SANDFISH HATCHERY TECHNIQUES By Natacha Agudo

PROVINCE DES ÎLES LOYAUTÉ SANDFISH HATCHERY TECHNIQUES

By Natacha Agudo

© Copyright Australian Centre for International Agricultural Research (ACIAR), the Secretariat of the Pacific Community (SPC) and the WorldFish Center, 2006

All rights for commercial / for profit reproduction or translation, in any form, reserved. ACIAR, SPC and the WorldFish Center authorise the partial reproduction or translation of this material for scientific, educational or research purposes, provided that ACIAR, SPC, the WorldFish Center and the source document are properly acknowledged. Permission to reproduce the document and/or translate in whole, in any form, whether for commercial / for profit or non-profit purposes, must be requested in writing. Original ACIAR, SPC and the WorldFish Center artwork may not be altered or separately published without permission.

Secretariat of the Pacific Community Cataloguing-in-publication data Sandfish hatchery techniques / by Natacha Agudo

1. Holothurians – Spawning – Handbooks, manuals, etc.

I. Agudo, Natacha II. Title.

639.8 AACR2

ISBN 978-982-00-0175-6

ACIAR, GPO Box 1571, Canberra, ACT 2601, Australia SPC, B.P. D5, 98848 Noumea Cedex, New Caledonia WorldFish Center, c/SPC, B.P. D5, 98848 Noumea Cedex, New Caledonia Cover design and layout by Muriel Borderie Photos by Natacha Agudo, Cathy Hair and Steve Purcell Printed at SPC Headquarters, Noumea, New Caledonia ii ABOUT THIS MANUAL

Sandfish is arguably the most commercially valuable of the tropical species of sea cucumber that are processed into bêche-de-mer. It is widely distributed throughout the Indo-Pacific, occurring in shallow inshore areas where it is easily accessible to coastal fishers. A-grade bêche-de-mer processed from sandfish commands some of the highest prices on the international market. But these same attributes also make it vulnerable to overexploitation. Sadly, this has happened in most places where it occurs. While sandfish was an important component of bêche-de-mer fisheries 20 to 30 years ago, its contribution to bêche-de-mer exports is now relatively small, even trivial.

Not surprisingly, there is widespread interest in restoring the production of sandfish, especially where it promises to deliver benefits to coastal fishing communities with few other options for earning livelihoods. Although improved management of capture fisheries, through measures designed to safeguard the remnant spawning adults, will always be key to restoring production, aquaculture has the potential to help restore production of this valuable species in three ways: h through production and release of cultured juveniles in restocking programmes to increase the number of spawners, but only where such releases are predicted to add value to other forms of management; h through ‘put and take’ sea ranching operations, where cultured juveniles are placed in the wild to be regathered at a larger size with no intention of allowing them to spawn; h through farming cultured juveniles in earthen ponds and sea pens.

This manual is designed to help government agencies and members of the private sector interested in implementing any of these ways of increasing production of sandfish by outlining the basic methods for spawning and rearing juvenile sandfish. It builds on the pioneering work done in 1988 at the Tuticorin Research Centre of CMFRI (Central Marine Fisheries Research Institute) in India and is based largely on methods developed and applied by the WorldFish Center (formerly ICLARM) in Solomon Islands, Vietnam and New Caledonia.

The information in the manual will enable hatcheries to produce sandfish suitable for release in the wild in relatively large numbers (tens of thousands) regularly. However, it does not pretend to be fully comprehensive. Rather, it is a reflection of current knowledge. We hope that it will soon be made out of date by those of you who apply and improve the methods described here.

Figure 1. Bêche-de-mer.

iii ACKNOWLEDGEMENTS

The preparation of this manual was made possible through dedicated research on various aspects ofthe culture and ecology of sandfish by two groups of people: firstly, those who contributed to basic knowledge on spawning and larval rearing, and the ecology of sandfish – Stephen Battaglene, Chantal Conand, Jean-François Hamel, D.B. James, Claude Massin, Annie Mercier, Andrew Morgan, Rayner Pitt, Chris Ramofafia and J. Evizel Seymour; and secondly, my colleagues at the Worldfish Center, staff at the Northern Fisheries Centre, Queensland Department of Primary Industries and Fisheries, and trainees from Conservation International, Papua New Guinea (Priscilla Eka, Pamela Mua), who helped me to refine and develop the basic methods.

I would also like to thank Warwick Nash, Johann Bell, Steve Purcell, Cathy Hair, Gale Semmens and Richard Knuckey for their encouragement, comments on the content and editing, and Steve Purcell and Cathy Hair for their photos.

This manual was funded by the Australian Centre for International Agricultural Research (ACIAR), the Secretariat of the Pacific Community (SPC) and the WorldFish Center.

iv CONTENTS Basic biology of sandfish...... 7 How to identify sandfish...... 7 More about the biology of sandfish...... 8 Where do sandfish occur?...... 8

Broodstock...... 9 When should broodstock be collected?...... 9 How many broodstock are needed? What size should they be?...... 9 Transport of broodstock to the hatchery...... 9 Maintaining broodstock in tanks, sea pens and earthen ponds...... 10 Ways to increase spawning success...... 11

Spawning...... 13 Preparing broodstock for spawning...... 13 Inducing sandfish to spawn...... 13 Observing the behaviour of broodstock...... 14 Steps to take when spawning fails...... 15 The first female has just spawned! How should fertilisation be managed?...... 15 Collection of eggs from the spawning tank...... 16 Estimating egg density...... 17

Larval rearing...... 19 Transferring fertilised eggs to larval tanks...... 19 Life cycle of sandfish...... 19 How should larvae be reared?...... 22 The first doliolaria larvae appear! What do they need to settle?...... 25 Feeding pentactula larvae and juveniles...... 26 Review of daily tasks during larval rearing...... 27

Nursery...... 29 Culture of early juveniles...... 29 Detaching and counting juveniles before transfer to nursery tanks...... 30 The two stages of the nursery phase...... 31 Review of tasks during the nursery phase...... 32

Grow-out of juveniles...... 33 In net pens in ponds...... 33 In ponds...... 34 In sea pens...... 35

Problems and possible solutions...... 36 Promising applications for hatchery-reared sandfish...... 37 Annex 1: Algal culture...... 39 Maintenance of algal cultures...... 39 Preparation and inoculation of algal cultures...... 40

Further reading...... 43



BASIC BIOLOGY OF SANDFISH How to identify sandfish

The body of the sandfish is elongated, cylindrical and stout. The dorsal body surface is relatively smooth and has small papillae (i.e. sensory tube feet) with black dots; colour varies from grey to black with dark transverse wrinkles. The ventral surface of the body is flattened and is generally whitish in colour. The mouth is on the ventral surface at the anterior end of the body. It is oval in shape and has 20 short peltate tentacles. The anus is located dorsally at the posterior end of the body.

Sandfish are Echinoderms, related to starfishes and sea urchins. The precise taxonomy of sandfish is: Phylum Echinodermata Class Holothuroidea (with tube feet) Order Aspidochirotida (with tentacles peltate) Family Holothuriidae (with body usually circular and gonads single) Genus Holothuria (Metriatyla) Rowe, 1969 Holothuria (Metriatyla) scabra Jaeger, 1833 SH I

Figure 2. Sandfish. OLOGY OF SANDF I C B I

Adult size range: 12–36 cm length

Adult weight range: 200–1500 g BAS

 More about the biology of sandfish Sandfish can be sexually mature at a size as small as 200 g. There is no apparent relationship between Sandfish have the same general anatomy as other sea fecundity (egg production) and body size. cucumbers. The gonads (ovaries or testes) lie in one tuft and open dorsally at the anterior end of the body Like other sea cucumbers, sandfish can regenerate through a single gonopore (i.e. genital orifice). The some of their organs. After spending long periods out digestive system is composed of a mouth, oesophagus, of water, or being affected by the use of chemicals, stomach, intestine, cloaca and anus. Respiratory being handled during collection and transport, or trees, which sandfish use to obtain oxygen, lie in the when stressed by predators, sandfish may eviscerate posterior of the body and open to the cloaca. The body their internal organs. Regeneration of internal organs wall that is processed into bêche-de-mer accounts for occurs within 2 months. about 56% of total weight. Sandfish and other tropical sea cucumbers can produce Sandfish move with the help of tube feet densely numerous toxins from their skin and viscera. These distributed on the ventral face, and through muscular toxins inflict distress, loss of equilibrium and death in action of the body wall. fish, but do not affect humans.

Sandfish feed on detritus, i.e. organic matter in the Where do sandfish occur? mud or sand. They appear to feed continuously using the peltate tentacles surrounding the mouth to place sediment into the mouth. Sandfish are found in many countries in the Indo- Pacific, from east Africa to the eastern Pacific. They are Sandfish are usually observed partially buried in usually found between the latitudes of 30°N and 30°S. sediment. The daily burrowing cycle varies according to environmental conditions. The preferred habitats of sandfish are shallow tropical waters, usually less than 20 m deep, such as sheltered The growth rate of sandfish depends on environmen- areas with high levels of nutrients, including muddy tal conditions and the time of year. At medium size, substrata and seagrass beds. They can tolerate reduced sandfish grow on average 0.5 cm per month, corre- salinity (20 ppt) for short periods and so are sometimes sponding to 14 g per month. Under good conditions found in brackish water. they grow to a size of 300 g in one year. We still do not know how long sandfish live, but it may be around 10 years.

Sea cucumbers have tiny calcareous plates called spicules in their skin. Microscopic examination of spicules is used to distinguish species. Sandfish have many spicules Figure 4. Wild sandfish in a seagrass habitat. in the shape of tables and knobbed buttons.

Figure 3. Geographical distribution of sandfish.

 react by them. when you moving anddisturb touch with a thin, transparent mucous layer. Animals should shiny,and lesions.smooth be should appearance Skin Broodstock should be undamaged, with no visible skin insomesmaller places, e.g. 250–415gin Vietnam. around 500 g. However, the size of broodstock be may be should broodstock of weight average Ideally, the proportion ofanimalstospawn.induce asmall to required usually are individuals 30–45 of Batches What sizeshouldtheybe? How manybroodstockareneeded? Figure 5.Collectionof broodstock. to important is It year. occur. whentheseperiods determine each periods two be spawning can there countries, some In 3 months. of period summer short a to restricted is spawning 25°, spawn varies among throughout the year. As the latitude approaches for sandfish Equator,the sandfish to close countries countries.In season spawning The immediate for ready are spawning. animals that so season Broodstock should be collected during the reproductive When shouldbroodstockbecollected? BROODSTOCK observation whentheyspawn. by or gonads, the examine to animals of dissection or biopsy by determined be only females can Sex externally. and males distinguish to impossible is It

the temperature within the range of 27–30°C. should be protected against direct sunlight to maintain containers transport. The during containers insulated in placed be should bags seawater.The of L 1 with bags oxygen-filled in individually packed and gently for transport to the hatchery, animals should be cleaned vehicle a to boatTothe broodstock transferthe from are transferredtoplasticbags. left in the transport containers to defecate before they for be hours.Preferably,should 2 way animals than the more this held be to are animals in the if provided kept be be should should Aeration containers. insulated in seawater broodstock collected sea, At Transport ofbroodstocktothehatchery to avoid stress, which can induce premature spawning. itis preferable to maintain them in optimal conditions long for periods (over 30°C) 80 hours) without eviscerating. to However, (up water static in temperatures high and oxygen dissolved low tolerate can Sandfish insulated container. Figure 6.Broodstock packed individually inplasticbags inan h h h Avoid with practised been varying success. has hours many for towels tea- damp in sandfish individual of transport The Shocks duringroadtransport handle them gently – animals the of skin the Damaging evisceration in result can which water, of out periods long for holding and shocks temperature Sudden

 BROODSTOCK Maintaining broodstock in tanks, sea pens and In sea pens earthen ponds

Broodstock that are not yet mature can be kept in captivity until they ripen. Similarly, following spawning, broodstock can be maintained near the hatchery for future use. These animals can be held in tanks, sea pens or earthen ponds. In tanks

Figure 8. Sea pen.

Sea pens for holding broodstock should be located close to the hatchery so they can be monitored easily and regularly. Sea pens should be around 800 m2 and the stocking density of the broodstock should be <200 g/m2. Additional feed is not needed. Survival in Figure 7. Broodstock maintained in tank with sand. sea pens is often very high, but growth rates may be lower than in ponds depending on local environmental conditions. Broodstock tanks should have a flat bottom with a volume of 1000 to 4000 L and be placed outdoors in In earthern ponds shade. The tanks should contain a 10–15 cm layer of sand or mud. Feed for broodstock should be added at the rate of 50 g/day. Suitable feed ingredients are: prawn head waste, soya bean powder, rice bran and seagrass powder. Care should be taken to ensure that broodstock do not lose weight. Broodstock should be stocked at a density of 15–30 animals per 1000 L tank in static aerated water. Exchange the water each day.

Broodstock held at low density in tanks with continuously flowing seawater, fed powdered dried algal preparations and ground-up prawn pellets, can Figure 9. Earthen ponds. often be spawned more than once. Earthen ponds, typical of those used for maintaining shrimp broodstock (450 to 1500 m2), have proved suitable for holding sandfish broodstock. Pond sediments should be friable, sandy-mud without large rocks. Water depths of <1 m are best. Ponds should be filled at least 2 weeks before transfer of broodstock to ensure natural plankton development. The stocking density of the broodstock should be <250 g/m². There should be daily water exchange or continuous flow. It should not be necessary to add any feed. Water quality parameters (temperature, dissolved oxygen and salinity) should be measured regularly, and daily if possible. 10 h avnae ad iavnae of keeping the wild, eachlocation. for should beevaluated disadvantages and captivity,in broodstock from individuals collecting or advantages The inducedtospawn.often were sandfish collected newly example, for Islands, essential. Solomon not In is spawning before months however, several for tanks or ponds in Note,broodstock holding that ponds. in temperatures higher the of because presumably individuals, wild than season the in earlier spawn generally they that is ponds in broodstock holding of broodstock advantage Another wild. than the spawn to easier maintained in sea pens or animals taken directly from are years 2 to months several for tanks or ponds in held Animals Ways toincreasespawningsuccess Figure 10.Feeding broodstock. Heavy rain low dissolvedoxygen High temperaturesand areas in the sediment. The combination of these extreme conditions can be dangerous for sandfish, which are which sandfish, for dangerous benthic andslowmoving.Itcanleadtototallossofbroodstockinafew days. be can conditions extreme these of combination The sediment. the in areas anaerobic of development and layers, bottom the in especially oxygen dissolved in fall temperature, in increase an include can stratification of consequences The surface. the at water low-salinity of layer thin a of presence the problems: Possible Heavy rain can lead to stratification of water in the pond. Stratification can be detected by detected be can Stratification pond. the in water of stratification to lead can rain Heavy possible). If acycloneispredicted,transfertheanimalsfrompondstoindoortanks (where Remove thesurfacelayeroffreshwaterbyadjustingheight theoutletpipe. aeration onthebottomwhereanimalsarelocated. Set uppaddlewheelsorotherpondaerationequipment;installation shouldprovide Increase waterflow,preferablycontinuously24hoursperday. morning). Regularly checkwatertemperatureanddissolvedoxygen(especially intheearly H ow

to

overcome

these h Summary h h h h h

problems 500 g)areneededtoinducespawning. weight (average individuals 30–45 of Batches each location. of for evaluated be should wild, the from directly disadvantages them collecting or and broodstock, maintaining advantages The within therangeof28–36ppt. maintained be must Salinity rain. heavy to due stratification require by caused oxygen dissolved low broodstock hold and temperatures high avoid to management to used Ponds with continuouswaterexchange. m 800 of pens sea flow- (b) food; of supply a and seawater through with substratum, mud or sand a L) with animals/1000 (15–30 tanks (a) keeping in: by them conditioned be can Broodstock shocks other Avoid during transport. and temperature 27–30°C. in at changes using containers seawater, insulated with bags in individually oxygen-filled broodstock wild Transport the spawningseason. during collected be must and broodstock healthy ripe broodstock, wild on relying When c erhn od a dniis f 20 g/m <250 of densities at ponds earthen (c) 2 at densities of <200 g/m <200 of densities at 2 ; and ; 2

11 BROODSTOCK Figure 11. Broodstock in a clean spawning tank before induction (30–45 animals are recommended).

12 6. 5. 4. 3. 2. Figure 12.Close-upofbroodstock inspawning tank. 1. Preparing broodstockforspawning SPAWNING h h Notes s a ifrn gop f nml foreach of animals group spawning. different a Use failtospawn.if they broodstock ponds.return or Also tanks,pens sea When spawning is complete, return broodstock to and faeces, untilspawningstarts. Siphon the tank bottom, to remove any sediment in thetank. them place and animals 30–45 the clean Gently water moderately. ( temperature ambient at seawater chlorine UV-sterilised with disinfect and (sodium hypochlorite). Fill with tank 1-µm filtered and the Clean water constant temperature atnight. maintain to heaters and cover Use a bare, flat-bottomed tank up to 2 m < 0C, o hih o 3–0 m Art the Aerate cm. 30–40 of height a to 30°C), induced atothertimes. be also can but moon, new and moon full the Spawning has often been observed just prior to night onthedaybroodstockarecollected. and/or evening afternoon, the usually during occurs spontaneous spawning induce Spontaneous to spawning. sufficient transport often and is collection by caused Stress

3 . Provide h h Thermal stimulation Figure 13.Coldshock treatment. Inducing sandfishtospawn h h Water pressure h h h Gonad extractionmethod h h h ep ae tmeaue wti te ag of range the within temperatures water Keep heaters.tank orusingaquarium either by hour,adding warmed 1 seawater for to the 3–5°C spawning by temperatures water Raise eun rosok o h sann tn at tank spawning ambient water temperature. the to broodstock Return minutes. afew powerful jet ofseawaterfor for about half an hour before subjecting them to a shade the in tank the in dry to broodstock Leave at ambientwatertemperature. blended male fresh gametes to the spawning tank Use the sperm as a spawning stimulant by adding the sperm. the gonadsStore of at 5°C to extend the viability males.2 ripe Dissect a few animals to extract gonads 1 from to animals covered. the keeping temperature, ambient at with water new water replace stimulation, thermal After asabove.heat shock apply Then ambient. below 5°C by temperature water the plastic lower quickly sealed to ice add containing bags and tank spawning the in the before water of level hour the this,shock.reduce To do heat 1 for treatment shock cold a is temperature water ambient the If throughout thetank. temperature uniform maintain to water the Stir 28–32°C.

> 30°C,give

13 SPAWNING Possible problem: After two days of spawning attempts, broodstock can lose their mucus and the skin may show white lesions due to stress and handling. Avoid handling the animals too much.

Observing the behaviour of broodstock

Figure 14. Dry treatment.

Dry treatment h Leave animals completely dry, or in 2 cm of seawater in the tank for 30–45 minutes.˜ Keep them in the shade. h Refill tank with water at ambient temperature.

Figure 16. Pre-spawning behaviour.

The behaviour of sandfish often indicates that spawn- ing is imminent. Pre-spawning behaviours include: h rolling movements h rhythmic contractions h lifting and swaying of the front end of the body h climbing the tank walls

Figure 15. Spirulina bath.

Food stimulants h Add dried algae (Spirulina at a rate of 30 g per 300–500 L, or Algamac 2000 at a concentration of 0.1 g/L) for 1 hour. Stir the water. h After 1 hour, remove as much waste from the tank as possible and replace water with new water at ambient temperature.

Combined treatments Often a combination of treatments is needed to induce spawning. The best combinations are given below: Figure 17. Animal stands erect ready to spawn. Treatment combinations A B C 1. Dry treatment 1. Hot shock treatment 1. Dry treatment 2. Cold shock treatment 2. Spirulina bath 2. Hot shock treatment 3. Hot shock treatment 3. Spirulina bath 14 h h h Steps totakewhenspawningfails h h h h spawning success. information: thefollowing Collect the unsuccessful attempts. This helps to improve future Record all information about spawning trials, stopspawningifdisturbed.often spawn 2–3 times over a period of an hour or more, but the bulging gonopore (i.e. genital orifice). Femalescan from spurt body powerful short a their in eggs releasing before erect females Spawning disturbed. are they when even hours, to minutes several for spawn to Males sperm. side of stream from continuous a sway releasing side, and erect are males Spawning the firstmale releases sperm. after later) or sooner (sometimes hour 1 eggs release to start which females, before spawn usually Males niiul cn oeie rlae p o t 6 to 4 to million eggs. up release sometimes can individuals some although million, 2 to 1 i.e. this, of fraction a release generally females induced but female, per eggs million 17 to 9 from ranges fecundity Natural Sandfish usually spawnatnightinthe wild. usually Sandfish night. at later induction spawning are Commence testes ripe and white. milky translucent look ovaries Ripe ripe. are they if determine to microscope a under gonads the examine and animals few a dissect or biopsy spawn, not do but behaviour pre-spawning demonstrate broodstock When induction, different methodsofstimulation. try spawning during respond not do broodstock If individual broodstockweight with irregular shape, %fertilised, diameter) individual and regular (% development egg of observations each for comments time spawning thatspawned total numberofmalesandfemales

including including How shouldfertilisationbemanaged? The firstfemalehasjustspawned! h h h h deformities. to eggdevelopment andinducelarval damage cause and fertilisation of rate the reduce can Polyspermy fertilisation). multiple (i.e. polyspermy Note that too much sperm in the spawning tank causes Management ofmales Figure 18.Aspawning sandfish. 2. 1. delayed spawningsatnightby: or spontaneous from eggs losing of risk the Reduce for several hours. several for active stays Sperm spawn. males many how and releasingRecord sperm, thetimethatmales start the more vigorous spawners. Retain eggs. releases female first the tank until the in spawning males two or one Leave releasing begin they sperm. continue will releasing they sperm. Often after shortly containers smaller to them transfer and males most remove tank, spawning the in sperm excess prevent To through system. by siphoning and adding new water, or use a flow- due to excess sperm, reduce the amount of sperm If the water in the spawning tank becomes cloudy

temperature overnight. following water constant maintain to covers and heaters the diffusers, air with tanks the collection Provide morning. until overnight moderate aeration, to retain eggs in suspension 100- internal with aflow-throughsystem. ntlig scn tn i sre wt an with series in tank second a Installing tank spawning a in broodstock Maintaining µ m sieve at the outlet pipe, with pipe, outlet the at sieve m

15 SPAWNING Management of females Fertilised eggs have a swollen membrane. If many sperm continue to cluster around the egg (i.e. h Record the time that females begin spawning. polyspermy), irregular egg development will occur. This will be helpful in estimating the progress of egg stages. h Increase aeration to moderate levels to keep eggs in suspension. h Count and record the number of egg releases per female. Females usually spawn 2–3 times. Once females finish spawning (usually evident when they stop moving), remove all broodstock (males and females). Return the animals to their tank, sea pen or pond. Separation of males into a separate tank once they begin to spawn, and later addition of controlled amounts of sperm to the females’ tank, is a way to avoid polyspermy while maintaining a larger Figure 20. A fertilised egg, two cell, and four cell stages. number of fathers.

Avoid Collection of eggs from the spawning tank h Disturbing or moving females when removing h surplus males so that spawning of eggs is not Use a small beaker to collect a sample of eggs from interrupted. the water column to estimate the fertilisation rate and percentage of eggs with advanced cell Observation of egg development division. h h After the first eggs are released, sample them Ensure that the fertilisation rate is high and the from the water column in a small beaker. majority of eggs are at the advanced cell division h stage. This is at least 1 hour after fertilisation. Measure egg diameter, using a micrometer h eyepiece placed in the lens of a microscope. Wait at least 1 hour after fertilisation before h Record the measurements of the eggs and collecting all the eggs. other observations such as: egg stages and size, percentage of regular round eggs, and fertilisation rate. h Repeat these observations every 20–30 minutes.

Recently shed eggs are white, spherical and visible to the naked eye. The diameter of the eggs ranges from 80 to 200 µm, but this varies widely within the geographic range of sandfish.

Spermatozoa are not visible to the naked eye; they appear as small, active dark dots clustered together around the eggs.

Figure 19. Fertilised egg. Figure 21. Egg collector. 16 Figure 22.Methodofegg collection. h h h h h Useful tips Be patient! Collecting eggstakestime.Be patient!Collecting suspension inthesieve. in eggs Maintain dirt. and sperm excess remove to eggs bowl.Rinse the temperature,in ambient at seawater, filtered of flow gentle a Introduce that theeggsare notsquashedonto themesh. so sieve the of mesh the above is level water the 80 µm sieve placed in a bowl (Fig. 22). Make sure 50– a into tank the from slowly eggs the Siphon h eg i te ae clm ae cleaner are column water the in eggs the Start by siphoning eggs from the water column; more hatcherystaffforashorterperiod. requires This eggs. collect to needed time the reduce to bowls in sieves with siphons several use and water, shallow in spawning out Carry them fromthe bottom ofthetank. collect then required, are eggs more If tanks. larval the in eggs of batches these sediment). Distribute or faeces by surrounded not (i.e. h h h h h h Estimating eggdensity Figure 23.Transfer ofeggs intoalarval tank. h Avoid total numberofeggsspawned. Use egg densities all from buckets to estimate the eachtank.total numberofeggsneededfor the determining after quickly tanks larval the to buckets the from eggs distribute and Transfer Record data. all eachbucket.for rate. Estimatethefertilisation under a microscope. Calculate the average density chamber) (e.g.Sedgewick-Rafter cell counting a using sample, each for density subsamples.egg the Estimate 1-ml three take bucket, each For the eggsuniformly. distribute to gently buckets the in water the Stir arebuckets filled. the until buckets, L 10 clean beakers,into using Transfer the collected eggs regularly and carefully, High densities of eggs in the sieve and buckets and sieve the in eggs of densities High <2 millioneggsper10Lbucket. i.e. eggs/ml, <200 be should buckets the in density egg Average eggs. damage can this as

1 7 SPAWNING Summary

h Batches of 30–45 clean broodstock are placed in a spawning tank, filled with 1-µm filtered and UV-sterilised seawater. h Thermal shocks, extracts of male gonads, water pressure, dry treatment, and food stimulants used alone or in combination can induce spawning. h Pre-spawning behaviours include rolling movements, rhythmic contractions, and lifting and swaying of the anterior end of the body. Males usually spawn first and females usually start one hour after the first male. h It is important to record spawning data, i.e. induction method used, number of males and females, spawning time, and observations of eggs. h Females usually release eggs 2 to 3 times. Moderate aeration maintains the eggs in suspension. Samples of eggs are regularly taken from the water column to examine the stage of egg development.

h After >1 hour post fertilisation, the eggs can be siphoned gently from the water column into a 50–80 µm sieve placed in a bowl. Flow-through seawater should be used to rinse the eggs. The eggs are transferred to buckets for counting before being placed in the larval tanks.

Figure 24. Observation of eggs under a microscope.

18 h h h h h h h h Figure 25.Larval tank. Transferring fertilisedeggstolarvaltanks LARVAL REARING 1 eggperml. to 0.3 of density a beakers,achieve or to buckets using tanks, larval the into carefully eggs Pour daylight. and photoperiod natural to tanks larval expose with 1–2 fluorescent 24hours, tubes (400 lux) per tank. Alternatively, per illumination continuous artificial hours 12 of minimum a Maintain retain theheat, ifnecessary. the to night the tanks.for covers or lids up larval Set in temperature water constant a maintain to thermostats with heaters aquarium Immerse against failure. water gentle precaution a as diffusers air and two circulation.Use aeration tank medium each insure in diffusers to air central two Install 26 to 30°C, and salinity is between 32 and 36 ppt. Ensure the water temperature is within the range of seawater. Fill the tanks with 1-µm filtered and UV-sterilised rite), withfreshwater. thenrinse Wash the tanks with chlorine (sodium hypochlo outlet screens. bottoms and central drains. up 100-µm Set mesh Prepare cylindrical tanks (up to 2 m 3 ) with conical - Life cycleofsandfish o-edn dloai lra bfr stln as from spawningtopost-settlement. cultured sandfish settling before pentactula larvae. Figure of 26 the illustrates life cycle larvae doliolaria non-feeding into transforming larvae stage) (feeding ofthe auricularia consists of sandfish development larval The Figure 26.Life cycle ofcultured sandfish. h h h Avoid during thefirstdays. because larvae of mortality total of risk the reduce they preferable are densities egg Lower eggs. excess Discard density. egg initial High the tank walls,damagingorkillingthem. against them throw can which current, strong a as in larvae and eggs transfer the trap can during this aeration of levels High and buckets larval tanksasthiscanaffectsurvivalofeggs. between water of salinity and 1–2°C) (over temperature in differences Large Gastrula 1 9 LARVAL REARING Stage Time after fertilisation Fertilised egg 0 Blastula 40 min to 3 hours Gastrula 24 hours Auricularia larvae h early 2 days h mid 4 days h late 5–6 days Doliolaria larvae 10 days Pentactula larvae 12–13 days Juvenile 15 days

Figure 27. Gastrula stage. Size range: 700–750 µm

Auricularia larvae

Main features: h Transparent slipper-shaped larvae with ciliated bands (for locomotion) h A single pre-oral anterior lobe and anal posterior lobe h Digestive tract complete: mouth, oesophagus and stomach h Slow moving – continuous activity h Pelagic and actively feeding on microalgae

Duration of this stage: 8 days Figure 29. Mid auricularia larva.

Size range: 430–563 µm Size range: 853 µm–1.1 mm Diameter of hyaline spheres: 50–70 µm

Figure 28. Early auricularia larva. Figure 30. Late auricularia larva. 20 Figure 31.Doliolaria larva. ofthisstage:Duration 2–3days h h h h h h Main features: Doliolaria larvae Size range: 420–620µm Pelagic andnon-feeding larvae Fast moving size decreasing andsettlement before metamorphosis with phase transitional Short Diameter ofhyaline spheres: 60–80µm with5hyaline spheres on eachside Larvae features begintoform Rapid changes occur inside the body and all adult bands around body Dark-brown,ciliated 5 with larvae barrel-shaped Duration ofthisstage:Duration variable h h h Main features: Pentactula larvae Figure 33. Juvenile. h h h Main features: Juveniles Figure 32.Pentactula larva. Size range: 330–750µm (feed onbenthicdiatoms) Benthic, crawling andfeeding larvae Average initialsize: 1mm detritus) Settled andfeeding stage (feed onbenthicalgae and tank, (e.g. andsettlementsurfaces diatom plates) Moving and crawling over the edge and bottom of anddifferential growthRapid locomotion) (for foot posterior single a and end anterior the at tentacles 5 with larvae tubular-shaped Dark Growth to4–5mmin1week substrata Slow moving and strongly attached to settlement juveniles early endfor at theposterior feet tube long two with but adult as shape Same

21 LARVAL REARING How should larvae be reared?

Figure 34. Seawater filtration system.

Seawater Cleaning tanks Seawater should be sand filtered, then passed through h Siphon the tank base and yellow patches of dead 1-µm filter bags or cartridges and finally sterilised larvae daily for the first four days of larval rearing. by UV. Healthy larvae stay in the water column, whereas deformed or dead larvae are found in the lower h Seawater parameters should be maintained as water column or settled on the bottom. follows: h Siphon any pink patches resulting from the Temperature 26–30°C development of bacteria. Dead larvae, faeces from larvae and overfeeding produce bacteria during Oxygen (DO) 5–6 ppm the advanced stages of larval rearing. Salinity 27–35 ppt Illumination pH 6–9 h Place a lid or a cover on top of the larval tanks for Ammonia 70–430 mg/m3 the first two days to keep eggs and early larvae in darkness.

22 h h * Protocol 3: Protocol 2: Protocol 1: Water change From Day10 Day 8 Day 6 Day 4 Day 2 stage During and after late auricularia Day 6 Day 4 Day 2 to pentactulastage From Day2 bacterial infection but is not always successful. infectionbutisnotalways bacterial h h Avoid Handle the antibiotic carefully. Use protection for the hands and face (no direct inhalation). Erythromycin is used to prevent to used is Erythromycin inhalation). direct (no face and hands the for protection carefully.Use antibiotic the Handle Add ethylenediaminetetraacetic acid (EDTA) (5 g/m protocols water: changing for three fertilisation). are There after days (i.e.two 2 day until tanks larval the in water the change not Do larvae. for harmful EDTA renders heavymetalsharmless. be can metals heavy of concentrations high seawater; in present naturally metals heavy with bind to used use andstoringthemincontainerswithchlorinatedwater. Contamination from one tank to another by thoroughly rinsing all materials with freshwater before and after is it change); water 100% effective inkillingtheswimmingstagesofcopepodsbutnot theeggs. to (50 dilution rapid by followed hours 1–3 for ppm 1–3 at (common Trichlorfon) Dipterex name, insecticide the with treatment chemical by removed be can Copepods ciliates. without 1- with change water Practise algal cultures. from and changes, water during ciliates, and copepods as such organisms, undesirable Introducing

Partial water change Partial water change withtheantibioticErythromycin Complete water change pentactula stage. g/m 5 of rate a at change water each after EDTA Add each day.Useagentlewaterflow(maximum2L/min). 100- a using water the of 30% Change a 100- with ml/min 200 of rate flow a at system flow-through a using daily water Change density of0.1–0.5larvae/ml. g/m 5 of rate a at EDTA Add seawater. sterilised 1- with it Fill tank. empty the Clean beakers. using containers, aerated to sieve from periodically larvae Transfer L/min. 5 of rate flow maximum 100- a through completely tank the Drain Complete waterchange(100%)everyseconddayuntillateauricularia stage. Carry outadailywaterchange(30%). Add Erythromycin pentactula stage. g/m 5 of rate a at change water each after EDTA Add Use agentlewaterflow(maximum2L/min). Change 30% of the water using a 100- µ m meshoutletscreeninsidethelarvaltanks. µ m filtered and UV-sterilised seawater. Use healthy algal cultures algal healthy Use seawater. UV-sterilised and filtered m * atarateof2g/m 3 ) when larval tanks are first filled. EDTA is chemical a 3 µ µ aftereachwaterchange. m mesh outlet screen inside the larval tanks larval the inside screen outlet mesh m m mesh outlet screen inside the larval tanks. µ see mesd n bw, t a at bowl, a in immersed sieve m 3 . Transfer and stock larvae at a at larvae stock and Transfer . 3 3 of the added water until water added the of of the added water until water added the of µ m filtered and UV- and filtered m

23 LARVAL REARING Feeding

Figure 35. Feeding auricularia larvae.

h Start feeding at day 2. Feeding rates for microalgae commonly used for larval h Increase the quantity of microalgae gradually rearing: from 20 000 to 40 000 cells/ml. Continue feeding as long as auricularia larvae are still present in the Hatching Larval stage Feeding rate water column. day* (cells/ml) h Examine the gut content of larvae under a 2 Early auricularia 20 000 microscope and estimate the residual algae in the water to adjust the amount of food. Well- 4 Mid auricularia 20 000–25 000 fed larvae have guts that are brown or golden in colour. 6 Mid and late 25 000–30 000 h Provide food twice a day after the water change. auricularia 8 Late auricularia 30 000–40 000

For auricularia larvae, the main microalgae used are: *Day 0 is fertilisation. Chaetoceros muelleri, C. calcitrans, Isochrysis aff. galbana, Rhodomonas salina and Tetraselmis sp. Availability of algal species may vary slightly between locations. Avoid A mixture of algae is better than using a single species h High algal concentration (>40 000 cells/ml). for rearing larvae. C. muelleri and R. salina, given in This can inhibit growth and development of equal parts, are optimal for larval culture of sandfish. larvae and decrease the survival rate. In cases Also, Isochrysis aff. galbana given for the first days and of overfeeding or algal blooms in high light then mixed with Chaetoceros sp. four or five days later areas, reduce the amount of feed given, and use is an adequate diet. a high rate of water change.

24 h h theaddition of: include Methods used to induce settlement of doliolaria larvae achieve helps aggregate nearthesurface. otherwise light andwill to attracted are tanks larvae doliolaria because settlement larval be Covering must surfaces provided. settlement Suitable settlement. delaying time, long a for swimming continue pentactula larvae into metamorphose found, doliolaria not are conditions and suitable larvae.If on settle substratum favourable to a for look larvae Doliolaria The firstdoliolarialarvaeappear!Whatdotheyneedtosettle? h h Possible problems egas evs wih rmt a distinctive a layer.biofouling promote which leaves, 0.25– Seagrass of concentration a 0.5 g/m at 2000 Algamac Settlement surfaces conditioned in unfiltered seawater can introduce predators to the larval tanks. Avoid contaminating larvaltankswithunwantedorganisms,suchascopepods andprotozoa. tanks. larval the to predators introduce can seawater unfiltered in conditioned surfaces Settlement The biofilmonsettlementsurfacescomesoffeasilyafter4–5days inthelarvaltanks,shadyconditions. 3 /day. Figure 36.Settlementsurface structure conditionedwithcultured diatoms.

eteet ufcs hud e rnfre it the is observed. tanks when the first larva doliolaria larval into transferred be should surfaces Settlement h h h h There are four ways to prepare the settlement surfaces: screens,andrough surface tilessuspended thewater.in mesh plates, polypropylene), fibreglass or polythene Suitable settlement surfaces include: plastic sheets (PVC, 1-µm filtered seawater to promote natural natural to promote conditioning withdiatoms. seawater filtered 1-µm running in (50–75%) shade partial under tanks outdoor in days 4–10 for surfaces the Immerse m Paint the surfaces with on thesettlementsurfaces. coating fine a form to days 4–5 of period a over seagrass seaweed filtered of extracts Add sp ( diatoms of cultures in them Immerse 2 ., Navicula ), and then leave them to air dry before use.

( Thalassia hemprichii Thalassia sp . , or Platymonas Spirulina , nau acoroides Enhalus (1–2 g dry powder/ sp . Sargassum ) for a few days. Nitzschia sp . or )

25 LARVAL REARING Feeding pentactula larvae and juveniles

When conditions are suitable for settlement, doliolaria larvae usually disappear from the water column in around three days. At this stage, they are settled and begin to metamorphose into pentactula larvae. Pentactula larvae need food: fresh and dried algae are an important source of food for pentactula larvae, and for juveniles up to at least 50 mm in length.

h Feed with cultured diatoms (Nitzschia sp., Navicula sp.) daily from the doliolaria stage in larval tanks, and for the first month in nursery tanks. Add sodium metasilicate (5 g/m3) and fertiliser (7 g/m3) once a week to promote growth of diatoms in the tanks. h Supplement feeding with commercial dried algae (Algamac 2000, Spirulina). Daily feeding rates are summarised in the table below.

Hatching day Stage Algamac 2000 Spirulina 10 Doliolaria 12 Pentactula 0.25 g/m3 Pentactula 0.25 g/m3 After 12 and juveniles After 20 Juveniles 0.5 g/m3 After 30 Juveniles Up to 1 g/m3 Up to 1 g/m3

Other possible feeds: h A fine paste of sieved (40–80 µm) seaweeds Sargassum( sp., Halimeda sp.) and seagrass (e.g. Syngodium isoetifolium). h A fine-grade shrimp starter pellet at a daily rate of 1–1.5 g/m3 in the nursery tanks.

Figure 37. Early juveniles in larval tank. 26 h h h h h h h h h h h Review ofdailytasksduringlarvalrearing eoe h see t h ote. is i with it Rinse freshwater.chlorinated outlet. the at sieve the Remove to the desiredachieve rate. feeding tanks the to microalgae Add microscope. under a algae of concentration test the or Count beaker tube. small a in column water the of the When water change is finished, take a sample on thesieve. of larvae avoidaggregationto sieve immersed 100-µm the below diffuser air an changed,place is water the when protocol. days change On water the Apply andabnormallarvae. proportion ofnormal the and tank per larvae 10 of lengths the Record fix thelarvae. microscope.a under Formalin addedto usually is sample to a counting chamber. Examine the larvae beaker. small the a of Transferinto 1 ml transfer and them of few sieve.a 100-µm Pipette small a of larvae sample from the a water take column by development: gently submersing larval Observe • • inthetanks: density Estimate larval Turn theaerationon. back dead for larvae. check to microscope a under dirt residual the Examine sieve.100-µm a patches,into pink or including yellow any tanks, of bottom the siphon Gently freshwater.air diffuserswith Turn off the aerationfor afew minutes. Wash the tank bottom the through pipe. drain thecentral purge days, 3–4 first the For day, andafternoon. inthemorning a twice salinity and temperature,oxygen Record density per ml. larval averagetimes.Estimatethree this Do larvae. the count and tube water test a in the column of sample a take Alternatively, samples of1ml, three in larvae the Count chamber). Rafter place on and a aliquot counting chamber ml (e.g. 1 Sedgewick- a Pipette beaker. 250 ml a with column water the of sample a Take Figure 38.Recording water parameters h h Summary h h h h h as arclra ave eaopoe into metamorphose larvae auricularia days, aremotile andplanktonic. After 10–12 rearing Earlysandfishlarvae, calledauricularia larvae, transferring eggs. and salinity between buckets and larval tanks when temperature water constant Maintain oeae eain Saae ms b 1- be must Seawater aeration. moderate 2 to (up m cylindrical be should tanks Larval cucumbers, shaped like sea adults, appear from day 15. Juvenile slow-moving larvae. pentactula benthic, into metamorphose larvae Doliolaria surfaces. settlement other which larvae,on andtank the offloor andwalls the settle on doliolaria swimming active omril re age Agmc 2000, (Algamac Spirulina algae dried commercial ( diatoms on mainly feed juveniles and larvae Pentactula settlement surfacesencourage settlement. settlement conditioned and Stimulants larvae. pentactula suitable benthic into for metamorphosing before surfaces look and appear larvae doliolaria days, rearing 10–12 After ins thewatercolumn. present still are larvae auricularia as long as cells/ml 40 000 to increasing cells/ml, 20 000 of rate the at added are microalgae Cultured water change can be partial day or complete. at start feeding and change Water doliolaria stage. the during and days rearing two first the for used are covers have or Lids or photoperiod. day, natural a hours 12 for illuminated be should tanks Larval UV-sterilised. and filtered 3 wt cncl otm ad upid with supplied and bottoms conical with ) ). Nitzschia Nitzschia sp., Navicula p) and sp.)

. The 2. µ m 2 7 LARVAL REARING Figure 39. Preparation of nursery tank includes inoculation with diatoms and insertion of settlement surfaces (plates).

28 Figure 40.Nursery tanks. h h Procedure forconditioningnurserytanks: thejuveniles.is availablefor food ensure to transfer the to prior conditioned be to least 60 cm deep (maximum 1 m). Nursery tanks need at be should tanks nursery the concrete. in water The made of usually fibreglass, flexible liner or PVC-cloth m 10 to 6 of are tanks large tanks or pools bigger Nursery raceways, tanks. nursery into from tanks transferred larval are juveniles old, days 25–35 At Culture ofearlyjuveniles NURSERY (i.e. adepthof 60–70 cm). seawater, surfaces settlement the immersing fully Fill the tank with 1-µm filtered and UV-sterilised and bare, settlementsurfaces. clean Clean all tank surfaces. Install the aeration system 3 , fe cniinn, h nrey ak ae ed to ready juveniles. andearly receive pentactula larvae are tanks nursery the conditioning, After h h h (26–28°C). warm and constant temperature water the Keep aerationmoderate andmixthewaterdaily. other settlement surfaces and tank walls. Maintain allow a diatom coating to develop on the plates or to days 3–4 first the for flow water the Turnoff (7g/m general fertiliser in the tank. water Add sodium metasilicate of (5 g/m volume total the of 6–7% of rate a diatomcultures at fresh with water the Inoculate 3 ). on thelight. Switch 3 ) and a 2 9 nursery Detaching and counting juveniles before transfer to nursery tanks

Pentactula larvae and early juveniles are characterised by a very wide size range and are difficult to detach. The transfer procedure involves detaching juveniles from the larval tanks, estimating their abundance, and then putting them into the nursery tanks. h First, remove the settlement surfaces and attached animals from the larval tanks while holding a plastic tray underneath to catch any juveniles that fall off the plates. Transfer the plates and any loose juveniles to the nursery tanks. h Second, remove the juveniles from the walls of the larval tanks. There are two methods for detaching juveniles from tank walls. 1) Drain the larval tank completely through the bottom pipe and by siphoning the floor on to mesh sieves (0.3 to 1 mm) placed in bowls. Use a gentle jet of seawater to detach any remaining animals. 2) Add potassium chloride (KCl) to the water using a 1% concentration. Leave for 10 minutes and then replace with normal seawater. The juveniles will detach rapidly when the new seawater is added. Drain the tank onto mesh sieves placed in bowls. Use a spray of 1% KCl to detach any remaining animals. h Estimate abundance by direct counts of juveniles Figure 41. Detachment by draining. on plates and sieves. 1) For plates, count all juveniles on several individual plates, selected randomly. 2) For sieves, count only a half or quarter of the total surface where there are high densities of juveniles. h Use abundance estimates to calculate the survival from the egg to pentactula stage for each larval tank. Survival is usu ally variable and rarely higher than 1–2%. h Use abundance estimates to calculate the initial density of juveniles placed in nursery tanks.

Possible problem h The wide variation in the size distribution of juveniles can lead to brased estimates of abundance; thousands of pentactula larvae are too small to count without harming them. Estimates of abundance become more reliable as the juveniles grow larger.

Figure 42. Detachment by siphoning. 30 can be 0.2–0.8 mm/day after one month. be0.2–0.8mm/day after can the survival rate can be up to 50%, and the growth rate m per juveniles 700 and 500 between be should density initial problems, such avoid Tosurvival. and growth (0.3–1g) in 30 days. However, high densities can affect a size of about 1 g. Juveniles should grow to 10–20 mm Juveniles are maintained in bare tanks until they reach be juveniles, andhighdensity. duetohandling of transfer can the following week mortality the during Substantial expected phase. critical and The early juvenile stage ( First nurseryphase(firstmonth) Figure 43.Nursery tankwithsettlementsurfaces. The twostagesofthenurseryphase 2 of tank floor. At a density of 500 juveniles per m per floor. juveniles tank 500 of of density a At < 5 mm length) is a vulnerable 2 , they arethey when 50% exceeds usually juveniles of rate survival mm/day, g/m 0.5 225 reach densities is if down slow rate will but growth the months, 2 After growth individuals. ofsmaller m to be reared at optimum densities of 100–300 juveniles/ After 1–2 months of grow-out on sand, juveniles need mud (e.g. orfood algae). dried with enriched then and cleaned be should sand The tanks. nursery of bottom the on distributed evenly is mm) (3–5 sand of layer sediment.thin of A amounts large ingest now can they because sand on grow to transferred are juveniles g, 1 or mm 20 of size a At Second nurseryphase 2 . Removal of large juveniles will an increase in the in increase an will juveniles large of . Removal > 20 mm. 2 .The

31 nursery Review of tasks during the nursery phase

Daily tasks h Record temperature, oxygen and salinity twice a day, in the morning and afternoon. h Wash the screen placed at the tank outlet. h Change the water, using filtered seawater (1 µm for the first two months, 10–25 µm later), with constant flow-through of 6 L/min (100–200%). In case of restricted seawater supply, flow-through can be reduced to 3 L/min (40–50%) every night. High flow rates are generally better than low rates. h Stop the water flow for a few hours for feeding. Feed with diatom cultures, dried algae, seaweed paste and/or fine-grade starter shrimp pellet. Weekly tasks h For the first weeks, leave animals in shade; they Figure 44. Copepod contamination. avoid bright conditions and prefer shady sides of settlement surfaces. h Add diatom cultures during the first month. h Add sodium metasilicate and general fertiliser. Summary Mix the water. h Invert all settlement surfaces. Diatoms grow in h Juveniles can be transferred to nursery tanks abundance on the upper side but not on the lower after 25–35 days. side. Pentactula larvae and juveniles stay at the h Nursery tanks are raceways, pools or tanks 3 bottom. of 6 to 10 m . They must be conditioned h Take a sample of 30 juveniles per nursery tank before transferring the animals. Conditioning to estimate the growth rate. Measure total and consists of developing a diatom coating on the individual weights and calculate the average. tank walls and settlement surfaces. Examine the physical condition of a few animals h Water exchange is flow-through or partial, under a microscope. with filtered seawater. h Transfer of juveniles from larval tanks to Monthly tasks nursery tanks requires detaching, counting h Estimate the number of juveniles per nursery and grading the animals. Spraying the tank to grade densities. Sample over 5% of the walls of the tank with seawater after tank area, including the settlement surfaces, using complete draining by siphoning, or chemical small quadrats (20 cm x 20 cm). In cases of high detachment using potassium chloride, are density, collect juveniles with a siphon and hand two methods of detachment. nets. Distribute surplus juveniles to a new nursery h There are two nursery phases. During the first tank. phase, the juveniles are held in bare tanks. After about one month, when they are 20 mm or 1 g, they are transferred to tanks with sand substrata (second nursery). Possible problem h Grading consists of reducing the density of h Unfiltered seawater can result in better juveniles over time. Initial densities range growth than filtered seawater, but predators from 500 to 700 juveniles per m2 in bare such as copepods can quickly contaminate tanks; optimum densities vary from 100 the tank. Copepods compete for food with the to 300 juveniles per m2 in tanks with sand juveniles. (second nursery).

32 or survival. Growth averaged rate 0.08–0.1g/day over 3weeks. used,be good).as not improveis growthgrowthnot but did nets bag the to Additionsandy-mudsubstrate of was low, good growth was obtained by adding ground shrimp pellets (chicken manure or juveniles/m 150 of Larger juveniles (1–2 g) can be placed in ‘bag nets’ (4 m seagrass. artificial juveniles/m 150 m (1 ‘hapas’to transferred were mm 5 as small as g),but (0.5–1 long mm 15–25 juveniles tanks, nursery in limitations space overcome to Caledonia, New In In netpensinponds Figure 45.Hapas (1m place toplace. from vary to expected be can Caledonia. Results New and Vietnam in experiments from are results following The GROW–OUT OFJUVENILES h h h Possible problems and bagnetsoffthesubstrata. Measurethetemperature,oxygenandsalinity ofthepondwaterregularly. hapas the of floor the raise sediment, anaerobic and temperature high levels, oxygen low of risks reduce To Nets becomedirtyafterseveral weeksinponds.Brushnetsregularly to allowfreewaterflow. net material. Some netting materials are too abrasive for sandfish juveniles, or too fragile to be durable. Tentex is a suitable Weight range 0.5 2 1 . Survival averaged 97% over 23 days and growth rate was higher (0.1 g/day) in hapas with with hapas in g/day) (0.1 higher was rate growth and days 23 over 97% averaged .Survival – 2 – 2 g ). 2 . Feeding was not necessary in ponds with good natural productivity. When productivity productivity productivity. natural When good with ponds in necessary not was .Feeding 1 g Bag net1 Hapa 1m 2 net pens with fine mesh) in earthen ponds at densities of densities at ponds earthen in mesh) fine with pens net Net pens – 2 4 m net pens with coarse mesh) in earthen ponds at densities 2 , 1mdeep Figure 46.Bag nets(4m 2 , 1mdeep 2 ). 660 Net mesh Sargassum 1 mm – 670 µ m sp. can also

33 GROW–OUT OF JUVENILES Figure 47. Sandfish juveniles grown in an earthen pond.

In ponds Possible problems h Quality and structure of sediment in ponds, In Vietnam, large sandfish (50–500 g) stocked in ponds and seawater supply, will affect growth and had growth rates of 2.2–3.2 g/day. These growth rates survival. Poor environmental conditions can varied inversely with stocking density in the range lead to excessive growth of filamentous algae 106–170 g/m2. Survival was high (88–97%) until and anaerobic sediment. the start of the wet season when massive mortality h Stratification of water from heavy rain leads occurred due to stratification and lethally low salinity. to low oxygen levels, high temperatures and inadequate salinity (below 20 ppt). In New Caledonia, juveniles of 1 g have been reared h Poor environmental conditions in ponds can in earthen ponds at a density of 1.4 juveniles/m2. The lead to development of skin lesions. growth rate observed over 1 year averaged 0.8 g/day (24 g/month).

1.2 28

1.0 26

24 0.8

22 0.6

20 0.4

18 (g/day) rate Growth

0.2 16

Mean water temperature (°C)

0 0.0 1 2 3 4 5 6 7 8 9 10 11 12 Grow out period (months)

Figure 48. Growth rate of juveniles of 1 g reared in earthen ponds in New Caledonia, and mean temperature of pond water. 34 mesh;2 Figure 50.Juvenile sandfish (2.5to12g). density high at stocked (500 g/m juveniles Vietnam, In in earthen ponds. and survival of sandfish in sea pens is much lower than m mesh;mm4 0.6m screens or palmirah rafters erected in shallow water with sandfish. These include 25 m Various sea pens and cages have been used to grow out Figure 49.Seapenof500 m In seapens i nt rw Rdcn te est t 390 g/m to density the Reducing grow. not did 2 netlon cages with 5 mm mesh. In general,mesh.Ingrowth mm 5 with cages netlon

m 2 i sa es uvvd el 9% but (98%) well survived pens sea in ) 2 velonscreen cages with mm7mesh; and 2 2 rectangular iron cagesmmwith 2 2 . 2 pens made from bamboo 2

4 juveniles/m 500 m Caledonia,NewIn juvenilesg1–20survival placedin and broken coral. substratum silty-sand a with deep m 1.5–2.5 water in placed was and mesh mm 8 had pen sea pen.The g/day over 5 months when reared in a 2000 m 2000 a in reared when months 5 over g/day of juveniles/m 0.73 growth at stocked g and 84 of weight (90%) average an with g/day. Juveniles survival 1.7 high in resulted h h h h h Summary 2 sea pens in shallow seagrass beds at densities of aaeet s eurd bt rwh and growth survival arefarlowerthanin ponds. but Less required, pens. is sea in management stocked be can Juveniles into earthenponds. directly released be can g) (>1 juveniles Large of risks off reduce to kept unfavourable environmentalconditions. pond be the of should bottom nets the bag and Hapas with coarsemeshinearthenponds. At 1 g, juveniles can be reared in large bag nets ponds. earthen in mesh) fine with pens (net hapas to transferred be can mm 5 as small as Juveniles 2 was 5–9 % after 18 months. 2 rw t rt o 1.05 of rate a at grew 2 sea

35 GROW–OUT OF JUVENILES 36 PROBLEMS AND POSSIBLE SOLUTIONS h h h h PROBLEMS ANDPOSSIBLESOLUTIONS or seabed cages can be used to replace nursery tanks. beusedtoreplace can or seabedcages nursery phase. nursery second the For nursery,for second animals the largerremove to grading ponds in nets bag often result in substantial costs. However, space can be saved by using additional conditioned plates and by tanks: nursery for space Limited UV treatment by andsterilisation chlorination-dechlorination. However, keeping larger cultures free of contamination by copepods requires systems for adequate filtration,Production of large volumes of algae: lightisalsoan Useadvantage. instead ofdaylight artificial stress. temperature-induced reduced through survival larval higher to lead and rearing larval of efficiency the improve will day each changes water partial only require that tanks larval larger temperature.of Use tanks: rearing of Size spawning season months. by afew sandfish. the ranching extend sea to developed and be farming to for need scope Methods the limits This eggs: Obtaining Figure 51. Earthenpondsuitable for installation ofhapas andbag nets. In many countries, eggs can only be obtained during a relatively short spawning season.spawning short relatively a during obtained countries,be many In only can eggs Small larval rearing tanks requiretanks arerearing andin prone labour considerable variation to larval Small As larger nursery tanks are required for the first nursery stage,can nursery this first the requiredfor are tanks nursery larger As Algal production be increased can by using large outdoor cultures. Figure 52. Hatchery-reared sandfish. h h h h h for pondofsandfish: following questions assessthepotentialfarming The needtobeanswered tofully pens. However, sandfish should not be reared in ponds together with shrimp because shrimp prey on sandfish. The availability of cultured juvenile sandfish also provides potential for farming this species in earthen ponds or sea h h h programmes. ranching sea and enhancement restocking, for stock used be could juveniles survive,hatchery-reared them of proportion high a ensure that wild the in sandfish cultured releasing for developed be can methods Provided SANDFISH PROMISING APPLICATIONS FORHATCHERY-REARED Are somerearingsandfish? sedimentsbetterthanothersfor ofsedimentsis needed,If enrichment cycleto whatisthebestmethod? production in the later added, to be have food maintain good growth rates? does or out, bethinned to need sandfish Do shrimp ponds? harvested filled and Do sandfishhaveeffect abeneficialonthesedimentsof recently farm to used previously those shrimp? including ponds into sandfish stocking for density optimal the is What animals are placed inthewildtobegathered atalarger size ‘put andtake’ operations. population.Instead,the within age-classes particular or biomass spawning augment to animals released the allowing of intention no there is that in enhancement stock from and restocking differs ranching Sea ofjuveniles.well by supply inthenatural overcoming shortfalls reasonably operating still is that fishery a of productivity the increase to designed is enhancement Stock wherecan toalevel once againprovide it regular,fishery depleted yields. substantial severely a of biomass spawning the restore to juveniles cultured releasing involves Restocking 3 7 PROMISING APPLICATIONS FOR HATCHERY-REARED SANDFISH Figure 53. Large volumes of cultured microalgae in 150 L bags in an indoor culture room with artificial light.

38 h h h h Maintenance ofstockcultures h h h organisations: thefollowing from available are cultures new start to algae quality High Purchase ofstockalgalcultures Maintenance ofalgalcultures ANNEX 1:ALGALCULTURE Figure 54.Stock cultures ofalgae inErlenmeyer flasks. longer than2weeks). Sub-culture the stock cultures once a week (or no to day maintain thealgae every insuspension. hand by cultures stock the Swirl loss ofalgae. to reduce the risks of contamination and complete species each of cultures stock of series two Keep 24°C). room (20– anair-conditioned in light artificial flasks to conditions, close static and Erlenmeyer sterile in ml) (250 in cultures stock the Store Association, Citadel Hill, Plymouth, PL 2PB, Biological Collection, U.K. Culture Marine Plymouth Marine Laboratory, Oban,PA37 Dunstaffnage Argyll 1QA, Scotland Ltd, Services Research Culture Collection of Algae and Protozoa, SAMS Box 1538, Hobart, Tasmania, 7001Australia CSIRO Marine and Atmospheric Research, GPO h h Conditions required for large volumes of algae (500 L) h h h h h of algae <500L) (volumes room algal the for required Conditions h Possible problem o otor utrs ih aua daylight, natural with provide continuous mixing cultures of water and protection outdoor For continuous mixingofwater. and light artificial provide cultures, indoor For ofalgae, transfer sterile ifpossible. Use a laminar flow or simple glasscabinet for the and carboys bags. flasks, large for aeration Provide 14hoursperday.‘daylight’) (‘cool white’ tubes fluorescent byProvide light or by airconditioning. 24°C and 20 between temperature air Maintain i.e., have a room exclusively for algal culture. Avoid contamination by cultures of other organisms, against rain. air linebeforeitenterstheculture. contamination, place a filter of 0.2–1 line due to high levels of moisture. air To avoid this the through introduced be could Ciliates µ m in the 3 9 ANNEX 1: ALGAL CULTURE Preparation and inoculation of algal cultures

Preparation or purchase of culture media Preparation of flasks and 10-L carboys The most common medium used in the culture of h Wash the culture vessels with detergent, rinse and microscopic algae is Guillard’s f/2 medium. However, leave to dry. when using this medium the quantity of all ingredients h Fill flasks and carboys with 0.2–1 µm filtered should be doubled; f/2 medium becomes f medium. and UV-sterilised seawater at low salinity (25– This f medium is more appropriate for culturing 30 ppt). Rhodomonas salina. h Add the culture medium. h Prepare the f/2 (Guillard’s) medium (nitrate, h Add the glass tubes (aeration line), cotton and phosphate, trace metal mix, ferric citrate, foil caps. vitamins). A solution of sodium metasilicate h Autoclave at 121°C for 20 minutes. is added only for cultures of diatoms. Use the f h Store flasks and carboys for at least 24 hours before medium for Rhodomonas salina culture. use to overcome the temporary rise in pH. h Alternatively, the media can be purchased from: h Inoculate with algal cultures. Culture Collection of Algae and Protozoa, SAMS Research Services Ltd, Dunstaffnage Marine Laboratory, Oban, Argyll PA37 1QA, Scotland. h Store the media in a dark, clean place in a fridge.

Figure 55. Carboys (20 L). 40 Figure 56.Algal culture ina500Ltank. h h h h Preparation of20-Lcarboys,bagsandtanks residual chlorine. Add sodium thiosulphate (stock Turn on the aeration for 10–15 minutes to remove upto24hourswithoutaeration.for 0.2 ml/L a for minimum of 4 hours, or overnight UV-sterilised and Add chlorine (12.5% active) at a concentration of filtered seawater. 1-µm with Fill to dry. Wash with diluted chlorine (10%), rinse and leave diatoms usethesilicateforproductionofanexternalshell. Chaetoceros as such cultures diatom for added is metasilicate Sodium h h Inoculate withalgalcultures. (3.75g/100L). also add sodiummetasilicate such as Aquasol (2.5 g/100 fertiliser L). agricultural For add minutes,diatom cultures, several After test. chlorine for ml/L chlorine.a residual Conduct 0.2 neutralise to hour 1 of rate a at g/L) 250 at solution sp. , Navicula , sp. and Nitzschia and sp. ;

41 ANNEX 1: ALGAL CULTURE Inoculation method for flasks and carboys Summary h Carry out inoculations in a laminar flow or simple glass cabinet. h Keep two series of stock cultures for each h Swab the cabinet work area and hands with 70% species of algae in Erlenmeyer flasks. alcohol. h Subculture once a week (or no longer than 2 h Place the culture volumes required for transfer weeks). in the cabinet close to the flame of a Bunsen h Maintain the cultures in flasks, carboys and burner. bags in an air conditioned room (20–24°C) h Flame the mouths of the flasks before and after with lighting 14 hours per day. Swirl the stock transfer. cultures by hand daily and provide regular h Working close to the flame, remove the cap of mixing for large volumes of cultures. the new flask and quickly add nutrients and algal h Place a filter in the air line before it enters the inoculum. culture to avoid contamination by ciliates. h Replace the cap immediately. h Preparation of an algal culture, and the h Label the flask with the date and the name of the sterilisation method used, depend on the algal species. culture volume. h Sodium metasilicate should be added to the Culture volumes Turnover time media for the culture of diatoms. 250 ml Erlenmeyer flasks 4–7 days h Inoculation and transfer of algal cultures 2 L flasks 3–7 days require a clean and sterilised environment. h For outdoor cultures, water temperature must 10–20 L carboys 4–7 days not exceed 32°C. 150 L bags 7–14 days 500 L tanks 4–7 days

42 Massin, C., 1999. Reef-dwelling holothuroidea James, D.B., Gandhi, A.D., Palaniswamy, N., Rodrigo, James, D.B., 2004. Captive breeding oftheseacucumber, James, D.B., 1999. for andculture technology Hatchery James, D.B., 1996. Culture ofseacucumber. Hamel, J.-F., Conand, C., Pawson, D.L, Mercier, A., 2001. Conand, C., 1989. aspidochirotes du holothuries Les Bell, J.D., Rothlisberg, P.C., Munro, J.L., Loneragan, Battaglene, S.C., Seymour, J.E., Ramofafia, C., 1999. Battaglene, S.C., Seymour, J.E., 1998. Detachment Battaglene, S.C., 1999. Culture oftropicalseacucumbers FURTHER READING 201. beche-de-mer. andexploitation as Echinodermata): Itsbiology The seacucumber Paris, 393p. exploitation. EtudesetthèsesORSTOM, 49: 1-370. fisheries.invertebrate enhancementofmarine andstock Restocking N.R., Nash, W.J., Ward, R.D., Andrew, N.L., 2005. Holothuria scabra Survival and growth of cultured juvenile sea cucumbers, Aquaculture Holothuria scabra and gradingofthetropicalseacucumbersandfish, Quarterly ICLARM restoration stock andenhancement.for 329: 4-144. Sulawesi, Indonesia. ofthespermondearchipelago(Echinodermata) Research Institute Special Publication cucumber J.X., 1994. and culture techniques ofthesea Hatchery Fisheries Technical Paper cucumber aquaculture andmanagement. Holothuria scabra Naga,Quarterly The ICLARM the seacucumber, 120-126. Research Institute Fisheries Central Marine lagon deNouvelle-Calédonie : biologie, écologieet Holothuria scabra 159: 263-274. Advances in Marine Biology Advances inMarine . , settlementsubstrates. juveniles from , India. from Advances insea Aquaculture Holothuria scabra Holothuria scabra 22(4): 4-11. Zoologische Verhandelinger Leiden Advances in Marine Biology Advances inMarine 463: 385-395. . 178: 293-322. Central Marine Fisheries Fisheries Central Marine 22(4): 12-16. Jaeger, inIndia. (Holothuroidea: No. 57. Naga, The Bulletin FAO 48: 41:131-

Ramofafia, C., Battaglene, S., Byrne, M., Pitt, R., Duy, N.D.Q, 2004. Breeding and rearingof Pitt, R., Duy, N.D.Q., 2003. How toproduce 100tonnes Pitt, R. 2001. Review of sandfish breeding and rearing Morgan, A.D., 2001. Aspectsofseacucumber broodstock Morgan, A.D., 1998. andspawningofthe Husbandry Shelley, C.C., 1985. Growth of Purcell, S., 2005. restocking for technologies Developing Pitt, R., Thu, N.T.X., Minh, M.D.,Phuc, H.N., 2001. Pitt, R., Duy, N.D.Q., Duy, T.V., Long, H.T.C, 2004. management. Advances inseacucumberaquaculture and the seacucumber 18:15-17. of sandfish. methods. Bulletin Bêche-de-mer Information management (Echinodermata: Holothuroidea). Queensland, 20November 1998. Science, inMarine Master ofScience of University Holothuroidea). Thesis submittedforthedegree of sea cucumber International Coral ReefCongress,International Coral 5: 297-302. in Papua NewGuinea. Proceedings oftheFifth potential(asbeche-de-mer) and theirfisheries Holothuria scabra andZeitlinger,Swets Lisse: 369-375. 2000, to5February January Dunedin, NewZealand, Conference,of the10thInternationalEchinoderm 28 scabra development inthetropical seacucumber Bulletin in NewCaledonia. sandfish: Update on the WorldFish-SPC project Bulletin and pensin Vietnam. intanks, sandfishgrowthPreliminary trials ponds Bulletin Information monodon ( Sandfish 333-346. . 2000 Proceedings InBaker(ed.) Echinoderms 22: 30-33. 15:17-27. ) co-culture tankstrials. SPC Holothuria scabra SPC Bêche-de-mer Information Bulletin SPC Bêche-de-mer Information

Bêche-de-mer Information Bulletin FAO Fisheries Technical Paper Holothuria scabra (Holothurioidea: Echinodermata) Holothuria scabra SPC Bêche-de-mer Information SPC Bêche-de-mer Information 20: 12-22. SPC Bêche-de-mer Information SPC Bêche-de-mer Information ) with shrimp ( ) withshrimp Actinopyga echinites (Echinodermata: SPC Bêche-de-mer 13: 2-8. Nam. in Viet 2001. Larval Penaeus Holothuria 463: 14: 14-21. and SPC

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