum (Plate 4.56).
Chelae make a first appearance in this stage, forming at the ends of the second pair of pereiopods (Plate 4.58).
265 Monal Lal MSc THESIS: CHAPTER FOUR ______
Zoea XI
Figure 4.20 Lateral view of M. lar zoea XI.
A diagram showing a lateral view of zoea XI is displayed in Figure 4.20 above, with a lateral view photograph of a specimen shown in Plate 4.59.
The rostral tooth count for this stage is six to seven, or five to six teeth along the dorsal carina (Plate 4.60). Individuals which possess five to six teeth do not have a post-orbital rostral tooth. This stage possesses fourteen to eighteen segments in the antennal flagellum (Plate 4.61), and six to eight segments in the antennular flagellum.
As all pleopods are now virtually fully formed, complete development of the appendices internae is seen (Plate 4.63) The chelae on the ends of the second pair of pereiopods are now larger, and used by the larva in feeding (Plate 4.62). It was difficult to ascertain whether chelae on the first pair of pereiopods had developed at this stage on the live specimens examined.
The basal segment of the fifth pair of pleopods now begins to develop setae on its rear margin. During this stage, there are four setae present.
266 Monal Lal MSc THESIS: CHAPTER FOUR ______
Zoea XII
Figure 4.21 Lateral view of M. lar zoea XII.
A diagram showing a lateral view of zoea XII is displayed in Figure 4.21 above, with a lateral view photograph of a specimen shown in Plate 4.64.
The rostral tooth count for this stage is eight or seven teeth along the dorsal carina (Plate 4.65). Individuals which possess seven teeth do not have a post-orbital rostral tooth. This stage possesses fifteen to twenty segments in the antennal flagellum (Plate 4.67) and nine segments in the antennular flagellum (Plate 4.66).
The chelae on the second pair of pereiopods (second cheliped) further enlarge from the previous stage, and have developed more setae along the pollex and dactylus (Plate 4.68). Chelae are also now evident on the first pair of pereiopods (first chelipeds).
The basal segment of the fifth pair of pleopods possesses eight setae on its rear margin (Plate 4.69).
267 Monal Lal MSc THESIS: CHAPTER FOUR ______
Zoea XIII
Figure 4.22 Lateral view of M. lar zoea XIII.
A diagram showing a lateral view of zoea XIII is displayed in Figure 4.22 above, with a lateral view photograph of a specimen shown in Plate 4.70.
The rostral tooth count for this stage is nine or eight teeth along the dorsal carina (Plate 4.65). Individuals which possess eight teeth do not have a post-orbital rostral tooth. This stage possesses around twenty-nine or more segments in the antennal flagellum (Plate 4.73) and fourteen segments in the antennular flagellum (Plate 4.72). The antennal flagellum is at least twice the length of the antennal scale or scaphocerite.
The second chelipeds have enlarged, and the larva can be seen capturing Artemia nauplii using these while feeding (Plate 4.74). Although smaller, the first chelipeds are also noticeable. The basal segment of the fifth pair of pleopods possesses eleven setae on its rear margin (Plate 4.75).
A characteristic habit of this larval stage not noticed in the earlier larval stages was the tendency to sit on the bottom of the LRT and walk for short distances using the pereiopod endopods. This was one clue that impending metamorphosis to PL was near.
268 Monal Lal MSc THESIS: CHAPTER FOUR ______
First post-larva, PL1
Figure 4.23 Lateral view of M. lar PL1.
A diagram showing a lateral view of the first post-larva is displayed in Figure 4.23 above, with a lateral view photograph of a specimen shown in Plate 4.76.
The rostral tooth count for this stage is nine or eight teeth along the dorsal carina (Plate 4.77). Individuals which possess eight teeth on the dorsal carina do not have a post-orbital rostral tooth. The ventral carina of the rostrum which up to this point has not possessed any teeth, now bears one a short distance from the apex of the rostrum near the eye (Plate 4.78).
This stage possesses around forty or more segments in the antennal flagellum and sixteen or more segments in the antennular flagellum. The antennal flagellum is at least three times the length of the antennal scale or scaphocerite.
The telson now appears triangular from above, with the rear margin coming to a point (Plate 4.79). This has been a gradual change from the rectangular telson of stage V larvae, and the tail fan now bears a resemblance to that of the adult.
The second chelipeds are now greatly enlarged and are the largest pair of legs the animal now possesses (Plate 4.80). In some individuals, rudimentary natatory exopods were visible after the moult to PL1 had been completed (Plate 4.81), whereas in others they were absent. This feature, along with the benthic behaviour characteristic of other 269 Monal Lal MSc THESIS: CHAPTER FOUR ______
Macrobrachium spp. PL was confirmation that this was indeed the post-larval stage for M. lar and not another larval stage.
Three out of the five PL1 produced were maintained in isolation after having been acclimated to freshwater, and were found to moult into PL2 after a period of 5 days from metamorphosis into PL1. A summary of the distinguishing characteristics for the zoea larvae and PL1 of M. lar are included in Table 4.4 below.
Table 4.4 Summary of readily discernable features characterising the larvae and PL1 of M. lar. * = larval stages which may incorporate more than 2 – 3 instars. ** = larval stages which may incorporate more than 3 – 4 instars. C. = around, d.c. = dorsal carina and v.c. = ventral carina. Stage Features I Rostral teeth (d.c.) = 0 Sessile eyes Non-articulating 6th abdominal somite/telson join Antennal flagellum segments = 0 II Rostral teeth (d.c.) = 0 Supra-orbital spine pair present Pterygostomian spine pair present Stalked eyes Partially articulating 6th abdominal somite/telson join Rudimentary uropod exopods seen Antennal flagellum segments = 0 III Rostral teeth (d.c.) = 1 Fully-articulating 6th abdominal somite/telson join Rudimentary uropod endopods seen; exopods now emergent Antennal flagellum segments = 3 IV Rostral teeth (d.c.) = 2 Uropod endopods now emergent Antennal flagellum segments = c.3 Antennular flagellum segments = 0 V Rostral teeth (d.c.) = 2 Telson now rectangular Fifth pereiopod now emergent Antennal flagellum segments = c.4 Antennular flagellum segments = c.1 VI* Rostral teeth (d.c.) = 2 Pleopod buds emergent (#3 & #4 always + #5 sometimes) Antennal flagellum segments = c.5 Antennular flagellum segments = c.1 270 Monal Lal MSc THESIS: CHAPTER FOUR ______
VII** Rostral teeth (d.c.) = 2 – 3 (2 if animal has no post-orbital tooth) More pleopod buds emergent (#2 & #5); #3 & #4 have elongated. Antennal flagellum segments = c.6 – 8 Antennular flagellum segments = c.1 VIII* Rostral teeth (d.c.) = 3 (2 if animal has no post-orbital tooth) Pleopods biramous (#3 & #4 always + #5 sometimes) & setae now present - usually on those that are biramous Pleopod pair #1 now emergent as a bud Antennal flagellum segments = c.8 Antennular flagellum segments = c.2 IX* Rostral teeth (d.c.) = 4 (3 if animal has no post-orbital tooth) All pleopods now biramous and have setae Buds of appendices internae first seen on pleopods #3 and #4. Antennal flagellum segments = c.9 Antennular flagellum segments = c.3 X* Rostral teeth (d.c.) = 5 (4 if animal has no post-orbital tooth) Chelae on pereiopod #2 now developed and visible Antennal flagellum segments = c.10 Antennular flagellum segments = c.4 XI** Rostral teeth (d.c.) = 6 – 7 (5 – 6 if animal has no post-orbital tooth) Appendices internae now clearly visible on pleopods with most advanced development (usually #3 & #4) – buds of appendices internae usually first seen at stage IX. Chelae on pereiopod #2 now more prominent Antennal flagellum segments = c.14 -18 Antennular flagellum segments = c.6 - 8 Setae present on rear margin of pleopod #5 basal segment = c.4 XII* Rostral teeth (d.c.) = 8 (7 if animal has no post-orbital tooth) Antennal flagellum segments = c.15 - 20 Antennular flagellum segments = c.9 Setae present on rear margin of pleopod #5 basal segment = c.8 Chelae on pereiopod #1 now evident XIII* Rostral teeth (d.c.) = 9 (8 if animal has no post-orbital tooth) Antennal flagellum segments = c.29+ (more than 2× length of antennal scale) Antennular flagellum segments = c.14 Setae present on rear margin of pleopod #5 basal segment = c.11 PL1 Rostral teeth (d.c.) = 9 (8 if animal has no post-orbital tooth) Rostral teeth (v.c.) = 1 Forward swimming behaviour and benthic habit Exopods with natatory setae on pereiopods greatly reduced or absent (ref. to photo) cf. to pereiopods themselves Prominent chelae on second pereiopods Antennal flagellum segments = c.40+ (more than 3× length of antennal scale) Antennular flagellum segments = c.16+
271 Monal Lal MSc THESIS: CHAPTER FOUR ______
As mass cultures were used for rearing the larvae of M. lar in this study, it was not possible to distinguish the exact number of instars the larvae passed through before metamorphosing into PL1. In order to determine the number of instars within each stage, it would be necessary to culture individual larvae in isolation with frequent observations to detect exactly when a moult occurred, and to see if this moult produced a new stage or another instar belonging to the same stage.
Each successive instar appeared to last between 2 to 3 days from hatch until the larvae moulted into zoea V, with each instar corresponding to a new developmental stage. It is likely that this intermoult duration continued through the remainder of larval development; however a minimum of two instars may occur for each new larval stage beyond zoea V.
Two particular stages, viz. zoea VII and XI, may include more than two and up to four instars, as some larvae at these stages in development showed nearly identical morphological features, and had increased in size relative to other larvae within the same stage. Any changes noted in morphology were subtle and very minor, e.g. additional setae on the antennal scale, pleopods and pereiopod exopods. This appears to be evidence of mark time moulting, and in the case of some zoea XI larvae, terminally additive staging.
Overall, the larvae of M. lar may moult through a minimum of twenty two and maximum of thirty one instars or more before being ready to metamorphose into PL1.
272 Monal Lal MSc THESIS: CHAPTER FOUR ______
4.3.4 Guide to identifying the larvae of M. lar
A pictorial guide to the identification of the larvae of M. lar was produced, using easily discernable features which would allow for the rapid identification of larval stages for the aquaculturist. This guide is included overleaf as an insert.
273 Monal Lal MSc THESIS: CHAPTER FOUR ______
I
Sessile eyes Join between 6th abdominal somite and telson non-articulating II
Stalked eyes Join between 6th abdominal somite and telson partially articulating Rudimentary uropod exopods seen developing in the telson III
3 segments in the antennal flagellum 1 tooth present on dorsal carina of rostrum Uropod exopods emergent and rudimentary endopods seen in telson IV
2 teeth present on dorsal carina of rostrum Uropod endopods emergent and telson becoming more rectangular in shape V
4 segments in the antennal flagellum Fifth pereiopod (walking leg) now emergent Telson now almost completely rectangular VI
5 segments in the antennal flagellum Pleopod buds emergent (#3 & #4 always + #5 sometimes) VII
Around 6 to 8 segments in the antennal flagellum More pleopod buds emergent (#2 & #5); #3 & #4 have elongated. 2 to 3 teeth present on dorsal carina of rostrum (2 if animal has no post-orbital tooth)
274 Monal Lal MSc THESIS: CHAPTER FOUR ______
VIII
8 segments in the antennal flagellum 2 segments in the antennular flagellum 3 teeth present on dorsal carina of rostrum (2 if animal has no post-orbital tooth) Pleopods biramous (#3 & #4 always + #5 sometimes) & setae now present - usually on those that are biramous Pleopod pair #1 emergent as a bud IX
9 segments in the antennal flagellum 3 segments in the antennular flagellum 4 teeth present on dorsal carina of rostrum (3 if animal has no post-orbital tooth) All pleopods biramous now and the exopods bear setae X
10 segments in the antennal flagellum 4 segments in the antennular flagellum 5 teeth present on dorsal carina of rostrum (4 if animal has no post-orbital tooth) Chelae now present on the second pair of pereiopods XI
14 to 18 segments in the antennal flagellum 6 to 8 segments in the antennular flagellum 6 to 7 teeth present on dorsal carina of rostrum (5 to 6 if animal has no post-orbital tooth) Appendices internae now visible on pleopods with most advanced development (usually #3 & #4) XII
15 to 20 segments in the antennal flagellum 9 segments in the antennular flagellum 8 teeth present on dorsal carina of rostrum (7 if animal has no post-orbital tooth) XIII
29+ segments in the antennal flagellum 14 segments in the antennular flagellum 9 teeth present on dorsal carina of rostrum (8 if animal has no post-orbital tooth)
PL1
40+ segments in the antennal flagellum 16+ segments in the antennular flagellum 9 teeth present on dorsal carina of rostrum (8 if animal has no post-orbital tooth) 1 tooth present on ventral carina of rostrum Prominent chelae on the second pair of pereiopods
275 Monal Lal MSc THESIS: CHAPTER FOUR ______
4.4 Discussion
4.4.1 Embryonic development of M. lar larvae
The embryonic development of M. lar appears very similar to that which has been described for other species of Macrobrachium, such as M. olfersii (Müller et al., 2003) and M. rosenbergii (Manush et al., 2006).
Other investigations on the embryonic development of M. lar have reported similar findings regarding the various stages of embryogenesis (Nandlal, 2010; Kubota, 1972), with the overall finding that the pattern of embryonic development in this species is not remarkably different.
As a result of the data gathered on the stages of embryonic development reached at the series of sampling intervals, a reference point for further work on maintaining M. lar brooder females has been established. Directions for future research may include investigations on whether increased temperatures are able to accelerate embryonic development to reduce the amount of time ovigerous females need to maintained in captivity, and on improved broodstock nutrition to enhance larval quality, among other topics of interest.
4.4.2 Larval development of M. lar
As a result of being able to rear the larvae of M. lar through all zoeal stages to the first post-larva, a complete description of the morphological development stages has been realised for this species. This will be of use in further larviculture and ecological investigations involving this and possibly other Macrobrachium species.
Overall, the general pattern of larval development shares similarities with patterns that have been documented for other Macrobrachium species which have a typical or prolonged/normal type of development (Alekhnovich & Kulesh, 2001; Jalihal et al., 1993).
276 Monal Lal MSc THESIS: CHAPTER FOUR ______
A number of species of Macrobrachium have been studied which produce larvae that have a known requirement for conditions of oceanic salinity (30 – 35 ‰). The number of larval stages described for some of these species includes M. sp. which has 12 (Ngoc-Ho, 1976), M. equidens which has 10 (Ngoc-Ho, 1976), M. grandimanus which has 9 (Shokita, 1985), M. intermedium which has at least 10 (Williamson, 1971) and M. olfersii which has at least 12 (Dugger & Dobkin, 1975).
A common feature among the species mentioned is the presence of at least ten larval stages (nine in the case of M. grandimanus), with M. olfersii and M. sp. having 12 stages. This is similar to the results of this study, which describes 13 stages for M. lar.
4.4.3 Larval development characteristics
From the results of the current study, it appears that the larval development of M. lar is extended, with larvae reaching PL1 between 77 and 110 days of culture at a very low (0.08 %) survival rate. This is supported by the findings of Atkinson (1977, 1973), who was able to rear larvae to the eleventh zoeal stage after 89 days.
This pattern of extended development has been attributed to the phenomena of mark time moulting and terminally additive staging exhibited by mid and late stage larvae, probably due to sub-optimal culture conditions. The onset of mark time moulting appears to occur from stage VI onwards, and terminally additive staging may occur in zoea XI. The work of Atkinson (Atkinson, 1977, 1973) also appears to show evidence of mark time moulting in descriptions of the ninth and tenth zoeal stages.
Further work is required to establish exactly which culture conditions are responsible, and indications are that these may include conditions of salinity and larval nutrition. It would be interesting to repeat larviculture trials at 35 ‰ to see what differences in development duration, if any, occur at this salinity. The question of larval nutrition should also be addressed, as suggested in Chapter 2 of this thesis.
277 Monal Lal MSc THESIS: CHAPTER FOUR ______
During earlier larval rearing trials at salinities which are now known to be sub-optimal for this species, viz. 20 ‰ and 25 ‰ (see Chapter 2); the time taken to develop to particular stages was reduced, and in some cases larvae exhibited signs of skipped staging. Given that PL were able to be produced at 30 ‰, this may indicate a minimum threshold at which larval development may be completed.
Evidence of mark time moulting has also been observed in other Macrobrachium spp., particularly those which inhabit marine or partly-marine conditions as adults; viz. M. equidens (Ngoc-Ho, 1976), M. rosenbergii (Valenti et al., 2010; Gomez Diaz & Kasahara, 1987) and M. vollenhovenii (Müller et al., 2003).
This observation of inherent developmental plasticity has been related to the wide marine dispersal capacity of the larvae of some species, and is an important factor to consider (Shokita, 1985). Other Macrobrachium species which share a wide (albeit less extensive) Indo-Pacific distribution with M. lar, and that have been demonstrated to show variability in developmental patterns and pathways, include M. grandimanus (Shokita, 1985) and M. equidens (Ngoc-Ho, 1976).
Studies of the population structure of M. lar using genetics in Japan (Imai et al., 2007) and in PICTs (Nandlal, 2010; Mather et al., 2006) have shown high genetic diversity over large geographical spatial scales. This implies a high level of gene flow between widely separated habitats, and lends weight to the requirement of this species for a long-lived pelagic larva which is able to colonise habitats far removed from its place of hatch.
The implications of phenomena such as mark time moulting are important in developing commercial-scale hatchery operations for Macrobrachium spp, as extended larval development duration dramatically increases hatchery operating costs.
In comparison to the results for M. lar obtained during this study, larval survival rates at the present time till metamorphosis for M. rosenbergii are between 40 – 50 % in flow- through hatchery systems, 60 – 80 % in Thai backyard hatcheries and between 60, 75 and 80 % in experimental and commercial recirculation systems; with development durations
278 Monal Lal MSc THESIS: CHAPTER FOUR ______of 29 to 35 days (Valenti et al., 2010). Bearing this in mind, when post-larvae for this species were first produced in the laboratory, the larval survival rate till metamorphosis was reported to be 16 – 17 % (Ling, 1962, 1961).
Based on the results of this study, commercial-scale hatchery operations for M. lar do not appear to be feasible in the short to medium term. In the longer term, further research into the improvement of larval survival and reduction in larval phase duration is required before commercial-scale larviculture will become viable.
4.5 Conclusion
As a result of the investigations carried out into the development of a suitable larviculture technique as described in Chapter 2 of this thesis, larvae of M. lar were able to be successfully reared and described from hatch till metamorphosis into PL1. Key features of embryological development were also able to be identified and described, which will be of use in determining time till hatch of ovigerous females maintained in the laboratory. A guide to identification of the larval stages of this species has been produced, which will be relevant to future larviculture or ecological research with this species.
The larval development of this species is extended, requiring at least 77 days before metamorphosis of the first post-larva, with very low survival rates up to metamorphosis.
Evidence of mark time moulting and terminally additive staging was seen in the development pattern of the larvae, which likely contributed to the lengthy development duration. The prolonged larval development observed is attributed to sub-optimal culture conditions, the most significant of these likely to be salinity and larval nutrition.
While the results of this study represent a significant breakthrough in efforts to domesticate M. lar, further research and development work will be required before it can become viable for aquaculture at a commercial level. Particular issues which need to be
279 Monal Lal MSc THESIS: CHAPTER FOUR ______addressed to achieve this include improving larval survival rates and decreasing development time till metamorphosis.
280 Monal Lal MSc THESIS: CHAPTER FIVE ______
CHAPTER FIVE
GENERAL CONCLUSION AND RECOMMENDATIONS
5.1 Review of objectives
The major objectives of this thesis and the extent to which they were achieved are summarised below. For detailed descriptions of the investigations carried out to achieve the following objectives, please refer to the relevant chapters.
i) To develop and describe a new larval rearing technique by which larvae of M. lar will be able to be successfully reared from hatch, through all larval developmental stages until they metamorphose into post-larvae.
The investigations described in Chapter 2 of this thesis detail the development of a new larviculture technique which was successful in rearing the larvae of M. lar from hatch until metamorphosis into PL1. This achievement places the University of the South Pacific as possibly the first institution to report rearing this species through all larval development stages to PL.
ii) To simply describe the embryonic development stages of M. lar with the view of determining time till hatch of ovigerous females maintained in captivity.
Key features of embryological development were identified and described in an investigation described in Chapter 4, which will allow the determination of time till hatch of ovigerous females maintained in the laboratory.
iii) To rear and describe larvae of M. lar through all larval developmental stages until metamorphosis into post-larvae.
Through the investigations detailed in Chapter 2, a total of five post-larvae were produced, metamorphosing at 77, 78, 85, 101 and 110 days of culture respectively after moulting
281 Monal Lal MSc THESIS: CHAPTER FIVE ______through 13 distinct zoeal stages. Overall percentage survival to PL1 was 0.08%. Morphological descriptions of the larval stages are provided in Chapter 4 of this thesis.
iv) To produce a simple larval development guide for identifying the developmental stages of M. lar larvae.
A guide to the identification of the zoea larvae of M. lar from stages I to XIII and PL1 is provided in Chapter 4, in a practical form that will be of assistance to future M. lar hatchery technicians or plankton ecologists.
v) By means of a series of controlled experiments, determine the optimal ranges of salinity and temperature required by the larvae of M. lar for successful laboratory-scale culture.
As a result of the investigations carried out in Chapter 3 of this thesis, it has now been established that the larvae of M. lar are able to hatch in either freshwater or brackish- water of approximately 10 ‰, but require gradually increasing salinities post-hatch reaching 30 – 35 ‰ which probably needs to be maintained from stages V – VI until metamorphosis into PL. A temperature of 30 ± 0.5 maximum larval survival and growth rates.
The general implications of the findings detailed in this thesis, and recommendations for further research into assessing the aquaculture potential of M. lar, are discussed in the following sections.
282 Monal Lal MSc THESIS: CHAPTER FIVE ______
5.2 Outlook for pilot-scale hatchery production
Based on the results of this study, pilot commercial-scale hatchery operations for M. lar do not appear to be feasible in the short to medium term, because the larval development of M. lar is extended, with larvae reaching PL1 between 77 and 110 days of culture at a very low (0.08 %) survival rate.
This pattern of extended development is attributed to the phenomena of mark time moulting and terminally additive staging exhibited by mid and late stage larvae, probably due to sub-optimal culture conditions. Further work is required to establish exactly which culture conditions are responsible, and indications are that these may include salinity and larval nutrition.
While baseline salinity and temperature requirements for the species have been established by the investigations carried out in Chapter 3, there is scope for fine-tuning the experiments in order to determine an optimised salinity acclimation protocol for larviculture. This may be particularly important for the early larval stages, because in most cases, 50 % of mortality occurred in the first 12 days of culture.
The primary issues which remain to be resolved if commercial-scale culture is to be made feasible are improvement of larval survival and reduction of larval phase duration. Further work with incorporating biofloc into the culture system and trialling micro- encapsulated feeds may yet prove that large scale larviculture for this species is possible. It took time for M. rosenbergii larviculture to become commercially successful, and with M. lar there is currently much room for similar improvements to be made.
For the present, the capture of wild juveniles will have to be relied upon for developing the culture of M. lar in ponds. However, it may be premature to discontinue efforts to understand the larval requirements of this species, because they may yet lead to successful mass-culture techniques through which production of sufficient numbers of PL for pond-stocking could be realized. As a counter-balance to its greatly extended larval
283 Monal Lal MSc THESIS: CHAPTER FIVE ______life, the apparent lack of dependence of M. lar larviculture on the increasingly scarce and expensive global reserves of Artemia offers an intriguing cost advantage over the more carnivorous M. rosenbergii.
5.3 Study constraints and limitations
A major constraint in designing and executing experiments to determine the culture requirements of M. lar larvae was the very low survival rate of larvae. This limited the sample size for various measurements and hence reduced the statistical power of experiments. Larvae also exhibited very high mortality when maintained in small containers over extended periods of time, so culture on smaller scales was not feasible.
A secondary constraint was the relatively low larval yield from ovigerous females maintained in the laboratory. Hatches were often prolonged, requiring a greater amount of labour to maintain larval rearing tanks, broodstock tanks and live feeds for the larvae simultaneously.
Unfortunately due to time, budgetary and resource constraints, it was not possible to attempt another culture run after producing PL. It would have been ideal to optimise conditions as much as possible during a repeat run to determine whether survival rates could be increased. This will have to await further research with the larviculture of this species.
5.4 Recommendations for future research
A priority area for further research is the improvement of larval survival rates up to PL to a level where commercial-scale production may be feasible. The current study recorded an overall survival of 0.08 % to the PL stage, which will need to be improved upon significantly to compare with the survival rates of M. rosenbergii larvae which average upwards of 40 to 80 %.
284 Monal Lal MSc THESIS: CHAPTER FIVE ______
Another area for further investigation involves shortening larval development time to compare with M. rosenbergii larvae. This study recorded the first and last PL produced metamorphosing on the 77th and 110th days of culture respectively. With M. rosenbergii, first PL are usually seen between the 20th and 25th day of culture, with all surviving larvae metamorphosing into PL by day 35 and 40 of culture. If development time could be reduced by half from that observed in the current study, this would compare well with the performance characteristics of M. rosenbergii larvae.
A key to improvement of larval survival and development duration may involve larval nutrition. Investigations on the importance of biofloc in the diet of the larvae are warranted, along with studies on the nutritional requirements of the larvae. Observations made during the current study showed that larvae had a distinct preference for biofloc over Artemia nauplii as a live food item, and if this is able to be experimentally proven, will imply cheaper hatchery operating costs for M. lar larviculture over M. rosenbergii,as less Artemia nauplii will be consumed and biofloc are able to be produced naturally in the larval rearing tanks.
Fine-tuning of the culture environment salinity and temperature parameters are also worth investigating, particularly for early larval stages, along with attempting to grow the larvae in a clearwater system. Further work on the salinity requirements of the larvae will identify if there is an optimal regime by which the salinity of the culture medium should be increased to maintain maximal larval survival and growth. Successful culture of larvae in a clearwater system will demonstrate if the nutritional requirements of the larvae are sufficiently understood to allow culture in a system which does not contain additional sources of live feed, as a greenwater system does.
There is also scope for further research into the broodstock management and maintenance of M. lar, as comparatively little is known about issues such as sexual maturation and fecundity in captivity, appropriate stocking densities and sex ratios, social hierarchical interactions and optimal broodstock nutrition among others for this species.
285 Monal Lal MSc THESIS: CHAPTER FIVE ______
The problems with asynchronous and prolonged hatching by ovigerous females encountered during this study also require further attention. One aspect of this is the determination of how much control that female M. lar are able to exert over the time and period over which their larvae hatch and their causative mechanisms if any exist. M. lar are reported to have levels of fecundity which are not much lower than those which have been documented for M. rosenbergii, and if larval yields at hatch can be improved from the levels observed during this study, this would be a positive outcome for further larviculture efforts with this species.
286 REFERENCES ______
Abbott, R. T., & Dance, S. P. (2000). Compendium of Seashells: A full-colour guide to more than 4,200 of the World's marine shells. El Cajon, California, USA: Odyssey Publishing.
Adams, T., Bell, J., & Labrosse, P. (2001). Current Status of Aquaculture in the Pacific Islands. Paper presented at the Aquaculture in the Third Millenium. Technical Proceedings of the Conference on Aquaculture in the Third Millennium, Bangkok, Thailand. http://www.spc.int/coastfish/Reports/Misc/SPC_aqua_rev.pdf
Alekhnovich, A. V., & Kulesh, V. F. (2001). Variation in the parameters of the life cycle in prawns of the Genus Macrobrachium Bate (Crustacea, Palaemonidae). Russian Journal of Ecology 32 (6), 420-424.
Alo, G., Pickering, T., & Gereva, S. (2011). Capture-based culture techniques for Macrobrachium lar for poverty alleviation in remote villages of Santo Island, Vanuatu Paper presented at the Giant Prawn 2011 and Asian Pacific Aquaculture 2011 Conferences, Kochi, Kerala, India.
Anger, K., & Moreira, G. S. (1998). Morphometric and reproductive traits of tropical caridean shrimps. Journal of Crustacean Biology, 18(4), 823-838.
Anger, K. (2001). Crustacean Issues 14: The Biology of Decapod Crustacean Larvae (Vol. 14). Meppel, Netherlands: CRC Press.
Anger, K., Hayd, L., Knott, J., & Nettelmann, U. (2009). Patterns of laval growth and chemical composition in the Amazon River prawn, Macrobrachium amazonicum. Aquaculture, 287, 341-348.
287 Aquafauna. (2007). Algamac® 3000 and 3050 flake brochure. California, USA: Aquafauna Bio-Marine Inc.
Armstrong, D. A., Chippendale, D., Knight, A. W., & Colt, J. E. (1978). Interaction of ionized and un-ionized ammonia on short-term survival and growth of prawn larvae, Macrobrachium rosenbergii. Biological Bulletin, 154, 15-31.
Arthur, J. R., Hurwood, D., Lovell, E. R., Bondad-Reantaso, M. G., & Mather, P. B. (2004). SPC Aquaculture Technical Papers: Pathogen and Ecological Risk Analysis for the Introduction of Giant River Prawn, Macrobrachium rosenbergii, from Fiji to the Cook Islands. Noumea, New Caledonia: SPC.
Atkinson, J. M. (1973). The larval development of the freshwater prawn Macrobrachium lar (Fabricius) reared in the laboratory. Master of Science in Zoology Thesis, University of Hawaii, Honolulu.
Atkinson, J. M. (1977). Larval development of a freshwater prawn, Macrobrachium lar (Decapoda, Palaemonidae), reared in the laboratory. Crustaceana, 33 (2), 119-132.
Avnimelech, Y. (2009). Biofloc Technology. A practical guide book. Baton Rouge, Louisiana, USA: World Aquaculture Society.
Bagenal, T. B. (1967). A short review of fish fecundity. In S. D. Gerking (Ed.), The biological basis of freshwater fish production (pp. 89-112). Oxford, UK: Blackwell Scientific Publications.
Barbier, J., Jimmy, R., & Nandlal, S. (2006). Final Report for Mini-project MS0402: Monoculture of the freshwater prawn, Macrobrachium lar, in Vanuatu and integrated prawn-taro farming in Wallis & Futuna (pp. 17). Canberra, Australia: ACIAR.
288 Brillon, S., Lambert, Y., & Dodson, J. (2005). Egg survival, embryonic development, and larval characteristics of northern shrimp (Pandalus borealis) females subject to different temperature and feeding conditions. Marine Biology, 147, 895-911.
Brock, V. E. (1960). The introduction of aquatic animals into Hawaiian waters. International Review of Gestational Hydrobiology, 45 (4), 463-480.
Brown, J. H., New, M. B., & Ismael, D. (2010). Biology. In M. B. New, W. C. Valenti, J. H. Tidwell, L. R. D'Abramo & M. N. Kutty (Eds.), Freshwater Prawns Biology and Farming (pp. 18-39). Chichester, West Sussex, United Kingdom: Wiley-Blackwell.
Chan, T. Y. (1998). Shrimps and Prawns. In K. E. Carpenter & V. H. Niem (Eds.), FAO Species Identification Guide for Fishery Purposes: The Living Marine Resources of the Western Central Pacific Volume 2 (Cephalopods, Crustaceans, Holothurians and Sharks) (pp. 851-865). Rome, Italy: Food and Agriculture Organization of the United Nations (FAO), South Pacific Forum Fisheries Agency (FFA) and Norwegian Agency for International Development (NORAD).
Cheah, S. H., & Ang, K. J. (1979). Short Communication - III. Preliminary trials on juvenile Macrobrachium rosenbergii production under modified static 'greenwater' conditions. Pertanika, 2(1), 69-71.
Chen, S.-M., & J.-C.Chen. (2003). Effects of pH on survival, growth, moulting and feeding of Giant Freshwater Prawn Macrobrachium rosenbergii. Aquaculture 218, 613-623.
Cheng, W., & Chen, J.-C. (1998). Enterococcus-like infections in Macrobrachium rosenbergii are exacerbated by high pH and temperature but reduced by low salinity. Diseases of Aquatic Organisms 34 103-108.
289 Cheng, W., & Chen, J.-C. (2000). Effects of pH, temperature and salinity on immune parameters of the freshwater prawn Macrobrachium rosenbergii. Fish & Shellfish Immunology, 10, 387-391.
Cheng, W., Chen, S.-M., Wang, F.-I., Pei-I., H., Liu, C. H., & J.-C.Chen. (2003). Effects of temperature, pH, salinity and ammonia on the phagocytic activity and clearance efficiency of Giant Freshwater Prawn Macrobrachium rosenbergii to Lactococcus garvieae. Aquaculture 219 111-121.
Ching, C. A., & Velez Jr., M .J. (1985). Mating, incubation and embryo number in the freshwater prawn Macrobrachium heterochirus (Wiegmann, 1836) (Decapoda, Palaemonidae) under laboratory conditions. Crustaceana 49 (1), 42-48.
Cholik, F. (1999). Review of mud crab culture research in Indonesia. In C. P. Keenan & A. Blackshaw (Eds.), Mud Crab Aquaculture and Biology. Proceedings of an international scientific forum held in Darwin, Australia, 21-24 April 1997 (pp. 14-20). Canberra, Australia: ACIAR.
Choudhury, P. C. (1970). Complete larval development of the Palaemonid shrimp Macrobrachium acanthurus (Wiegmann, 1836) reared in the laboratory. Crustaceana, 18(2), 113-132.
Choudhury, P. C. (1971a). Laboratory rearing of larvae of the palaemonid shrimp Macrobrachium acanthurus (Wiegmann, 1836). Crustaceana, 21, 113-126.
Choudhury, P. C. (1971b). Responses of larval Macrobrachium carcinus (L.) to variations in salinity and diet (Decapoda, Palaemonidae). Crustaceana (20), 113-120.
Choudhury, P. C. (1971c). Complete larval development of the Palaemonid shrimp Macrobrachium carcinus (L.), reared in the laboratory (Decapoda, Palaemonidae). Crustaceana, 20 (1), 51-69.
290 Choy, S. C. (1984). On the freshwater Palaemonid prawns from the Fiji Islands (Decapoda, Caridea). Crustaceana, 47(3), 271-277.
Cohen, D., Finkel, A., & Sussman, M. (1976). On the role of algae in larviculture of Macrobrachium rosenbergii. Aquaculture, 8, 199-207.
Collart, O. O., & Rabelo, H. (1996). Variation in Egg Size of the Fresh-Water Prawn Macrobrachium amazonicum (Decapoda: Palaemonidae). Journal of Crustacean Biology 16 (4), 684-688.
Correia, E. S., Suwannatous, S., & New, M. B. (2004). Flow-through Hatchery Systems and Management. In M. B. New & W. C. Valenti (Eds.), Freshwater Prawn Culture. The farming of Macrobrachium rosenbergii (pp. 52-68). Oxford, UK: Blackwell Science.
Daniels, W. H., Cavalli, R. O., & Smullen, R. P. (2010). Broodstock Management. In M. B. New, W. C. Valenti, J. H. Tidwell, L. R. D'Abramo & M. N. Kutty (Eds.), Freshwater Prawns Biology and Farming (pp. 40-54). Chichester, West Sussex, United Kingdom: Wiley-Blackwell. de Grave, S., Cai, Y., & Anker, A. (2008). Global diversity of shrimps (Crustacea: Decapoda: Caridea) in freshwater. Hydrobiologia 595, 287-293.
Dhont, J., Wille, M., Frinsko, M., Coyle, S. D., & Sorgeloos, P. (2010). Larval Feeds and Feeding. In M. B. New, W. C. Valenti, J. H. Tidwell, L. R. D'Abramo & M. N. Kutty (Eds.), Freshwater Prawns Biology and Farming (pp. 86-107). Chichester, West Sussex, United Kingdom: Wiley-Blackwell.
Dobkin, S. (1971). A contribution to knowledge of the larval development of Macrobrachium acanthurus (Wiegmann, 1836) (Decapoda, Palaemonidae). Crustaceana, 21 (3), 294-297.
291 Dobkin, S., Azzinaro, W. P., & van Montfrans, J. (1974). Culture of Macrobrachium acanthurus and M. carcinus with notes on the selective breeding and hybridization of these shrimps Paper presented at the Proceedings of the 5th Annual Meeting of the World Mariculture Society.
Dugger, D. M., & Dobkin, S. (1975). A contribution to knowledge of the larval development of Macrobrachium olfersii (Wiegmann, 1836) (Decapoda, Palaemonidae). Crustaceana, 29 (1), 1-30.
Estudillo, C. B., Duray, M. N., Marasigan, E. T., & Emata, A. C. (2000). Salinity tolerance of larvae of the mangrove red snapper Lutjanus argentimaculatus during ontogeny. Aquaculture 190, 155-167.
FAO. (1997). Aquaculture Development. FAO Technical Guidelines for Responsible Fisheries. No. 5. Rome, Italy.
FAO. (2000). Code of conduct for responsible fisheries. Rome, Italy: Food and Agriculture Organization of the United Nations.
FAO. (2009). The state of world fisheries and aquaculture 2008. Rome, Italy: FAO Fisheries and Aquaculture Department, Food and Agriculture Organization of the United Nations.
FAO. (2010). The state of world fisheries and aquaculture 2010. Rome, Italy: FAO Fisheries and Aquaculture Department, Food and Agriculture Organization of the United Nations.
Fielder, D. R. (1970). The larval development of Macrobrachium australiense Holthuis, 1950 (Decapoda, Palaemonidae), reared in the laboratory. Crustaceana, 18, 60-74.
292 Gomez Diaz, G., & Kasahara, S. (1987). The morphological development of Macrobrachium rosenbergii (de Man) larvae. Journal of the Faculty of Applied Biological Science, Hiroshima University, Fukuyama, 26 43-56.
Gore, R. (1985). Moulting and growth in decapod larvae. In F. R. Schram & A. M. Wenner (Eds.), Crustacean Issues 2: Larval Growth (Vol. 2, pp. 1-65). Rotterdam, Netherlands: A. A. Balkema.
Greenwood, J. G., Fielder, D. R., & Thorne, M. J. (1976). The larval life history of Macrobrachium novaehollandiae (de Man, 1908) (Decapoda, Palaemonidae), reared in the laboratory. Crustaceana, 30 (3), 252-286.
Hall, S. J., Delaporte, A., Phillips, M. J., Beveridge, M., & O’Keefe, M. (2011). Blue Frontiers: Managing the Environmental Costs of Aquaculture (pp. 104). Penang, Malaysia: The WorldFish Centre.
Hanson, J. A., & Goodwin, H. L. (1977). About Macrobrachium species. Paper presented at the Shrimp and Prawn Farming in the Western Hemisphere: Proceedings of the 2nd workshop on the culture of Macrobrachium held at Charleston, South Carolina, USA in June 1976, Charleston, South Carolina, USA.
Hayd, L. A., Anger, K., & Valenti, W. C. (2008). The moulting cycle of larval Amazon River prawn Macrobrachium amazonicum reared in the laboratory. Nauplius, 16 (2), 55-63.
Haynes, A. (1999). The long term effect of forest logging on the macroinvertebrates in a Fijian stream. Hydrobiologia 405, 79-87.
Hickman Jr., C. P., Roberts, L. S., Larson, A., L'Anson, H., & Eisenhour, D. J. (2001). Integrated Principles of Zoology (13 ed.). New York, USA: McGraw Hill Higher Education.
293 Holthuis, L. B. (1950). Subfamily Palaemonidae. The Palaemonidae collected by the Siboga and Snellius Expeditions with remarks on other species. The Decapoda of the Siboga Expedition. Part 10. Siboga Expedition Monographs, 39a(9), 1-268.
Holthuis, L. B. (1980). FAO Species Catalogue Volume 1 - Shrimps and Prawns of the World: An annotated catalogue of species of interest to fisheries (Vol. 1). Rome, Italy: FAO.
Holthuis, L. B., & Ng, P. K. L. (2010). Nomenclature and taxonomy. In M. B. New, W. C. Valenti, J. H. Tidwell, L. R. D'Abramo & M. N. Kutty (Eds.), Freshwater Prawns Biology and Farming (pp. 12-17). Chichester, West Sussex, United Kingdom: Wiley-Blackwell.
Holtschmit, K.-H. , & Pfeiler, E. (1984). Effect of salinity on survival and development of larvae and post-larvae of Macrobrachium americanum Bate (Decapoda, Palaemonidae). Crustaceana 46 (1), 23-28.
Imai, H., Ikeda, H., & Cheng, J.-H. (2007). Genetic diversity and population genetic structure of Macrobrachium lar and M. formosense in the Ryukyu archipelago. [Abstract]. University of the Ryukyus Repository, 1.
Imamura, T., Seeto, J., Williams, L., Mow, A.-M., Vadiga, R., & Lal, M. (2009). Freshwater prawn and crab hatchery in Fiji with rotifer culture Rua Cell System. Suva, Fiji Islands: Japan International Cooperation Agency (JICA) and University of the South Pacific.
Ismael, D., & New, M. B. (2004). Biology. In M. B. New & W. C. Valenti (Eds.), Freshwater Prawn Culture: The Farming of Macrobrachium rosenbergii (pp. 18-40). Oxford, United Kingdom: Blackwell Science.
294 ITIS. (2004). Integrated Taxonomic Information System (ITIS) Report. Macrobrachium lar (J. C. Fabricius, 1798). Taxonomic Serial No.: 96308 (Database Report). Retrieved 17/6/2011 http://www.itis.gov/servlet/SingleRpt/SingleRpt
Jalihal, D. R., Sankolli, K. N., & Shenoy, S. (1993). Evolution of larval development patterns and the process of freshwaterization in the prawn genus Macrobrachium Bate, 1868 (Decapoda, Palaemonidae). Crustaceana, 65 (3), 365-376.
Jenkins, A. P., Jupiter, S. D., Qauqau, I., & Atherton, J. (2010). The importance of ecosystem-based management for conserving aquatic migratory pathways on tropical high islands: a case study from Fiji. Aquatic Conservation: Marine and Freshwater Ecosystems, 20(2), 224-238.
Jennings, S., Kaiser, M. J., & Reynolds, J. D. (2006). Marine Fisheries Ecology (5 ed.). Oxford, UK: Blackwell Publishing.
Johnson, S. K., & Bueno, S. L. S. (2004). Health Management. In M. B. New & W. C. Valenti (Eds.), Freshwater Prawn Culture: The Farming of Macrobrachium rosenbergii. London, United Kingdom: Blackwell Science.
Karplus, I., & Sagi, A. (2010). The Biology and Management of Size Variation. In M. B. New, J. H. Tidwell, L. R. D'Abramo & M. N. Kutty (Eds.), Freshwater Prawns Biology and Farming (pp. 316-345). Chichester, West Sussex, United Kingdom: Wiley-Blackwell.
Katre, S., & Pandian, T. J. (1972). On the hatching mechanism of a fresh water prawn Macrobrachium idae. Hydrobiologia, 40 (1), 1-17.
Khan, S., Khanam, S., & Ali, S. (1984). Development of early larval stages of Macrobrachium birmanicus (Schenkel, 1902) (Crustacea, Decapoda, Palaemonidae). Bangladesh Journal of Zoology, 12 (2), 79-89.
295 Knowlton, R. E. (1970). Effects of environmental factors on the larval development of Alpheus heterochaelis Say and Palaemonetes vulgaris (Say) (Crustacea, Decapoda, Caridea) with ecological notes on larval and adult Alpheidae and Palaemonidae. Doctor of Philosophy Thesis, University of North Carolina.
Kubota, W. T. (1972). The biology of an introduced prawn Macrobrachium lar (Fabricius) in Kahana Stream. Master of Science in Zoology, University of Hawaii, Honolulu, Hawaii.
Kutty, M. N., & Valenti, W. C. (2010). Culture of other freshwater prawn species. In M. B. New, J. H. Tidwell, L. R. D'Abramo & M. N. Kutty (Eds.), Freshwater Prawns Biology and Farming (pp. 502-523). Chichester, West Sussex, United Kingdom: Wiley-Blackwell.
Lal, M., Pickering, T., & Seeto, J. (2011). Laboratory larval rearing of Macrobrachium lar in Fiji Islands. Paper presented at the Giant Prawn 2011 and Asian Pacific Aquaculture 2011 Conferences, Kochi, Kerala, India.
Larned, S. T., Chong, C. T., & Punewai, N. (2001). Detrital fruit processing in a Hawaiian stream ecosystem. Biotropica 33 (2), 241-248.
Law, A. T., Wong, Y. H., & Abol-Munafi, A. B. (2002). Effect of hydrogen ion on Macrobrachium rosenbergii (de Man) egg hatchability in brackish water. Aquaculture, 214, 247-251.
Lee, C. L., & Fielder, D. R. (1981). The effect of salinity and temperature on the larval development of the freshwater prawn, Macrobrachium australiense Holthuis, 1950 from South Eastern Queensland, Australia. Aquaculture 26, 167-172.
296 Ling, S. W. (1961). Notes on the life and habits of the adults and larval stages of Macrobrachium rosenbergii (De Man). Indo-Pacific Fisheries Council Proceedings, 9(2), 55-61.
Ling, S. W. (1962). Studies on the rearing of larvae and juveniles and culturing of adults of Macrobrachium rosenbergii (De Man). Indo-Pacific Fisheries Council Proceedings Current Affairs Bulletin, 35, 1-11.
Lober, M., & Zeng, C. (2009). Effect of microalgae concentration on larval survival, development and growth of an Australian strain of giant freshwater prawn Macrobrachium rosenbergii. Aquaculture, 289, 95-100.
Luton, C. D., Brasher, A. M. D., Durkin, D. C., & Little, P. (2005). Larval drift of amphidromous shrimp and gobies on the island of Oahu, Hawai'i. Micronesica, 38(1), 1-16.
Maciolek, J. A. (1972). Macrobrachium lar as a culture prawn in the tropical insular Pacific. Proceedings of the Annual Conference of the Western Association of State and Game Fish Commissioners, 52, 550-558.
MacLean, M. H., & Brown, J. H. (1991). Larval growth comparison of Macrobrachium rosenbergii (de Man) and M. nipponense (de Haan). Aquaculture 95, 251-255.
Mallasen, M., & Valenti, W. C. (2006). Effect of nitrite on larval development of giant river prawn Macrobrachium rosenbergii. Aquaculture, 261, 1292-1298.
Manush, S. M., Pal, A. K., Das, T., & Mukherjee, S.C. (2006). The influence of temperatures ranging from 25 to 36°C on developmental rates, morphometrics and survival of freshwater prawn (Macrobrachium rosenbergii) embryos. Aquaculture 256, 529-536.
297 Manzi, J. J., & Maddox, M. B. (1977). Algal supplement enhancement of static and recirculating system culture of Macrobrachium rosenbergii larvae. Helgol. Meeresunters, 28, 447-455.
Manzi, J. J., Maddox, M. B., & Sandifer, P. A. (1977). Algal supplement enhancement in Macrobrachium rosenbergii (De Man) larviculture. Proceedings of the World Mariculture Society, 8(207-223).
Marquet, G., Keith, P., & Vigneux, E. (2003). Atlas des poissons et des crustacés d’eau douce de Nouvelle-Calédonie. Paris, France: Muséum National D’histoire Naturelle.
Mather, P. B. (Producer). (2002). An assessment of the patterns of genetic diversity and stock structure in wild populations of the Giant Freshwater Prawn (Macrobrachium rosenbergii): A resource for improving culture stocks in Indonesia and the Philippines. [Abstract] Retrieved from http://www.aciar.gov.au/project/FIS/2002/083
Mather, P. B., Duffy, A., & Nandlal, S. (2006). Evaluation of the extent of population structuring in wild stocks of the indigenous species of giant freshwater prawn (Macrobrachium lar) in the Pacific. Paper presented at the Aqua 2006, Florence, Italy. Powerpoint retrieved from https://www.was.org/Documents/MeetingPresentations/AQUA2006/WA200 6-530.pdf
Monaco, G. (1975). Laboratory rearing of larvae of the palaemonid shrimp Macrobrachium americanum (Bate). Aquaculture, 6, 369-375.
Moraes-Valenti, P., & Valenti, W. C. (2010). Culture of the Amazon River Prawn Macrobrachium amazonicum. In M. B. New, J. H. Tidwell, L. R. D'Abramo & M. N. Kutty (Eds.), Freshwater Prawns Biology and Farming (pp. 485- 501). Chichester, West Sussex, United Kingdom: Wiley-Blackwell.
298 Mossolin, E., & Bueno, S. L. S. (2002). Reproductive biology of Macrobrachium olfersii (Decapoda, Palaemonidae) in Sao Sebastiao, Brazil. Journal of Crustacean Biology 22 (2), 367-376.
Müller, M. R., E. M. Nazari, & Simões-Costa, M. S. (2003). Embryonic stages of the freshwater prawn Macrobrachium olfersii (Decapoda, Palaemonidae). Journal of Crustacean Biology, 23(4), 869-875.
Nandlal, S. (2005). Monoculture of the native freshwater prawn Macrobrachium lar in Vanuatu, and integrated with taro in Wallis and Futuna. SPC Fisheries Newsletter #112 - January/March 2005, 40-44.
Nandlal, S. (2010). A new species for culture in the Pacific: evaluation of the potential of the indigenous Macrobrachium lar (Fabricius, 1798). Doctor of Philosophy Thesis, University of the South Pacific, Suva, Fiji Islands.
Nandlal, S., & Pickering, T. (2006a). Freshwater prawn Macrobrachium rosenbergii farming in Pacific Islands countries Volume 2: Grow-out in ponds. Secretariat of the Pacific Community: Secretariat of the Pacific Community.
Nandlal, S., & Pickering, T. (2006b). Freshwater prawn Macrobrachium rosenbergii farming in Pacific Islands countries Volume 1: Hatchery operation. Noumea, New Caledonia: Secretariat of the Pacific Community.
New, M. B. (2010). History and global status of freshwater prawn farming. In M. B. New, W. C. Valenti, J. H. Tidwell, L. R. D'Abramo & M. N. Kutty (Eds.), Freshwater Prawns Biology and Farming (pp. 5-11). Chichester, West Sussex, United Kingdom: Wiley-Blackwell.
Ngoc-Ho, N. (1976). The larval development of the prawns Macrobrachium equidens and Macrobrachium sp. (Decapoda, Palaemonidae), reared in the laboratory. Journal of Zoology London, 178, 15-55.
299 Pickering, T. (unpublished). Duty Travel Report for Vanuatu. 15-27 March 2011 for ACIAR DABL Freshwater Prawn Macrobrachium lar Capture-culture Project (pp. 14). Suva, Fiji Islands: SPC.
Pickering, T., & Forbes, A. (2002). Marine Studies Technical Report: The Progress of Aquaculture Development in Fiji (1 ed., pp. 40). Suva, Fiji Islands: Marine Studies Programme, University of the South Pacific.
Pickering, T. D., Ponia, B., Hair, C. A., Southgate, P. C., Poloczanska, E. S., Patrona, L. Della, Teitelbaum, A., Mohan, C. V., Phillips, M. J., Bell, J. D., & Silva, S. De. (In Press). Vulnerability of aquaculture in the tropical Pacific to climate change. In J. D. Bell, J. E. Johnson & A. J. Hobday (Eds.), Vulnerability of Tropical Fisheries and Aquaculture to Climate Change. Noumea, New Caledonia: Secretariat of the Pacific Community.
Pillai, D., Johnson, S. K., & Bueno, S. L. S. (2010). Health Management. In M. B. New, W. C. Valenti, J. H. Tidwell, L. R. D'Abramo & M. N. Kutty (Eds.), Freshwater Prawns Biology and Farming (pp. 256-277). Chichester, West Sussex, United Kingdom: Wiley-Blackwell.
Ponia, B. (2004). FAO Fisheries Report No. 731: Report of the Workshop on the Implementation of the 1995 FAO Code of Conduct for Responsible Fisheries in the Pacific Islands: A Call to Action. Nadi, Fiji, 27-31 October 2003. In FAO (Ed.), Appendix K: Responsible Aquaculture in the Pacific Islands: The 1995 FAO Code of Conduct for Responsible Fisheries and its Application to the Pacific Islands (pp. 160). Rome, Italy: FAO. Ponia, B. (2010). A review of aquaculture in the Pacific Islands 1998 - 2007: Tracking a decade of progress through official and provisional statistics Aquaculture technical papers. Noumea, New Caledonia: SPC.
Raabe, C., & Raabe, L. (2008). Caridean Shrimp. Glossary of Anatomical Terminology. Retrieved 18/6/2011, 2011, from
300 http://www.chucksaddiction.com/shrimpanatomy.html
Rahman, M. M., Wille, M., Cavalli, R. O., Sorgeloos, P., & Clegg, J. S. (2004). Induced thermotolerance and stress resistance in larvae of the freshwater prawn, Macrobrachium rosenbergii (de Man, 1879). Aquaculture 230, 569- 579.
Richards, A., Lagibalavu, M., Sharma, S., & Swamy, K. (1994). Fiji Fisheries Resources Profiles - FFA Report No.94/4: Fiji Fisheries Division, Suva, Fiji Islands.
Sandifer, P. A. (1973). Effects of temperature and salinity on larval development of the grass shrimp, Palaemonetes vulgaris (Decapoda, Caridea). Fisheries Bulletin, 71, 115-123.
Sandifer, P. A., Hopkins, J. S., & Smith, T. I. J. (1975). Observations of salinity tolerance and osmoregulation in laboratory-reared Macrobrachium rosenbergii post-larvae (Crustacea: Caridea). Aquaculture 6, 103-114.
Sethi, S. N., Ram, N., Dube, K., Prakash, C., & Venkatesan, V. (2011). Breeding, fecundity and ovarian development of freshwater prawn Macrobrachium lar in Andaman and Nicobar Islands. Paper presented at the Giant Prawn 2011 and Asian Pacific Aquaculture 2011 Conferences, Kochi, Kerala, India.
Shakuntala, K. (1977). The relation between body size and number of eggs in the freshwater prawn, Macrobrachium lamarrei (H. Milne Edwards) (Decapoda, Caridea). Crustaceana, 33 (1), 17-22.
Shokita, S. (1973). Abbreviated larval development of the fresh-water prawn, Macrobrachium shokitai Fujino et Baba (Decapoda, Palaemonidae) from Iriomote Island of the Ryukyus. Annotationes Zoologicae Japonenses, 46 (2), 111-126.
301 Shokita, S. (1985). Larval development of the Palaemonid prawn, Macrobrachium grandimanus (Randall), reared in the laboratory, with special reference to larval dispersal. Zoological Science 2, 785-803.
Shokita, S., Takano., M., Nandlal, S., & Vereivalu, T. (1984). Environmental survey of rivers and biology of inland water prawns in Fiji. Unpublished report. Ministry of Primary Industries, Suva, Fiji Islands.
Shokita, S., Takeda, M., Sittilert, S., & Polpakdee, T. (1991). Abbreviated larval development of a fresh-water prawn, Machrobracium niphanae Shokita and Takeda (Decapoda: Palaemonidae), from Thailand. Journal of Crustacean Biology, 11 (1), 90-102.
Short, J. W. (1998). Pictorial key to Australian Macrobrachium. Brisbane, Australia: Queensland Museum.
Short, J. W. (2004). A revision of Australian river prawns, Macrobrachium (Crustacea: Decapoda: Palaemonidae). Hydrobiologia 25 1-100.
Short, J. W. (n.d.). Pictorial key to the River Prawns, Macrobrachium of Southern Papua New Guinea: Gondwana Scientific.
Silverthorn, S. U., & Reese, A. M. (1978). Cold tolerance at three salinities in post- larval prawns, Macrobrachium rosenbergii (de Man). Aquaculture 15, 249- 255.
Smith, G., Salmon, M., Kenway, M., & Hall, M. (2009). Description of the larval morphology of captive reared Panulirus ornatus spiny lobsters, benchmarked against wild-caught specimens. Aquaculture, 295, 76-88.
Smith, G. G., Ritar, A. J., Thompson, P. A., Dunstan, G. A., & Brown, M. R. (2002). The effect of embryo incubation temperature on indicators of larval viability
302 in Stage I phyllosoma of the spiny lobster, Janus edwardsii. Aquaculture, 209, 157-167.
Sokal, R. R., & Rohlf, F. J. (1973). Introduction to Biostatistics. New York, United States of America: W. H. Freeman and Company.
Soundarapandian, P., Prakash, K. S., & Dinakaran, G. K. (2009). Simple technology for the hatchery seed production of giant palaemonid prawn Macrobrachium rosenbergii (de Man). International Journal of Animal and Veterinary Advances, 1 (2), 49-53.
Subramanian, S., Sambasivam, S., & Krishnamurthy, K. (1980). Experimental study on the salinity tolerance of Macrobrachium idae larvae. Marine Ecology - Progress Series, 3, 71-73.
Suzuki, H. (2001). A comment on the geological formation of the Mishima Islands (Takeshima, Ioujima and Kuroshima) as inferred from their freshwater crustacean faunas. Kagoshima University Research Center for the Pacific Islands. Occasional Papers, 34, 137-140.
Takano, M. (1987a). Freshwater prawn culture in Fiji. A manual for seed production of Macrobrachium rosenbergii. Suva, Fiji Islands: Fiji Japan Aquaculture Research and Development Project, Japan International Cooperation Agency.
Takano, M. (1987b). Potential of other Macrobrachium Species for Aquaculture Fiji Aquaculture Symposium: An In-house Review of Aquaculture Development Activities in Fiji. Suva, Fiji Islands.
Tayamen, M., & Brown, J. H. (1999). A condition index for evaluating larval quality of Macrobrachium rosenbergii (de Man, 1879) [(Short Communication)]. Aquaculture Research 30, 917-922.
303 Tiwari, K. K., & Pillai, R. S. (1973). Shrimps of the genus Macrobrachium Bate, 1986 (Crustacea: Decapoda: Caridea: Palaemonidae) from the Andaman and Nicobar Islands. Journal of the Zoological Society of India, 25, 1-35.
Treece, G. D., & Yates, M. E. (2000). Laboratory Manual for the Culture of Penaeid Shrimp Larvae. Texas, USA: Marine Advisory Service, Sea Grant College Program, Texas A&M University.
Uno, Y. , & Kwon, C. S. (1969). Larval development of Macrobrachium rosenbergii (De Man) reared in the laboratory. Journal of the Tokyo University of Fisheries, 55 (2), 179-190.
Valenti, W. C., & Daniels, W. H. (2004). Recirculation Hatchery Systems and Management. In M. B. New & W. C. Valenti (Eds.), Freshwater Prawn Culture. The farming of Macrobrachium rosenbergii (pp. 69-90). Oxford, UK: Blackwell Science.
Valenti, W. C., Daniels, W. H., New, M. B., & Correia, E. S. (2010). Hatchery Systems and Management. In M. B. New, W. C. Valenti, J. H. Tidwell, L. R. D'Abramo & M. N. Kutty (Eds.), Freshwater Prawns Biology and Farming (pp. 55-85). Chichester, West Sussex, United Kingdom: Wiley-Blackwell.
Willführ-Nast, J., Rosenthal, H., Udo, P. J., & Nast, F. (1993). Laboratory cultivation and experimental studies of salinity effects on larval development in the African River prawn Macrobrachium vollenhovenii (Decapoda, Palaemonidae). Aquatic Living Resources, 6, 115-137.
Williamson, D. I. (1971). Larval development in a marine and a freshwater species of Macrobrachium (Decapoda, Palaemonidae). Crustaceana, 282-289.
Wong, J. T. Y. (1987). Responses to salinity in larvae of a freshwater shrimp, Macrobrachium nipponense (de Haan), from Hong Kong. Aquaculture and Fisheries Management, 18, 203-207.
304 Wong, J. T. Y., & McAndrew, B. J. (1990). Selection for larval freshwater tolerance in Macrobrachium nipponense (de Haan). Aquaculture 88, 151-156.
Yen, P. T., & Bart, A. N. (2008). Salinity effects on reproduction of giant freshwater prawn Macrobrachium rosenbergii (de Man). Aquaculture 280 124-128.
Young Uhk, S. G. (2006). Collection and identification of freshwater prawn (Palaemonidae; Macrobrachium spp.) juveniles in creeks and rivers of Fiji for aquaculture, a preliminary study. Electronic. School of Marine Studies, Faculty of Islands and Oceans, University of the South Pacific. Suva, Fiji Islands.
Zheng, Z., Jin, C., Li, M., Bai, P., & Dong, S. (2008). Effects of temperature and salinity on oxygen consumption and ammonia excretion of juvenile miiuy croaker, Miichthys miiuy (Basilewsky). Aquaculture International 16, 581- 589.
305 APPENDICES ______
Appendix 1.1 Glossary of anatomical terms for Caridean prawns. Terms in parentheses are the plural forms. Adapted from Raabe & Raabe (2008). # Term Definition 1 Abdomen The tail, consisting of six body segments and the telson/uropods. 2 Antenna (antennae) Long, paired, usually flagellate appendage projecting from the front of the cephalothorax. 3 Antennal flagellum Multi-articulate, whip-like terminal part of the antenna. (Antennal flagella) 4 Antennal peduncle Three basal segments of the antenna, from which the flagellum arises. 5 Antennal spine Spine situated on the anterior margin of the carapace just ventral to the orbital margin. 6 Antennular Multi-articulate paired filaments (sometimes flattened flagellum and lamellate) of the antennule. (antennular flagella) 7 Antennular Three basal segments of the antennule, from which the peduncle flagella arise. 8 Antennule Short, paired, usually flagellate appendages projecting from the front end of the cephalothorax. 9 Anterior The front or frontal area. 10 Article Any one of the subdivisions of an appendage segment. 11 Basial spine Spine projecting from a thoracic appendage. 12 Biunguiculate Having two of or being forked. 13 Branchia Respiratory organ (gill) associated with an appendage (branchiae) or with the body wall. 14 Branchiostegal Short spine on or near the anterior margin of the spine carapace ventral to the antennal spine and dorsal to the anteroventral angle of the carapace. 15 Carapace The "head shield" cuticular structure arising from the posterior margin of the cephalon, extending anteriorly and posteriorly, and covering the cephalothoracic somites of the body. 16 Carina (carinae) A ridge or keel of the exoskeleton. 17 Cephalothorax Anterior part of the body consisting of the fused cephalon (head) and thorax, bearing all the appendages except the pleopods and uropods. 18 Chela (chelae) Pincer formed by the two distal podomeres of a pereiopod in which the movable finger (dactyl) opposes a fixed finger formed by a distal extension of the propodus. 19 Chelate Appendage ending in a chela (claw). 20 Cheliped Any chela (claw)-bearing thoracopod; typically refers
306 to first pair(s) of pereiopods. 21 Cornea Faceted, usually pigmented portion of the eye. 22 Coxa (coxae) First or proximal podomere of a typically seven- segmented appendage. 23 Coxal spine Spine projecting from the coxa of a thoracic appendage. 24 Dactyl Terminal podomere of a typically seven-segmented appendage. 25 Discoidal Cleavage or furrow. 26 Endopod Mesial ramus of a biramous appendage, especially one arising from the basis or from the protopodite of the pleopod. 27 Endite Lobe of several proximal podomeres of various appendages. 28 Epigastric tooth Tooth on the carapace situated above the gastric region behind the first (posteriormost) rostral tooth. 29 Epipod Lateral exite of the coxa of a thoracic appendage, sometimes branchial in function. 30 Epistome Transverse plate anterior to mouth area. 31 Exopod Lateral ramus of a biramous appendage, arising from the basis, or from the protopodite. 32 Exuviae The shed exoskeleton or molt. 33 Eyestalk Peduncle or unfaceted part of the eye supporting the cornea. 34 Flagellum (flagella) Multi-articulate, usually whip-like terminal part of the antennule or antenna. 35 Genitalia The external reproductive structures. 36 Glabrous Smooth, glossy. 37 Hepatic spine Lateral spine situated near the anterior margin of the hepatic region of the carapace. 38 Integument Outer covering or exoskeleton. 39 Ischial spine Spine projecting from the ischium or third segment of the thoracic appendage. 40 Labrum Upper lip or unpaired structure arising anterior to the mouth and often covering it. 41 Mandible One of the heavily calcified jaws lying anterior to (beneath, in ventral view) other mouth-parts. Each mandible is a stout, muscle filled structure and comes in several variations. It may carry both an incisor process for biting and cutting and a molar process for crushing and grinding food. Often present is a small, segmented palp (mandibular palp) equipped with setae which may function in cleaning the mouthparts. The various combinations of palp, incisor process and molar process are important features in the taxonomy and classification of the caridean shrimp. 42 Maxillae Posterior to the mandibles are two pairs of maxillae which play a role in food handling. 43 1st maxilla The second mouthpart and fifth cephalic appendage.
307 (maxillule) 44 2nd maxilla Paired mouthpart appendages of the fourth and fifth (maxilla) cephalic somites. The lateral blade of the maxilla (scaphognathite) extends back into the gill chamber, also known as the gill bailer. 45 Maxillipeds One of a pair of three sets of thoracic appendages, arising posterior to the primary mouthparts. The two anterior pairs are often modified for feeding, while the third pair is often pediform, resembling the pereiopods. 46 Merus (meri) Fourth segment from the proximal end of a typically seven-segmented appendage. 47 Orbital spine Spine projecting from the ventral extremity of the orbital margin. 48 Palm Portion of the chela proximal to the propodal finger. 49 Pereiopod One of the five posterior paired appendages or legs of the cephalothorax. These limbs have the typical coxal and basal segments of a biramous crustacean limb and a stick-like endopod of five segments: ischium, merus, carpus, propodus and dactylus (dactyl). The first two pairs of caridean pereiopods are equipped with chelae (claws). The chelae are used for food searching and handling, aggression and defense and grooming. In many caridean species one of the two pairs is stout and robust while the other pair is slender and delicate with the chelae carrying tufts of setae which can be used for grooming. The posterior (or last) three pairs of pereiopods are the walking legs, allowing the shrimp to step in all directions as well as supporting the body when at rest. There is considerable variation in the length and thickness of the walking legs amongst the species and usually correlate with the general body robustness and exoskeleton thickness. 50 Pleurobranchia Gill attached to the body wall (pleural membrane), (pleurobranchiae) dorsal to the articulation of the appendage. 51 Pleurite One of the lateral flaps on each of the anterior five abdominal segments. 52 Podomere Any one of the segments of an appendage, such as a segment of a pereopod or maxilliped. 53 Postantennal spine Spine located on the anterolateral area of the carapace (on the posterior part of the antennal region). 54 Postcervical spine Spine located immediately posterior to the cervical carina. 55 Postcervical sulcus Sub-vertical carapace groove located posterior to the cervical sulcus. 56 Posterior The polar opposite of Anterior (front) or the back end. 57 Postorbital spine Spine situated near the orbital margin posterior to the antennal spine. 58 Pterygostomian Marginal spine arising from the anteroventral angle or spine border of the carapace. 308 59 Ramus (rami) A branch of an exopod or endopod. 60 Rostrum (rostra) Anteromedian projection of the carapace between the eyes. 61 Scaphocerite Laterally rigid lamellate exopod of the antenna; the antennal scale. 62 Segment Division of an appendage. 63 Somite Each of the main divisions of the body. 64 Spermatophore The sperm-carrying, variously complex mass, issuing from the male petasma during copulation. 65 Statocyst Sensory organ of awareness of rotation and position located at the base of the first antenna. 66 Sternite Ventral part of a thoracic or abdominal somite. 67 Sternum Ventral surface of the cephalothorax or abdomen. 68 Stylocerite Pointed scale arising from the lateral base of the first segment of the antennular peduncle. 69 Sulcus (sulci) Groove. 70 Suprahepatic spine Spine arising from the edge of the cervical carina dorsal to the hepatic spine. 71 Supraorbital spine Spine located posterior to the orbital margin of the carapace. 72 Suture Either transverse or longitudinal, weakly sclerotized line or seam on the carapace. 73 Telson Terminal unit of the abdomen bearing the anus. 74 Tergum (terga) Arched dorsal part of each of the anterior five abdominal somites. 75 Uropod Paired, biramous appendage attached to the sixth abdominal segment, usually combining with the telson to form a tailfan. 76 Ventral surface The side closest to the ground or the "under-side".
309 Appendix 2.1 Daily record and LRT population estimation sheets.
EC = Egg Custard GW = Green Water BD = Benthic Diatoms TANK # Date Salinity Temp Water Larval Count SC = Squid Custard D = Diatoms R = Rotifers Tx. Stage FEEDING SCHEDULE ‰ C 7AM 9AM 11AM 1PM 3PM 5PM 7PM 11PM MESH SIZE & COMMENTS
310 Individual Counts Date Tank Volume Volume Counted in: Total Population Comments
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 Average
310 Appendix 2.2 Composition data for Multivitamin capsule.
Amount/Unit Active Amount/Unit Active Ingredients Units Units (Tablet) Ingredients (Tablet) Biotin 30 mcg Potassium 40 mg Riboflavin Boron 150 mcg 2mg (Vitamin B-2) Calcium 162 mg Selenium 25 mcg Chloride 36 mg Silicon 0.00 mg Thiamin Chromium 25 mcg 1.70 mg (Vitamin B-1) Copper 2 mg Tin 10 mcg Folic Acid (Folate) 400 mcg Vanadium 10 mcg Iodine 150 mcg Vitamin A 5000 IU Iron 18 mg Vitamin B-12 6 mcg Magnesium 100 mg Vitamin B-6 3 mg Manganese 2.5 mg Vitamin C 60 mg Molybdenum 25 mcg Vitamin D 400 IU Niacin/Niacinamide 20 mg Vitamin E 30 IU (Vitamin B-3) Nickel 5 mcg Vitamin K 25 mcg Pantothenic Acid 10 mg Zinc 15 mg (Vitamin B-5) Phosphorus 125 mg
311 Appendix 2.3 Algamac 3050TM composition and nutritional data.
312