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DEVElOI)MENTOFIIATCIIERY FACILITIES FOn TilEBREEDING AND LAltV AL REARING OFSELECTED MACROBRAC/IIUM SPECIES

MYRON PAUlCORT

Dlssertatlon presented In porth" fulfilment of the requirements for the degree MASTEn OFSCIENCE In ZOOLOGY In the FACULTY OF NATURAL SCIENCE at the RAND AFRIKAANS UNIVERSITY

SUPERVISOR: Prof11.1. SCIIOONDEE CO-SUPERVISOR: DrJ.T. FERREIRA ij.... ;, . ~.

0248054/4/11 BIBLIOTEEK :;.';:-:: "au

AUGUST 1983 DEDICATED 1'0 MY PARJ::NTS AND CIIERYL FOR TIIHIR CONSTANT SUPPORT AND ENCOURAGEMHNT TADLE OFCONTENTS Page

CIiAPTER ONE INTRODUCTION ...... 1 CIiAPTER TWO LITERA'rURE REVIEW ••••••...... •.•.•..•...... ••..••• 4 2.1 CflOiCEOFCULTURESYSTEMS . 6 2.2 LARVAL REARING FACILITIES . 14 2.3 HOLDING FACILITIES FOR BREEDING STOCK •••••••••••• 19 2.4 IIOlDiNG FACILITIES FOR POST·LARVAE ••••••••••••••• 20 2.5 MANAGEMENT OF ADULTS FOR BREEDING PURPOSES •••• 21 2.6 LARVAL DEVELOI)~fENTAL FORMS •••••.•••••••••••••• 2S 2.7 LARVAL REAfUNG AND FACTORS AFFECTING DEVELOP· r.tENT ...... •..••••.•.•.....••.•.•...... •.•.••• 28 2.8 POST·LARVAL REARING . 36 2.9 DISEASES AND OTHER FACTOltS AFFECTING SURVIVAL OF PRAWNS IN CULTURESYSTEMS . 41 2.1 0 SU~I~IARY ••••••••••••..•••••••••••••.••••.••••••••• 48 CHAPTER TIIREE MATERIALS AND METHODS . 52 3.1 INFRASfRucrulm ...... • 52 3.2 INVESTIGATION INTO TilEUSE OF SOLAR ENERGY AS A SOURCE OF IIEAT FOR CULTURE WATER . 55 3.3 ANALYSIS AND MANAGEMENT OF WATER CONDITIONS •• 56 3.4 LARVAL REARING FACilITIES . 59 3.5' HOLDING FACILITIES FOR BREEDING STOCK •••••••••••• 65 3.6 1I0LDlNG FACILITIES FOR POST-LARVAE . 69 3.7 COLLECTION AND MANAGEMENT OF ADULTS FOR BREEDING PURPOSES •...... ••..•.•.•....••.....••••• 69 3.8 PRELIMINARY STUDY OFLARVAL DEVELOPMENTAL FORAfS ...... • ...... •...... ••••••• 12 3.9 LARVAL REARING PROCEDURES AND MANAGEMENT PAACflCES •.•.•...... ••.•...... ••••••• 7S 3.10 INVESTIGATION OF TIlE SALINITY PREFERENCES OF TilE EARLY LARVAL FORMS •••••••••••••••••••••••••••• 82 3.1t POS'f·LARVAL REARING ...... •• 88 I, Paso

3.12 TREATMENT OF DiSEASES . 91 3.13 SU~IAIARY •••••••••••••••••••••••••••••••••••••••••• 92 CHAPTER FOUR ItESU LTS ...... •...... ••.. 9S 4.1 PJlYSICQ.CHEMICAL PARAMETERS OF WATER QUALITY •• 9S 4.2 COLLECTION AND IDENTIFICATION OF TilE INDIGENOUS SPECIES ....•••••.•...... •.....•...... •••••••• 103 4.3 POTENTIAL OF SOLAR ENERGY AS A SOURCE OF HEAT FOR CULTURE \YATElt ...... •...•. 106 4.4 SURVIVAL AND REPRODUCTION OF TilE ADULTS UNDER LADORATOny CONDITIONS . 106 4.5 LAIWAL REARING PROGRAMME ••••••••••••••••••••••• 113 4.6 POST·LAnvAL REARING •••••••••••••••••••••••••••••• 140 4.7 TREATMENT OF DISEASES . 142 4.8 SU~Ir.fARY •••••••••••••••••••••••••••••••••••••••••• 144 CHAPTER FIVE DISCUSSION ...... •••.••••.•...... •••...•.•...... •••••••••• 146 LITERATURE CITED .....•...... •...... •••.••• 166 j

ACKNOWLEDGEMENTS

J would like to take this opportunity toexpressmy gratitude to the (ollowing people and lnstltutions, without whose aid this study would not have been completed.

• Prof(ssor 11.1. Schoonbee for acceptingme liSa candidate (or this study and for his support and encouragement over the course of the project.

• Dr J.T. Ferreira (or his sound, practical advice, when most needed.

• The C.S.I.R.and the Rand Afrikaans University for financial support.

• The Department of Zoology at the Rand Afrlkallns University and especially Dr J.G. van As, his postgraduate students andMr T.Brockman. (or their assistance.

• Dr K.M. Calgcr and the staffof the Deportment of Zoology at the University of Zululand for their valuable assistance In thecollcction of wild prawns.

• Professor L.B. lIolthuls (Lelden, Netherlands), forhis co-operation in ldentl­ fying the indigenous species.

• Md. llesp and Mr J. Dell, for the development of anelectronic temperature control unit.

• Dr V.Hamilton·Atwell for the preparation of material for electron microscope studies.

• Marilyn Kohli, Sharon Cort, Dee Wingate and Richard Benjamin (or their help inrounding the manuscript off.

• Mrs L. Lamblsnon for her assisttnce in the coUeclion of prawns from the Limpopo river.

• Mrs GJ. Best, for her patience in typing thismanuscript.

• Mr W. Makhura (or hiJ assistlnce during the course of theproject. u

SUMMARY

• LDboralory scale hatcherylacllllies were developed. lesled and usedlor breeding and rearlng/lve lndtgenous Macrobrachfum species aswell as Ihe glanl Malaysian prawn, M. rosenbergll. Tire culture systems chosen comprised closed water re­ circulation systems employinggravel biological fllters lor water purtflcation, and synll,ellc sea salts were usedlor prt:parl"g a brackish water culture mediumlor larval rearing.

• Analysis 01culture water lromholding and rearlng systems Indicaled that thes« culture systems generall)' provided satisfactorycondttion: ofwater qualll)'.

• An Invesllgallon into the use 01solar energylor heall"g culture water was con­ ducted. Results Indicated Ihat th« apparatus used could easily be tncorporated into Ihe infrastructure 01 a hatchery. sttuated In aregion expertenctns dry sunny wlntm, 10 provide an additional source01 energy lor hcatlng culture water.

• Wild praw"s were collecteda"dmaintalnedIn thelaboratory 10 supply larvae lor arcarl"g programme. Thes« survived and reproduced In the laboratory, but with decreasing survivaland fecundity overa period01time lor certain 01the species. This appeared 10 be the result 01aggressive Interactions andpossibly Inadequale nuttitlon:

• Adult:01the Indigenous species were identifled IIslng available literalure bUI wert onlypositively identtfled 01alaler stage by Proieuor L.B. Holthuts, (Letden, Netherlands) 10 whom examples were sent.

• Jnlllal attempts01 rearing Ihe larvae 01lireIndigenous species were unsuccessful, with poor survival and development resultingfrom lowsalintties. However, during Ihts period M. rosenbcrgil andM. rude larvae were reared successfully 10 pOSI· larvallorm.

• Experiments were conducted 10eSlabliJh th« salinUI prelerences 01tht early larvallorms olll,e IndlgmouJ species. TI,e resulls ollheseexpertment« sl,owed Ihallhe early larvalforms require salinities ofmore Ihan 8SO100 lor survival anddevelopment. ill

-, • Followlllg the solitr/ty experiments, larvae wer« ,eD1ed In large numbers at Increased sal/nlty levels. with tilt StlueJJ/1I1 completion ollan'al developmenr In three o/theIn­ dlgenolls species. lIowtvtf,ln twoolt"tse speclts.percentage sun/val topost·

IOn'al/onn WQS low and devtlopment exttnded over 0 Ion,period oltlme.

•A ",ellmlnar)' study o/the mo,pllOloglcal development 01larvae o/tlre Indigenous

species WQJ conducted. ReJu11J Stlggesud t"at the morpllologlcal development 01 IQn'ae /0110 wedslmllar pomms as for other Macrobrllchlum species. '. SAM EVATflNG

• Labonuonum ondersoek Is ultgevotT na teelt!as/llleite vir v)'llnhumse Macrobrachium spestes, sowei as virdie reuse Maltlsleue gamaat, t-bcrobrachium rosenbergll. Die kultuursisteme waarop beslultIs. "el bestaan ult geslole waler­ hmlrkuleringslsleme. walUl)'det/s gebmlk gem4Qk Is van 'n bl%Ariese ,nils /llttr vir sulwtringsdoelelndes. SlnletltSe seesout ts gebrnlk am brakwater kultuurmedla ' vir die ullbroel van lanves tulll temaak.

• At/aUtst van die water waarln dlt volwasse garnalt aangtJrou Is, sowel as vir die ultbroelslsreme, toon aan dot dlt waterkwalltell van hltrdle betrokke sutem« oar die ulgemttn goed gebly her.

• 'n Ondmoek na die gebrulk van so"lIgencrgle In die mllltling ).'an die wattr waorlt/ c/lt gamale aangehouts, dill daarop dar Mtrdle betrokke stelsel nu/tlg tn­ gtslult kan word In die infrastruktuur as bykomsllge bron van en ergle, veral gt· durend« die wolketose wlntermaande In die blnntland

• Wildt gamale Is versamelen aa"gelrou onderlaboratctlum toestande virdi« voor­ slenlng van larwes lye/ens die teeltprogram. 'n Groor penentaste hlervan heldie lomande oorleef en oak voortgeplant In dte labomtotium, maar In di« gtval van 'sommlge spesles het gamalebegin afsterfoar 'n perlode van tyd, war oak gepaard. gegaan helmel 'n a/name II/ voortplantlng. Faktor« "'01 hler 'n rolgcspeeillelis gerdtllli/isur as aggresstew« tnterakste: mer 'n gebrtk aan die korrekte votdsel as 'nmoontlik« bykomstige !aklor.

• Volwasst eksemplare van die lnheemse gamaaaoorte wat gebmlk Is, Isaanvankllk gerdtllli/iseer mel behulp l'an beskikbare ltteratuur. Vir bevesllgende Idenllfisering Iseksemptar« van a/ die sptsltsaan Professor L.B. /lollhuls. (Letden, NedtrlandJ, gesluur, wat 'n outoritelt oplIlerdle gebled Is.

• Aunvankllke poglngs am larwts van die Inheemse spesles grool te ntQQk deur 01 hulle onlwlkkclingSladlums was onsuksesvol, groolllks as ge).'olg van It lae sallnl· ttlte l'Qn die water wat gcbrulk Is. DII was tgrtr moont/lk om Iydens hltTdle periodt die larwcs van M. rosenbcrgii en M. rode met subes It laat ontwlkktl 101 posl!4rwt1. y

• EkJper/menle Is ven'olgms ultgevoer om die JDllnltellsvoorkellTe van die vloel lorwalevonn« van die betrokke tnneems« speslts vas It SId. VolgellS remllale verkry, kon aangeloon word dalVtral die vroee ianvale vormesal/nlteltsl'lakke van meer as 8 S 0/00 bmodlg vir oorlewlng en ontwlkkeUng.

• Na aJhandellng van die JD/lnlteltsekJperlmmle Is grool gelalle larwes ullgebroel onder verhoogde sa/ln/te/tsvlakkt walgesklk bevlnd Is. Dithet daartoe gelel dal In drle van die tnheemse spesles die lanvQQlolllwlkkellng volledlg kan plQQJvlnd 101 en melpostlarwale vorm. In twe« vall die dn« spesles Is daarnog probleme ondervlnd sodaI die oorlewlng ttl dieonlWlkkcllng napostlarwes nag tetevntellend was, en plaasgevlnd het oar 'n besondere lang perlodt.

• 'n Voarloplge ondersoek IIa die mor!ologlese onlwlkkellng van larwes van die be­ trokke tnneems« spestes Is ultgevoer. Die resultat« verkrJ' dill daarop dol die mor/olollest onlwlkkelln, van larwes 'n soortzelyk« patroon volg as virander Macrobrachlum spesies, watreeds elders beskry!lsIn die literatuur. J

CIIAPTER ONE

INTRODUcnON

The genus Alacrobrac/rlum (Bate, 1868) occurs throughout the tropics and in several sub­ tropical areas (Holthuis, 1980). Almost 1I11 the species spend part of their life cycle in fresh­ water (Holthuls, 1980) and the term "freshwater prawn" has been applied to representatives of the genus (Goodwin and Hanson, 1975). or the one hundred and twenty five known species, most are ofgood size and it islikely that they would be used as food wherever they occur(lfolthuls, 1980).

Freshwater prawn fisheries, us well liSunsophisticated prawn culture techniques have existed for centuries incertaln Asian countries and among Indian tribes In the tropical Americas (Costello, 197 I; Ung and Costello, 1976). Fishery practices for freshwater prawns have also been recorded for the African continent, mainly from West and East African countries (llolthuls, 1980; Prah, 1980). In South Africa a commercial bait fishery, which includes Macrobrachillm species was developed bythe Natal Parks Board In 1952 at LakeStLucia on the Natal Coast (Bickerton, 1978). Local people in the llrea are known to trap freshwater prawns in the rivers for food (Doug Cooke, perscomm. ).

Culture practices for the rearing of freshwater prawnsunder controlled conditions developed after the discovery of the catadromousnature ofMacrobrac1lillm rosenbergit (De Man), by S.W. Ling in 1961 (Hanson & Goodwin, 1977). The years that followed S:lW the development of massculturing techniques for M. rosmbergll in Hawaii, by Fujimura and co-workers (Malecha, 1979) as well as investigations of the culture potential of other Macrobrachlum species (Lin'g & Costello, 1976). M. rosenbergll. the giant Malaysian prawn, has been con­ sidered the most suitable species forcultivation, and Is being introduced into countries interested inculturing freshwater prawns (Rabanal, 1980). Ofthe one hundred and twenty five knownMacrobraclrlum speciesonly ahandful have been investigated for theirculture potential (Miylijlma, 1977).

With the exception of the proposed culture of Alacrobraclllum volltnhov~nll (Herklots, 18S7) in Ghana, very little Information appears to be available onthe utlUsation of freshwater prawn resource. In Africa, for the purpose of aquaculture (RlIbanal, J980). Holthuls (1980) reported that the commercial cultureofM. ros~nbt,,11 has been invesligated In Malawi, Mauritius and the Seychelles. Production ofM. ros~nbt,,11 reached a commercial level by 1980 in Mauritius (Thompson, 1980) and by 1981 at an inland produe­ lion unit forthe same species in Zimbabwe (Kenmuir, 198 I). 2

A well established market (or prawnsexists InSouth Africa. which in the past,relied 1lIrse­ lyon the supply ofmarine prawns from Mozambican waten. for along period o( time. However. Moumblcan waters arc At present out ofbounds to South AfriQn trawlers. and although a limited number of prawnsare Imported from Mozambique, the market issupplied with prawns from as fn afield asAustralia and Taiwan. AJ I result of the increasingcost of marine prawns and the world-wide depletion of marineresources, the development ora freshwater prawn Industry has become adefinite economic proposition In South Afric•. Commercial production units are being developed toward this end at present, but these rely on the Importation ofM. rosenbtrgll post-larvae for the stocking of grow-out and produc­ tion ponds,at a high cost and with high mortality risks. AJ a result, the development of local hatchery facilities for the production of freshwater prawn post-larvae has become a priority. While M. rosenberzl; is the species of choice for commercial culture, Nature Con­ servation ornclsls are concerned about the possible introduction of these prawns Into South African waters where they might pose a threat to the Indigenous species. in certain areas of the country. In addition. climatic conditions over much of South Africaare not entirely satisfactory for culturing this tropical species (Schoonbee, pen. comm.) and the indigenous species of South Arrican waters m.lY possibly provide I substitute In the more temperate regions of thecountry. It was thereforedecided that together with the development of freshwater prawn hatchery facilities at the Rand Afrikaans Unlverslty, the culture potential of the indigenous species would alsobe Investigated.

The following Indigenous Macrobraclllum species were collected for the purpose of study:

MacrobTaclllum Ieptdactylus (Hilgcndorf, 1897) Macrobraclllum rude (Hellcr, 1862) MacTobTach/um peterstt (Hilgcndorf, 1879) Macrobraclllum scabriculum (Heller. 1862) Macrobrach/um australe (Gu~rin, 1838)

The five Indigenous species vary In their distribution. with M. rude, M. lep/dactylus and M. scabriculum being dlstributed alona the east coast of Arrica and Madagascar, extendln, as far as India. M. eustrat« is distributedover a similar area, but extending as far as Polynesia (ffollhuis, 1980). The record of lhls species from the African eontlnent Is apparently the first accordlns to Professor Holthuls, (Leiden, Netherlands) who identified the material. M. petenll Is apparentlyrestricted In distributicn tothesoutheast coast of Africa (ltollhuis. 1950; Read, 1982). Available Informallon Indicates that, with the exception ofM. pelenl/ for which no data is available, the indlscnous species are used for food In the areas where they 3

occur, being fished to varying degrees (IIolthuis, 1980). Fishin, rorM. rude is regular In certain parts of India and Bangladesh (IIollhuis, 1980), and thespeciesis listed asa eultl­ vated species by Panikkar (1968). Studies or the field biolo,y ofM. rude have been conduc­ ted (Ling lind Costello, 1976), but nofurther data is available.

The proposed aims or the present study were as follows:

• The development and preliminary testing0/suitable systems /0' the holdllll 0/ prawn brttdlngstock andthe rearlng o/Iarvae under laboratory conditions. 17ru Included the trial rearing olM.rosenbergillanoae In the culture systems developed, and the Itstlng 0/solar energy apparatus lor "tatlng culture water.

• The determination 0/optimum environmental conditions lor th« ~xperlmtntal rearing 01 larvae ofth« five Indigenous species cotlecttd. wit" emphasis onthe salinity preferences ofth«larvae ofthes«species.

• The rearlng oflarvae ofth« five Indigenous species through alllh~ developmental forms lorth« purpose o/Identlflcatlon.

• The utilisation oftnformauon obtainedduring ti,e course 0/the pro/eet/ora planned comparative mass rearlng trtaloflarva« 01the Indigenous species and M. rosenbergii, under experimental hatcheryconditions. 4

CHAPI'.ER2

UfERATURE REVIEW Page

CHOICE OF CULTURE SYSTEMS 6 Culture systems.suitable for temperate climates 6 Closed water recirculation systems and biofiltration 6 Temperature 8 Salinity 9 pH 9

Dissolved oxygen •••••••••u .. 9 Design ofbiofilters 9 Management ofbiofdtration systems 12 LARVAL REARING FACIUTIES 14 Culture vessels 14 Filtration systems ...... 16 .Motive force ...... 17 Heating ...... 18 HOLDING FACILITIES FOR BREEDING STOCK 19 HOLDING FACILITIES FOR POST·LARVAE 20 MANAGEMENT OF ADULTS FOR BREEDING PURPOSES 21 Salinity. temperature and photoperiod ...... 21 Nutrition ...... 22

Stocking density and sex ratios ... ~.~ ~ .. ~~ .. ~ .. ~ ~ .. ~ ~ ~.~ ~. ~ ~~. ~ ~ .. 22

Breeding ••••~.~ ~.~.~ ~~~•••• n •• •• ~ ~ ~ ~~ ~~ • ...... ~ ~ .. 23 LARVAL DEVELOPMENTAL FORMS 25

Definition of terms • ~ ~.~.~ ~ •• •• ~ n ~ ~ .. ~ ~ ••••• .. . . . ~ ~ ••~ .. ~~ ~ ~. ~ ~ ••""."" 25

Phase ...... ~ .. ~ ~ ~ ~ ~ ~ .. ~ ~ .. 26 Form ...... 26 Instar and stage ...... _ . 26 Larval development in the Palaemouidae 26 LARVAL REARING AND FACfORS AFFECflNG DEVELOPMENT .•••.•• 28 Background ...... 28 Nutrition and diet ...... 29

Nutrition ...... - ~~ - .. 29 Diet .- __ .. 29 S

Page Qualitative aspects offeeds 30 Quantity and concentration offood 30

Frequency offeeding ...... , . 31 Salinity and temperature 32 pH ...... 34 .Water hardness 3S Photoperiod and light intensity 3S

Stocking density ...... ,u . 35 POST LARVAL REARING 36 Background ...... 36 Temperature and salinity ...... 37 Nutrition ...... 37 Stocking density ...... 39 Juvenile age and breakpoint 40 DISEASES AND OTHER FACTORS AFFECTING PRAWN SURVIVAL IN CULTURE SYSTEMS 41 Diseases 41 Epibionts ...... 42 Shell disease ...... 42 Black nodule ...... 43 White syndrome ...... 43 Fungal infections ...... 43 Parasitic infections ...... 43 .Predation and cannibalism ...... 44 Tolerance to toxic substances ...... 44 Ammonia ...... _.. 44 Nitrite ...... 45 Nitrate ...... 46 Carbon-dioxide and precipitants 46 Chemical treatment ofdiseases in closed recireuIation systems 47 SUMMARY ...... _.. 48 6

CHAPTERTIVO

UTERATURE REVIEW

2.1 CHOICE OF CULTURE SYSTEMS 2.1.1 Culture systems suitable for temperate climates

Forster and Wickins (1972) listed three possible ways to culture prawns in the Uuited Kingdom: .

• Extensively - for cold water prawns which could be cultured in outdoorponds.

• Intensively - for controlled rearingofthese prawns in outdoorponds.

• Very intensively - high stocking densities indoors, in a completely controlled environment.

Forster and Wickins (1972) considered the latter approach the most suitable for culturing fast growing tropical prawns such as M. rosenbergii in the United Kingdom. Sandifer and Smith (1978) were of the opinion that a combination ofthe second and third alternatives listed by Forster and Wickins (1972) offered the greatest immediate potential for the success­ ful commercial fanning ofM. rosenbergii in South Carolina, and in other areas with similar temperate climates. This combination would involve the indoor rearing oflarvae andjuve­ niles during the cooler months, so that larger juveniles, rather than newly metamorphosed . post-larvae, could be stocked into ponds, making better use ofthe summer growing season (Sandifer and Smith, 1976, 1978). Although the best available areas for freshwater prawn production in South Africa are sub-tropical in nature, environmental conditions occurring there are not usually entirely adequate for freshwater prawn culture, and the full utilisa­ tion ofthe summer growing season would require grow-out of post-larvae to a larger sizes Indoors.before stocking into ponds (Schoonbee, personal communication). Thus the ap­ proach recommended by Sandiferand Smith above,for temperate regions, was considered suitable for application at the Rand Afrikaans University, Johannesburg.

2.1.2 Closed water recirculation systems and biofiltration

Recirculating water systems in which most ofthe culture water is treated and re-used (Sandifer and Smith, 1978) are, according to available information, the systems ofchoice 7

for the intensive indoor rearing of prawns (Forster and Wickins, 1972; Sandifer and Smith, 1976,1978;\\'ickinsand Beard, 1978;McSweeney, 1977.)TIlcchoiceofrecirculating systems is based on the following advantages considered relevant to the present study:

• Minimal water requirements, due to re-use0/ culture water, and thus reduced reliance on large external supplies ofwater.

• Efflcientuse and conservation 0/heat energy, part/clllarl}' Ifthe buildings, Irouslng tire system are insulated.

• Control can be exercised over tire Introductiona/predators. diseases andpollu­ tants due to reduced reliance onlarge external SIIpplies ofwater.

• Eosin observatlon, feeding and treatment 0/cultured organisms. (Forster and Wickins, 1972; Wick insand Deard,1978;McSweeney, 1977; Sandifer and Smith, 1976, 1978).

Green water" and clear water static culture systems, which involve periodic exchangeof water, arc widely used for the production ofjuveniles in Mactobrachlum hatcheries at salinities ranging from 8-17 SO/00 (Sandifer et al.• 1977.) Clearly, large volumes of sea water would be required forsuch operations. Sandifer and Smith (1976, 1978) havedemonstrated the feasibility of using synthetic sea salts in recirculating systems, making it possible to establish hatcheries inland, indcpcndantof coastal sites and adjacent to inland freshwater grow out areas.

Purification of water in recirculating culture systems is achieved by the use of biofilters (Forster and \Vickins, 1972; Wickins and Beard, 1978; Sandifer and Smith, J978), which are considered by Forster and Wickins (1972)and Sandifer and Smith (1978) to be the most economical means ofpurification. TIle successful operation of closed recirculation systems depends to a large extent on the detoxification of excretory products, mainly ammonia (Armstrong et al.• 1976), by micro-organisms which utilisecompounds such as ammonia, urea, proteins and carbohydrates for their own growth and metabolism (Forster and Wickln!, 1972; Wickins and Beard, 1978). TIle end products of this process include nitrate and carbon

• Culture medium in which algae have been cultured prior to use. 8

dioxide which are unlikely to be toxic until they reach levels many times higher than those occurring naturally in sea water (Wickins and Beard, 1978). According to Stickney (1979) the use of blofilters in warm water aquaculture is considered to be more of an art thana science, since development of these systems isstill in a research phase.

Spotte (1979) described biological filtration liS consisting of three processes, resulting from the activity of bacteria, namely:

• mineralisation • nltrtflcatton • dlssimtlation

TIle followingInformation on biofiltralion Is summarised from SPOIlC (1979):

During mtneraltsation, heterotrophtc bacteria arc responsible for th« breakdown ofnitro­ genous organic compounds, and thetr uttltsatton as cntrs)' sources, wit" ammonia asan end product. TIre ammonia produced Is then oxldlsed during nttriflcation by autotrophic bac­ teria, sue" as Nitrobacter to produce nimte wille" Is then utilised b)'other autotrophs such as Nitrosomonas to producenitrate. Dissimilation, t"t reverse ofnttrlflcatton, occurs when anaerobes reduce tlr~ end products ofnitriflcatlon to lower states 0/oxidation, allowing tire release 0/some nitrogen to tile atmosphere. Dissimilation sho/lld not occur in "calthy wellaerated fllten.

According to Spotte (1979) the nitrificationprocess is affected bythe following environ­ mental factors: • temperatun • salinity • I'll • dtssolved oxygen

Someof the possible effects of these fllctors lire briefly reviewed here.

Temperature, Ofthe factors listed, temperature exerts the mM sl~nlficllnt effect on nltrifi· cation,with abrupt decreases in temperature causing time bgs in theconversion ofnutrients, while increases In temperature result in increased biochemical activity (Spotte, 1979). 9

Salinity. Although biofilter bacteria lire fairly tolerant of Iluctuaticns in salinity, large and abrupt changes in salinity may result inrepression of bacterial activity (Sporte, 1979).

pll. Biofiltration processes may lead to II decline in the pll of theculture medium (Wickins and Beard, 1978; Spotte, 1979; Stickney, 1979), with the mineralisation of organic carbon compounds and nitrification accounting for the bulk of the acid forming processes of bacteriological origin (Spotte, 1979). Decline in pll affects nitrification, which ceases in marine systems at pH 5 (Wickins and neared, 1978), According to Spotte (1979), Saek! (1958) found that low pll in freshwater aquaria inhibited ammonia oxidation, and thata

pH of between 7.l and 7,8 WIlS ideal for theactivity of nitrifying bacteria. Boyd (1979) re­ ported that nitrification in fish ponds was most rapid lit pll values between 7 and 8, at temperatures between 25 and 300C. According to Spotte (1979) Srna and Baggeley (1975) found that marine nitrificrs functioned best lit pll 7,45 wilhan effective range of 7 to 8,2.

Dissolved Oxygen. When functioning properly, a filter bedconsumes a considerable amount of oxygen, the consumption being mainly due to the activities of heterotrophic bacteria (Spottc, 1979). According to Spottc (1979) well aerated aquaria have predominantly aerobic bacteria while anaerobes proliferate under low oxygen concentrations, with therisk of toxic metabolites such as hydrogen sulphide, methane and ammonia being produced. Provision of an abundant supply ofoxygen to the filter bedisconsidered a key requirement for the filter system by McSweeney (1977).

Another key requirement of the biofiltcr isa sufficient area of bacterial substrate for the oxidation of nitrogenous wastes (McSweeney, 1977). This aspect is dealt with in the follow­ ingsection.

2.1.3 Design of Blofilten

Of the various biofiltration systems In use in aquaculture, Wickins and Beard (1978) re­ commend the use of percolating biofilters, as these are considered to be simpler and safer than, for example.activated sludge or sedimentation systems, Typically, the filter consists of a bed of gravel which provides an enormous surface area for theattachment of micro­ organisms (Wick ins lind Beared, 1978). However, the design or the filter system presents certain problems, of which Forster and Wickins (1972) nnd Wickins and Beard (1978) list three, namely: 10

• The prediction ofbiological loadings, In terms of excretory products and mole­ rials leached from the food, wit/cit can be placed on Illr system.

• The estlmatlon oflite tolerance oflite selectedspcclts 10 accumulations oflis ownandother waste products,

• nit determinatton 0/filter capacity In terms a/waste removalat known hy­ draullc" a"dbiological loadings.

The determination of the size of the filter is not entirely amenable to scientific methods (McSweeney, 1977) and reliable formulae for calculating size have still to be adequately developed (Stickney, 1979). Spotte (1979)presented a formula developed by Hirayama (l966n) for detcnnlning the carrying capacity of a biofilter, but noted that this shouldonly be used as a guide. The carrying capacity of the filter has been defined as the animal load that can be supported, and this is conceived of as the oxidising capacity of the filter (Spottc, 1979;\ViekirlS and Beard, 1978). This oxidising capacity should be equal to or greater than the pollution load of the anlmals in the system(Spotte, 1979). The sizeof the biofiltcr depends on many factors (Stickney, 1979) among which the following appear to be relevant:

• Sur/actarea (Spatie, 1979) and specifk: surface area (wtcktns and Beard, 1978) 0/ thelilltTmedium.

• Number and mass oforganisms (Spotte, 1979: Stickney, 1979).

• Flowrate through the fllter (Spotte, 1979;Stickney. 1979)or hydraulic load (Wlckinsand Beard, 1978).

• Filler volume (wtckins and Beard, 1978) or total volume ofwater In the system (Stickney, 1979).

• Pollution load (SpOilt, J979) or biologicalload{Fonter and Wick Ins, 1972).

• Hydraulic load. rate of transport of nutrients to filter bacteria. 11

McSweeney (1977) suggested that the carrying capacity of a given medium W:lS best deter­ mined experimentally, and that excessively large filters should beavoided. However, McSweeney (1977) also noted that as theproduction of waste varies with the age and size of prawns in intensive systems, the filterbed should be sized for maximum expected load­ ing. Stickney (1979)suggested that as a general rule it isbetterto have too much filter capacity than toolillie.

Spotte (1979) recommended that for aquarium filters, the surface area of the filter bed should equal thesurface area of the aquarium tank. In addition it should be a unifonn minimum depth of 7,6 cm of gravel, as the distribution of bacteria in the filter bed was a function of it's depth. with nitrification declining rapidly below a depth of 5 em. However, Spotte (1979) also reported that work done by Hirayama (1965)demonstrated that the apparent effects ofdepth arc misleading because the time required for water to pass through n filter bed is proportional to depth. TIlUs deeper filter beds require faster flow rates of water than shallow ones.

The size and shape ofgravel used is important, as smaller grains have a greater surface for bacterial attachment than the same mass of larger grains, and angular gravel has more sur­ faces than rounded gravel (Spotte, 1979). Inaddition, Spotte(1979) noted that the accumu­ lation of detritusina filtcr bed enhances itsmechanical filtering ability, as well as its nitrifl­ cation potential. Stickney 0979) however, considersgravel unsuitable for high density closed system aquaculture, due to clogging of the filter with detritus, and suggests that a medium with less surface area and more open space between particles ismore efficient than sand or gravel, occupying thesame amount of biofilter volume.

It was mentioned earlier (section 2.1.2) that biofiltrntion processes result in a decline inpH, and it is thus necessary to buffer the system against changes in pH by incorporating calcare­ ous material, such asoyster shells, in the filter medium (Wickins and Beard, 1978; Stickney 1979; Spotte, 1979). Although this procedure is satisfactory for freshwater aquaria, the buffering of marine systems is more complex, and the use of sodium carbonate or bicarbo­ nate is recommended by Spotte (1979).

TIle biological load isseen by Wickins and Beard (1978) as the quantity of soluble wastes to be removed at each cycle of a given volume of water through the filter. These authors noted that little was known about the nitrogen excretion of tropical prawns, but rates basedon approximate measurements ranged from I mg total ammonia nitrogen (NIf4-N) per gram of 12

prawn per day, (or 5111311 prawns of up to Sg live mass, to O,S mg total NII4 -N per gnm, per day, (or prawns of 10-20 g. TIle type :U1d quantity of solids and solubles leached(rom food lind converted 10 ammonia by micro-organisms depends on the composition of the diet, the efficiency of the binding agent.and the retention lime of Iood in the tank (Wick Ins and Beard, 1978). In addition to the amount of food entering thesystem, Spotte (1979) stressed the importance of lndividual body masses in considering carrying capacity of a filter system, :IS Ihis is not simply a functlon of total animal mass. Pollution load changes with growth of :mllllais so that an aquarium which can support a single 100 g fish may not necessarily support 10 fish of 109 mass each.

According to McSweeney (1977) the volume of culture tanks lind the rate of accumulation of nitrogenous wastes determine the optimal now rates through a /ilter bed, and the rille of removal of wastes should lit least be equal to Ihe accumulation rate of wastes. McSweeney (1977) suggested Ihal now rates should be specified as exchanges of tank volume per unit lime, und quotedresults of a questionnaire distributed by him, which showed that the water exchange rates In use in aquaculture systems ranged from four tank turnovers per day (used by the author and his co-workers) up to nine lind ten tank turnovers per d3Y.

Sandifer and Smith (1976) employed lin increasing rate of turnover in their recirculating systems for larval rearing. These authors began with a very low turnover in the first day,in­ creasing this to about two tank volumes per day from day 2 - 2S,and thereafter up to twenty-five tankvolumes per tank per day. Continuous rapid recirculation of water through larval rearing systems maintains excellent water quality, but rapidly flushes ATremia nauplii inlo the filter (Sandifer and Smith, 1978). These authors found that 3 slow continuous or periodic now, equivalent to two to five tank volumes per day was sufficient to maintain adequate water quality, while minimising theloss of larv;e! feeds such as Artemta: Spotte (1979) recommended a now rate of 0,7 x 10-3 ms-I throughanaquarium filt cr bed consist­ ing of uniformly sized, rough, angular gravel of 2-5 mm in diameter. A higher flow rateof water through thefilter bed than is required forcomplete nitrification, may result in exces­ sive levels of nitrite incultures (Armstrong et al., 1976).

2.1.4 Man3gemcnl of blofllrratlon systems

Management of new blofllters Involves theconditioning of the filters quickly and effectively (Spotle, 1979). Spoue (1979) describes a conditioned aquarium asone where t he filter bed bacteria lire In equilibrium wilh the routineinputof energy sources. 13 .

Proper colonisatlonof II biofilter depends partlyon the presence of A nutrient 103d, so Hut an unstocked or Iighlly stocked water system will develop theproper bacterial flora more slowly than a system Ih:S! is more hellvily stocked (Stickney, 1979), Suckney (1979) (cds that heavy slocking of the filter system inili:llly to bring about rapld colonlsation o( theblo­

tiller, is unwise liS thisplaces stress on theanirn;als (rom metabolite accumulation, before the tiller begins 10 operate III peak efficiency. Spotte (1979) on theother hand, feels that Ills

quicker and more practlca! to overcompensate I new filler bed in lin aquarium when con­ ditloning n. Tlut ls, 10 condltion it with II slighlly greater animal load than ir would ulti­ mately support, thus eliminating later increases in ammonia land nllrile when more animals lire lidded. Anlnuls wlth tolerance of high ammonia levels«or eXlIlllple carp or catflsh) should Ideally be used (or this purpose (Spotte, 1979).

The alternative is10 IIdd the species 10 bestocked In a itf3t1Ulll manner, concinu:,Uy monltor­ Inlt ammonla levels 10ensure Ihat rhe species tolerance Isnot exceeded during the condltlon­ ing process (Spotte, 1979). According to Spoue (1979) mlncralisatlon, nitrificatton and t1lsslmil:llion arc processes following one another, more or less sequenliallY,ln :a new aquarium, and the process of nitri~catlonC3n be used to determine when :a new aquarium becomes conditioned. Apparently the :a111mon13 level usuallysubsides within two weeks. at temperatures above ISoC, when ir should be less than 0,1 myJ2 NII4-N in a conditioned system (Spotte, 1979). The conditioning process can be accelerated by innoculating the new aquarium with an establlshcd population of filler bacteria, scraped from the surface of lin already cond ltlcncd filler (Stickney, 1979;Spottc, 1979), which has been maintained at the same temperature asthe new one (Spotte, 1979).

Imbalances, resulling from sudden increases Inanimal numbers, theirmasses and/or the quantity of food fed, often produce measurable increases in levels of ammonia and nitrite (Spotte, 1979), According to Spotte, theseincreased levelsmay be permanent if a con­ ditioned aquarium isoverloaded beyond lismaximum carrying capacity. Besides replaclna waterlost through evaporation, it is necessary to conduct partial water changes biweekly for the following reasons In aquarium bioflltratlon systems:

• Mallagmlrlll 0/ II/1raU I~vtll • Removal 0/ excess detritus (Spotte, 1979)

In addition 10 1'3rt131 water chanscf., m3nagcmenl of nilnle Icvcb may be accomplished by the culturing of pl.lnllin the w.Jrer system and huvcsring some of them pcriodic.1Uy (Spouc, 1979). 14

Spotte (1979)recommends a 10% partial change ofwaterata time, during which the sur­ face of the nllerbed isstirred gently to suspend the detritus in the water, enabling removal together with the 10% water to be replaced. It is important that the replacement water is at the same temperature, salinity and pllasthe aquarium water, sothat nitrification is not affected (Spotte, 1979). There should not bemore than a 10C difference in temperature, overa 24 hr period (Spotte, 1979). Replacement of water lost through evaporation in brackishor sea water aquariums should be conducted before thesalinity increases bymore than 0,2 S 0100 (Spotte, 1979). The replacement water should be tap water aged forat least three days under aeration, to remove any traces of chlorine (Spotte, 1979).

2.2 LARVAL REARING FACIUTIES

As previously mentioned (section 2.1.1) Intensive closed water recirculating systems, using artificial sea salts, have been recommended for hatchery units situated inland in temperate climaticzones, This review of larval rearing facilities hastherefore been approached with this consideration as a guide.

According to McSweeney (1977) a generalised intensive culture system comprises three m:uor components:

• Culture vessel • ~/ltratlon • Motive force lor circulating water

Into this culture system, factors suchasfood, heat and oxygen are introduced (McSweeney, 1977).

2.2.1 Culture vessels

Asurvey conducted amongst Macrobrachlum hatchery operators bySandifer et aL, (1977), revealed that tanks employed for rearing Macrobraclrlum larvae varied markedly from one hatchery to another. Containers ofvarious shapes and sizes, from earthenware jan to oquarium tanks, have been used by different laboratories for experimental purposes, while forlarge scale operations. larae rectangular concrete tanks arc nomlally used (Una and CosteUo, 1976). Asimilar situation apparently also exists incountries like Taiwan (Schoonbec, pm. comm.). 15

While McSweeney (977) feels that economics is the strongest factor affecting both material and design,Zielinski, et al., (1974) have shown that the actual shape of the contalner is im­ portant, and they presented criteria forthe design of 10lrval rearins tanks. Probably the most important consideration in the designoflarv:ll rearing tanks is the need for a circulation pattern, which will circulate food particles unlfonnly to allanimals IlS well as eliminate dead spaces inthe tank (Zielinski et al; (974). A circulation pattern is also important for maintaining uniform water quality conditions. such IlS temperature, oxygen and dissolved wastes throughout the tank (Zielinski tl al., 1974). These authors stated that the m3,n1tude of the flow regime should not be so strong liS to remove food particles from the &rasp ofthe larvae.

S3ndifer and Smith (1974) found that aeration alone was not sufficient to maintain suspen­ sion of food particles, and that by establishing clrculatlon patterns capable of suspending food particles, growth of larvae in closed reclrculatlnz systems was as rapid as in "green water" culture systems.' .

Settlingof food particles was found to be a major causeof mortalities in a comparative study ofdifferent larval rearing systems by Dugan et al., (1975). These authors found that larvae became trapped in the sedimenteddebris when moulting, with resultant mortalities. Although Dugan tl at, (1975) made attempts to remove bottom debris by siphoning, they experienced difficulties in the removalofdebris without removing larvae as well. In addition, the processwas very time consuming, and was considered inefficient. By sloping the floor of the tank downward.In the direction of fluid motion, settled food particlesand otherdetritus can be moved toa common collection point, where they can beremoved by siphoning or airlift (Zielinski et al, 1974). This approach was suggested by Dugan et al., (1975) asa possible solution to their problems experienced with flat-bottomed tanks, making siphoning of settled particles more effective and a less tlme-consumlng process. Zielinski et al., (1974) have found that there isan optimum combined condition offloor slope and flow current to produce particle motion in tanks, and have used jets of fluid to induce a flow current in tanks. In addition, these authon suggest that sinceaeration is required to maintain an ade­ quate oxygen level inthe tank, it can becombined withjet action or flow channeUsation to achieve optimum operating conditions.

Ina study of culture techniques (or M. rostnbtrrillarvae, Sick and Beaty (1974) found that theusc of conical tanks, designed with. niter and airlift, resulted inuvinp in labour, morc efficient utilisation offood and better survival of larvae. These authors listed the advantqes ofsuch containen u: 16'

• EQSe 01 tank maintenanceand prottctlonagainst mass loul/ngofthe tank,by keepln,the bottom area /0 aminimum. • No agitation oftire watercolumn by aln/ontl. • Btlltrsuspension offormula looddue /0 constam tumover ofthe water column. • No posslblt/I)' ofdead areaJ (artaJ ofno lood oroxygtnJ.

In their two-phase rearing technique, Dupn et al., (1975)obtained improved survival of larvae in the first phase, when using conical rearing tanks.

Results of a survey conducted amongMacrobrochlum hatchery operations by Sandifer et al, (1977) revealed that although tanks employed for.rearing },Iacrobraclrlum larvae varied markedly from hetchery to hatchery, cylindrical tanks with conical bottoms appeared to be the most efficient lind preferred design. One of the highest production levels reported in the survey was obtained using tanks of this design. According to Zielinski (1977) flow pat­ terns in cylindrical tanks appear to be more sensitive to thetype of Inflow and outfiow con­ ditions employed, than rectangular tllnks. Zielinski (1977)recommended the withdrawal of water through the top centre of the tank and its reintroduction tangentially. thus permit­ ting settlementof food particles in a smali area for easy removal.

The Interior of therellring tank appeanto be significant, with Sandifer et al., (1977) re­ porting that, although inconclusive, results obtained by them suggested dark interiors pro­ duce better results than light interiors. The conical tanks used byAQUACOP, in which very high production levels were obtained, as reported by the above authors, had the interior painted green. Read (1982) used beakers painted dark green In experimental studies,with the purpose ofaiding the orientation oflarvae towards their food. Chao and Liao (1977) reported the use of fibreglass tanks inTaiwan, which are painted black au tside and light grey inside. These authors recommend covering the sideand part of the top ofsemi­ transparent, white, plastic tanks with black plastic sheeting.

2.2.2 FUtl1tlon Iystems

Dialogical filters used with closed reclrc:ulatlna systems instudies reviewed, are usually com­ posed ofgravel or sand substrates(DupnelaL, 1975;S:mdlrerand Smith, 1976,1978; Menuveta and Plyatlratilivokul, 1980). The biological filter Is usually a separate unit al­ thought Dupn etaL, (1975) and Menasveta and Piyatiratltivokul (1980) Investigated system. with bullt-ln filten, in comparative studies of larval rearing systems. Dugan etaL, 17

(197S), in an IIttempt to reduce the accumubtion of bottom debris, incorporated IfIvel filters into rening tanks, but found tlut larvae were drawn ontothe filter bed when moult­ ing, with mortalitieus a result. Menasvetllllnd PiYlltirlllilivokul (1980) were satisfied with ", results obtained with their built-in biological filter system. nle difference in resultscould probably be ascribed to the fact that, allhough neither group of Investigators maintained water circuilitory patterns, MenllSVetallnd Piyatiratitivokul (1980) had airstones pl3ced atthe bottom of thetanks, thus agitating the water column and reducing the suction effectof the Oltcr.

Morc commonly, the fllter is a sepllrate unit which maybeconnected to one or marcrearin. tanks. The separate undergravel filter used by Dugan et al., (1975) consisted of two rectangu­ lar I 000 2 cement tanks, one a rearina tank and the othera OItration tank, connected by airlifts for water circulation. Menasveta (1980) used a rectangular tank which wasdivided into two compartments, one for filtration lind one for rearing, connected by airlifts for water cireulauon. Sandifer and Smith (1978) employed a single bioOltraUon unit, to whJch a battery of conical rearing tank. were connected by a pump for water circulation. The filler tank acts asI reservoir, and contains a heat exchanger. Water entering the filter unit passes through Illter bags (or removal ofparticulate matter, so thatwater passing through the biologicalfilter isfree ofsuspended material. Water is thenpumped back to the conical rearing tanka.

2.2.3 Motive force

According to McSweeney (1977) the energy source for thepump circulating water islm­ material aslong asasufficient now ismaintained. Results ofa survey conducted by McSweeney (1977), indicated that electrical pumps were chosen above airlift pumps. While they are somewhat less efficient than mechanical pumps,airlift pumps can be effectively used in culture systems for ciraalalion, aeration and waterexchange, and have certain ad­ vantagesover mechanical pumps (Sandifer and Smith, 1974). Advantages of airlin pumps arc:

• Lowtf COSIS Initially. Qlfd lowtrmaintenanc« CoslS lIS Iht~ an no mOlll1llpattI (Stmdl/trand Smith. 1974:Spollt, 1979J.

• Grtaltr tl/lclency DI low htad0/wattr and hilh submtrrtnce (SDndl/erGIld Smith, /974: Spottlt. J979J. 18

• Ease olflow ,ttguIDllon (Sandllerand Smith, 1974; Spou«, 1979).

• Easeollnstal/atlon (Spolle, 1979).

• The provision 01aeration while pumping(Spalie, 1979).

• Relative hannlttssness 10 animals accidentally entering thepump (SpOilt. 1979).

The airlift pump. (Fig. 48) which comprises a foot piece. eduction pipe and head piece, II operated by a supply of compressed airintroduced at the foot piece (Sandifer and Smith, 1974). Principles oroperation ofthe airlift pump are as follows (from Spotte, 1979):

When a pipeIs submerged In water In avertical position, water levels equtltbrate Inside and outstd« th« pipe. "'hen atr bubbles are Injected tnto II,e base 01 the pipe II,ey producta mixlure 01air and wate,that Is l/giller than wateralone. thus upselllng II,e equilibrium. When this happens, heavier water from outside moves Into th« pipe. and wl// spill out01 the headpiece as long as air Is Introduced Into th« pipe.

The main factor affecting the efficiency of airlift pumps isthe percentage submergence of the eduction pipe (Spone, 1979). In addition, Spotte (1979) states that efficiency decreases if the volume orairinjected exceeds the capacity of the eduction pipe. This Is detectable by gurgling sounds. As a general rule. Spotte (1979) mentions that doubling the diameter of the pipe increases its capacity 5-6 times. However, large diameter pipes (specifications not given)are more efficient at highdepths of submergence (70%). but smaller diameter pipes (no specifications given) are more efficient at lowsubmcrgences (50%) (Sandifer and Smith, 1974). Inaddition, the useful pumping range, within 10% of the maximum efficiency, is considerably greater for larger diameter airlifts(Sandifer and Smith, 1974).

The type of foot piece employed is apparently not important (Sandifer and Smith, 1974) but aeration by theairlift pump is enhanced by the cre'aUon of finer bubbles (Sandifer and Smith, 1974;Spotte, 1979).

2.2.4 Ileatina

Prnwn cullurists may depend on natural ambient tcmperatures or may increase temperatures by direct application of cncrgy. Authonsuch u Goodwin and Hanson (1975) notc that artificial heatinlisused in Intcnsivc systems. The necessity for supplemental heaUnl isI 19

factor limiUns thcdcvelopment ofintensive prawn culture systems in temperate climates (Sandifer and Smith, 1978).

Although many of the experimental studies referred to involved temperature control, details of the systemsused to heat water were not given. In laboratory studies, temperature control may be accomplished by the usc ofconstant temperature chambers (Oupn ~I al., 1975; Moreira, et al., 1982) incubators (Knowlton, 1974; lIubschman, 1975) temperature con­ trolled water baths (Sandifer, 1973; Wang and Williamson, 1977) orby the use ofimmcrsion heaten in the rearing containers (Uno and Kwon, 1969; Wang and Kuwabara, 1976). How­ ever, the lattcr authors reported high mortaUties as a result of theInclusion C?f immenion heaters in the rearins containen due to lethal temperatures occumna in the vicinity of the heater, Large scale rearing in intensive systems, where supplemental heaUns is required,could involve considerable heating costs (McSweeney, 1977; Sandifer and Smith, 1978; Wick Ins and Beard, 1978) and three maior alternative energy sources are being investigated, namely:

• WQSte htat • so!arhtat • gtothtnnalhtal

(Goodwin and Hanson, 1975; Wickins and Beard, 1978; Zielinski, 1977; McSweeney, 1977; Sandifer and Smith, 1978).

Any form of energy source is acceptable as long as it maintains therequired temperature without affecting water quality (McSweeney, 1977). McSweeney (1977) is of the opinion that the technology and economics exist to make solar heating practical in a commercial venture. Heatin closed recirculating systems isrequired onlytomaintain water temperature (Forster and Wicklns, 1972)and, if the whole system is housed in an insulated building. thisfurther reduces heat loss (Forster andWickins, 1972; Wickins and Beard, 1978).

In practice, the heating of replacement water must be taken into account as wen, as this determines the major part of heating costs, and depends upon theeffectivenessofmethod. used to remove uneaten food, faeces, suspended matter and soluble substances (Wickins and Beard, 1978).

2.3 HOLDING FACILmES fOR BREEDING STOCK

Adult Macrobrachfum prowns are easy to maintain in captivity, under laboratory conditions, and for prolonged periods of time (Una and Merican, 1961; Coslello, 1971; Dugan and 20

Frakes. 1972). By maintaining breeding stock in controlledenvironmental systems, I con­

Unuous supply of larvlle can be ensured for year-round hatchery production. even in tempe­ ..; rate climates(Sandifer and Smith. 1974).

Closed tank system. appear utisfactory for holding broodstoek indoors during winter, ACCOrdins to Sandifer and Smith (1976).who maintained broodstock indoon in these systems for approximately seven months. Diofilten maybescpante (Wicldns and Beard, 1978; Sandifer and Smith. 1978) or built intotanks (Dugan etal., 1975). Although tank conflguratlon does not seem to be criUcal. shelteror habitats arc Included to reduce losses resulting from aggressive behaviour andcannibalism (Dugan tt al., 1975; Sandifer and Smith, 1978).lIabitats arc discussed in more detail under post·larval facilities (Chapter 2, section 2.4).

TIle simplest fonn ofclosed system used forholding of broodstock appears to be that used by Dugantl al.•(1975) who used concrete burial vaults.These vaults were equipped with undergravel blologi~1 filters made from 20mm PVC piping beneath 5 ern ofdolomitic gravel. Water drculatlon through these filters was by means ofairUft pumps.

2.4 JlOLDING FACILITIES FOR POST-LARVAE

TIle scope of this project does not encompass the study of post·larval rearing or grow-out, Nevertheless, post-larvae produced during thecourse of this project were not discarded and facilities were provided to accommodate them. Only intensive rearing facilities are reviewed here, as the facilities used in this project fall under that category.

Avariety of containers has been used by workers involved inthe experimental rearing of post-larvae in closed recirculation systems. Kneale and Wang (1979) employed a system which consisted ofa tank divided into two compartments.one for juvenile renrins and one for filtration and healing. TIle circulationof water between these compartments was accomplished by airUft pumps. This typeof system wasused to evaluate the possibleeffects of temperature,artificial habitats and stocking density on the survival and growth of M. rostnbtlJiljuveniles. Dupn et aL, (1975) reared MacfObrtlchlum acanthurus (Wiegmann) and M. rosenbt,,11 post·lnrvlle in I 000 2rectansular tanks with undergravel biofi]ters.

An important consideration In the design of post·larval rearing facilities. appears to be the provision of adequate lU'tificial habitats orsubstrates. These are intended to reduce the 21

effective prawn density, and thus aggressive interactions, allowing for the full utilisation of a given watercolumn (Sandifer and Smllh, 1976, J978; Willis ~I 01., J976; M:mcebo, 1978; McSweeney, 1977; Wickins and Beard, 1978; Kneale and Wang, 1979). Willis eral., (1976)

I found that habitat complexity was required for the post-metamorphosis high density grow­ out ofMacrobraclJlum species, in intensive culture systems. Kneale and Wang (J 979) found that the inclusion ofhabitats in rearing tanks, at levels between one and three per tank did not appear to influence the survival ofJuvenlle M. rosenbtrgll, butconceded that habllat may influence growth rate. According toMcSweeney, (1977), most cuJturists rearing prawns intensively,make usc of some form of artincild substrate to increase surface area available to prawns. The simplest fonn ofsubstrate consists of vertical panels in the tank, while the most commonly used form consists of stacked horizontaJ I:lyers, and both arc usually made

of netting material soas not to impedewater flow (McSweeney I 1977). McSweeney considered vertical panels at least equally aseffective asthehorizontal type in the early stagel of growth, but possibly not as effectiveIn the later stages. Wicklns and Beard (1978) pre­ sented a hypothetical shelving concept, foruse in self-cleaning tanks, which consisted of a spirallingsubstrate surface. Sandifer and Smith (J 978) included noating plants, su~h as Elodla or E/c/rorn/a, in nursery t~nks equipped with artificial habitats, thus providing shelter, food and anadditional waste removal mechanism.

2.5 MANAGEMENT OF ADULTS FOR BREEDING PURPOSES

Several J,facrobrac1llum species are known to mate and spawn readily in captivity (Costello, 197J; Dugan and Frakes, J972; Goodwin and Hanson, 1975; Sandifer and Smith, 1978) and in a suitably controlled environment they will breed all year round (Goodwin and Hanson, J97S; Sandifer and Smith, 1978). A review ofcertain relevant considerations is presented here.

2.5.t Salinity, temperature ond photoperiod

Dugan et al., (1975) achieved year round production of prawn larvae, by maintaining the adults of four indigenous North American ItIacrobracIJlum species and M. ro$~nbtrgll in freshwater recirculation systems at 27,SoC under a 14 hr photoperiod. These authors found that continuous lighting had a detrimental effect on breeding stock and, although a 12hr photoperiod was found to be satisfactory, a 14 hr photoperiod was chosen, as this simula­ ted natural summer daylenlth conditions forthe prawns investigated. Wickins and Beard 22

(1974reportedimproved growth and reproductive potential ofM. rosenbergll after control" of pH and photoperiod was introduced, with pH between 6.4 and 7.4and a photoperiod of 8 Ius. Macrobrachillm rosenberztt breeding stock may be maintained in either fresh· water or slightly brackish water (2-8 S0/00) (Ung and Costello, 1976). Sandifer and Smith(1976, 1978) maintained M. rosenberrll broodstock at temperatures between 2SoC and 300 C and salinity from 0 to 4 S 0/00 inclosed recirculation systems.

Wang and Williamson (1977) maintainedM. rosenbergi! adults at 260C in a system de­ veloped for continuous production ofjuveniles. Read (1982) maintained. /It. peterst; fern Illes in a closed freshwater recirculation system at 240C and 16 hr photoperiod. Read (1982) also found that theonset of the reproductive cycle in M. petersll coincided with a rise In water temperature and increasein daylensth.

2.5.2 Nutrition

Mocrobrachtum adults are omnivorous (Du~n et al., 1975). Ung and Merican (1961) and Ling (1969b) listed a wide variety of fooos, both of animal and plant origin, on which adultsofM. rosenbtrgl/ could be fed. According to Ling and Merican (1961), M. rosenbergll will eat plant material if no better food is available. Ung and Costello (1976) recommen­ ded feedingadult /It. rosenberzti on fresh foods such as mussels, cockles, trash fish or com­ pounded fish foods, depending on which was cheaply available.

.. . Sandifer and Smith (1978) fed adult M. rosellbergl/ in closed recirculation systems once or twice per day ona commercial prawn feed at I- 2% of prawn biomass, with ad libitum supplemental feeds several times per week. Read (1982) fed /It. petmt! females on a fish· meal-maizemeal mix bound with gelatin, on alternate days. Dugan etat. (1975) fed adults of five Macrobracir/um speciesheld in closed recirculation systems ona variety foods, finally settling for trout chow fed on alternate days. Macrobraclrlum adults are nocturnal (Dugan et al., 1975) and M. rosenbergl/ adults tend to move into shallow water at night for feeding (Ling, 1962).

2.5.3 Stockingdensity and sex fIItlos

Direct references to stocking density for adults. held in communllitanks in laboratories, appearto be scarce. Una (1962, 1969b), recommended stocklna 5-10 adult M. fOStnbtrrtl 23 Ij

persquare metre of tank floor in tanks of40cm depth. S3ndlfer and Smith (1978) stocked 30 to 90 prawns, of30g or more in m3SS, per square metre of tank bottom. in tanks equipped wilh arlindal habitats. and connected to closed recirculation systems. The usc of artificial h:tbitats in the form ofPVC pipes and Hydnlla sp. W3S reported by Dugan It al, (1975), whlle Wang and Williamson(1977) provided sloping plastic shelters in tanks houslnS breeding stock, to provide cover for moultins females.

Fewermales are stocked than females. ata ratio of 1 male toapproximately five females (Ung. 1969b;S3ndifer and Smith, 1978; Chao and Uao, 1977), allhough Dugan et al, (1975) reported stocking one male to two females, as recommended by Ung (962).

2.5.4 Breeding

Mating in M. rosmbtrgfland M. acanthurus is preceded by a pre-mating moult by the female, following which the male will protect the female from aggressive attacks by other prawns (Lingand Merlcan, 1961 ; Ling, 1962, 1969b; Choudhury, 1971 b.) Newly moulted M. rosmbergfl females Invariably lay eggs 24hrs after the pre·mating moult. and where females arc kept separately from males, the sexes should be Introduced to each other for mating 8-12 hrs after pre-rna ting moult(Ling and Merican, 1961). Egg laying orspawnlns takes place approximately six hours aftermating in M. rosenbugil (Ling and Merican, 1961 ).

Sexually ripe female M. rosenbergti arc characterised by orange-coloured ovaries visible through the carapace, as well as the abdominal pleura which distend outwards to fonn a brood chamber (Ling and Merican, 1961). Ripe M. peterstt females show a dark green­ coloured ovary (Read, 1982). Egg colour In M. rosenbergit is orange Initially. ligbtenlng from about the 12th day ofincubation tolight-grey (Ling, 1962). ltIacrobraclrlllm carcinus (Linnaeus) eggs are the same colour as those ofM. rosenbergll while M. acanthurus eggs arc olive initially, changing to grey before hOltching (Dugan et al., 1975). MacrobraclJlum pelersll eggs are brown-green initially, changing to yellow and then grey in the later stages of incubation when the eye pigment of the larva is visible (Read, 1982).

Although berried M. rormbergll females m3Y be held in fresh or braclcish watcr (l.S - 12 SO/00) during incubation of the eggs (Ling and Merican, 1961), it is common practice according to Linland Costello(1976), tomaintain berried females inbrackish water of6 ­ 12SO/00. Sandifer and Smith (1978) m31ntained females at 12 S0/00 in hatching tanks 24

for the Incubatlcn period, thus the larvae are subjected to little or no temperature or salinity stressduring transfer from hatching to rearing t'lnks. Dugan et al..• (1975) hatched larvae in fresh and brackish water, and found that there were noadverse effects if larvae were hatched in freshwater and gradunlly acclimlttsed 0,' transferred directly to brackish water.

Development of fertilised eggs takes 16-20 days at 2SoC (Dugan etaL. 1975). The incu­ bation period forM. rosenbergll is 19 days at temperatures between 25 and 2SoC (Ling and Merlcan, 1961; Ling 1962, 1969b). Read (1982) found that the embryonic development of M. peterst! was mark~dly retarded at ISoC, and that at 300C theIncubation period was 12dnys. Read (1982) also found that ovary and egg maturation took place simultaneously InM. peterstt so that bythe time the eggs hatch, the ovary Is fully ripe again.

Various types of containers are used for hatching larvae in, varying from aquarium tanks (Ling and Costello, 1976), to specially designed hatching and collection tanks (Sandifer and. Smith, 1976, 1978; Dugan et al., 1975; Smith and Hopkins, 1977). Incubation and hatch­ ing may also be conducted directly In rearing tanks (Ling andCostello, 1976). Accordlna to Ling(1962), hatching tanks should be provided with aerators. Hatching ofMacrobrachlum eggs usually takes place at night (Choudhury, 1970a, 1971a, 1971 b;Wickins and Beard, 1974; Dugan et 01., 1975), and may be spread over two nights In M. rosenbcrgll (Wickins and Beard, 1974; Chao and Liao, 1977).

Macrobrachium rosmbrrgll females may be expected to lay eggs 3 to4 times per year (Ling, 19698; \Vickins and Beard, 1974; Wang and Williamson, 1977). Read (1982) deduced that since only one spawn per moult cycle was possible forM. peters//. the number of spawns per breeding season is Influenced bythe frequency of ecdyses. According to Una (19698) the moulting frequency ofM. rosenbergll adults depends onage, and the amount and quality offood eaten. Wickins and Beard (1974) found thatthemoulting frequency of M. rosenberg// was constant, with no proportional change with the age of the prawns.

Dugan and Frakes (1972) and Dugan et at, (1975) reported induced spawning ofcertain Macrobrtlchlum species by reducing the temperature in tanles containing ripe females rrom 27,S - 240C fora period or two weeks, anerwhich the temperature wasapin raised to 27,SoC. l1tIs resulted In morc or less synchronous spawningof the females. Dugan et tiL, (1975) suacsted Uat this technique could be used where limiled numben or broodstoclc are available and where It Isnecessary to obtain numerous lame at one time. Dugan et al., (1975) presented figures ofexpected fecundity ofcertain Macrobrach/um species, and noted that this may depend on thesize of the slage one larvae, as is the case forM. carcinus which produces more eggs than M. rosenbergll ofacomparable mass. and hu smaller stage one larvae.

According to Dugan etat, (197 S) the following m3SS to en ratios may be expected:

M. rosenbergll mass 80 & 70000 eggs M. carc/nus mail 7S B 120 000 to 140 000 eggs M. acanthunls mass 2S g - 30g 8000 to 18000eggs Macrobraclllllfn air/one (Smlth,1874) SOO to I 000eggs but up to 5 000eggs (nomass given)

Large M. rosenbergll females may produce up to I SO 000 eggs per spawning (Ling and Merican, 1961). Read (1982) recorded that M. petersii females with a carapace length in excess of 15 mm produced between I 500 and 3 000 larvae.

Sandifer and Smith (1978) found that the viability of larvae produced by a given female, frequently decreased after repeated sp3wnlng. which may have resulted either from dietary deficiencies (adults fed only prepared diet), or physiological faclors associated with re­ peated ovarian development and spawnings.

Smith and Sandifer (1979) reported the construction of breeding depressions by male M. rosenbergli in production ponds. the number and sizeof which were apparently related to the size and density of the adult maleprawns in the ponds. These authors noted that thts phenomenon had notbeen reported for wild stocks.

2.6 LARVAL DEVELOPMENTAL FORMS

2.6.1 Definition oftenm

Most Crustacea hatch atan early stage of their development and thus have to undergo an extended period oflarval development (Knowlton. 1974). during which the larva passes through a series of moults accompanied bygradual morphological changes. A great variety of names has been applied to these morphological changes inthe course ofdescriptive studies on crustacean larvae (Knowlton. 1974). and it is necessary todefine terms to be used In the following review: 26

Phase Williamson (1969) proposed three bllsic consecutive phases of development for the Decapoda and Euphausloeuo. namely: naupllus, zoe« and mtga/opa. Knowlton (1974) considered thenauplius, protozoea androta to be the three b3sic crustacean larval phases, which he referred to liS larvel "forms". Knowlton (1974) later adopted the use of the term form to signify the degree of morphogenesis during zoeal development, introducing ambI­ guity in the use of this term. Phase is employed in this review inthe sense that Williamson (1969) usedit. The use of the term mega/opa by Williamson refcntoan intennediate phase of development, between the local andjuvenile phases and Is also known as the post·/ana. From the literature referred to, it appean that the concept ofthe post-larv« as a distinct transitional phase between larval and juvenile phasesis favoured above that of mega/opa, and is adopted here for that reason.

Fonn. Form wlll beused to sIgnify only the degree of morphogenesis and not necessarily the number of moults, inaccordance with the approach adopted byKnowlton (1974).

Instarand st:lge. lnstan arc the periods between moults. Where the number of moults be­ tween hatchingand attainment of adult fonn arcconstant, the tnstar may be consideredas astage ofdevelopment (Meglitsch, 1967). Thus Instor andstage may be used synonymously. Refereces to Instars and stages will imply adistinct moult-related morphological fonn here, unless the term used istaken from a reference work.

Inthe Caridea thenaupltus and protozoea phases arc bypassed and the larva hatches asa toea (Knowlton, 1974; Wickins and Beard, 1978), which in tum passes through a number of morphological fonns before metamorphosing into apost·/arva (Knowlton, 1974).

2.6.2 Larval etcvelopment in the Palaemonldae

The larvaldevelopment of the Caridea isa prolonged process with six or more instm (Knowlton, 1974). Studies of the larvaldevelopment ofseveral representatives of the Palaemonlda« Indicate that development Is usually accompanied byadegree of variability, with respect to thenumber ofinstars and moulting history, morphological development and duration of development (Reeve, 1969; Sandifer, 1973; Pillal and Mohamed, 1973; Knowlton, 1974; Sandifer and Smith, 1979). Typically, among Pa/acmon/doe. some larvae of I specieswill metamorphose to posr·larvae after passing through (ewer developmental forms, or a shorter developmental time than the bulk of the population reared under IdentI­ cal conditions,while inothen metamorphosis to post-larvae ismarkedly delayed (Sandifer 27

and Smith, 1979). According to Pillai and Mohamed (1973)this variabiUty is common among certainspecies ofMacrobrachlum with respect to thenumber of moults and morphological forms. Initially, moulting and morphogenesis is retatively constant amons theCaridea: Thereafter, it becomes more irresuJar, and the probability ofindividual variation increases wllh each successive moult (Knowlton, 1914).

During the early larval stages ofMacrobrac1llum idella (Hilgendorf) and M. acanthurus, each moult isaccompanied by a new larval stage, but from the fifth fJt. acanthurusr and sixth (M. Idtlla) moults, each moult doesnot necessarily result inanew stage (Choudhury, 1910a; Pillai and Mohamed, 1973). Similar observations were made by Reeve (J 969) who found that a laboratory reared populatlon ofPalaemon sermtu: (Pennant) moulted syn­ chronously for the firsl five moults, and thereafter lesssynchronously. Variability in the number of stllges in the development of P. senatus was reported byReeve (1969). Sandifer (1913) reported similar findings for Palaemonetes vu/gans (Say). Knowlton (1974) recorded differences In morphology among conspcciflc Indlviduliis of P. vulgaris of the same moult, appearing at the third orfourth moult. In addltlon, Knowlton (1914) noted that variaUon In the nurnber of Instars has been recorded for three Palaemon and severnI Palaemonetes species.

It h3S been suggested by Knowlton (1974)that a certain autonomy existsbetween moult­ Ing lind morphogenetic processes. Thus the development of morphological features may be distributed over a varying number ofmoulls, with charactersshowing diffferent degrees of development indifferent larvae after the same number of moults (Reeve, 1969). Reeve (1969) also concluded that the fonn of the larva alone couldnot beregarded as indicative of ,he age or previous moulting history. MaCTobrachium idella and /tI. acanthurus larvae, for instance, may moult at regular intervals, without reaching the next morphological stage ormay moult with very smaU changes in morphology (Choudhury, 1970a; Dobkin, 1971 i Pillal and Mohamed, 1973). It was thus possible for Ling(1962) 10describe twelve larval stages in the development ofM. roscnbtrgli. which the same author later reduced to eight morphologically dislinci stages occurring over eleven moults. Uno and Kwon (1969) de­ scribe elevenstages for AI. rostnb~rglL Dobkin (1971) agreed with the examples chosen by Choudhury (1971 a)for his descrfprlon of the larval development ofAI. acanthurus, and found that specimens from South Florida appeared to be Idenlical wilh those described by Choudhury from jamaica, as far as mode of development and morphology arc con­ cerned. Nevertheless, Dobkin (1971) fclt that since variation normally occurs, and since moulting is accompanied by small changesinmorphology over mosl ofthe larval period, 28

the selection of A number of "morphological stages" for descriptive purposes b nece5S31ily an arbitrary proem, except in the case of the distinctive early stages.

2.7 LARVAL REARING AND FACTORS AFFEcnNG DEVELOPMENT

2.7.1 Background

Early attempts at rearing freshwater prawn larvae to juveniles under controlled conditions were unsuccessful for a number of years untll 1962, when the first M. rosenberstt juveniles were produced byS.W. Ling, following the discovery the year before by Ling thnt the prawn Is catadromous, with the early larval stages requiringsaline conditions for develop­ ment (Ling and Costello, 1976; Hanson and Goodwin, 1977).

TheInitlntion of prawn culture research In lfawnll In 1965 followed. with the development of techniques forthe mass rearing of prawns forcommercial seale prawn farming by Fujimura and co-workers (Ling and Costello. 1976). Within npproxlmately ten yep.n, Inte­ rest In freshwater prawn culture generated research and development projects Internatlonal­ Iy. involving otherMacrobracl,l"m species as well (Ling and Costello, 1976). The latter authors llst fifteen Macrobracl,lwn species u:i1ised at that time In the research and develop­ ment of prawn farming. Ofthose listed. twelve species had had laboratory life histories Investigated. However. except for M. rosenbugll, the rearing of Iarvae for the production ofjuveniles was still inan experimental stage for the species listed, with techniques used for the giant freshwater prawn fonning the basis for rearing the other species. Of the lndl­ genous speciesinvestigated in this study, only Itt. rude is known to be cultivated. (P:anlkkar, 1968). Panikkar (1968) did not supply any further infonnation. Ling and Costello (1976) reported that"'. rude had been Investigated p:artially, in the field. Read (1982) has reared M. ptlmll to post larval stage, as part of anecophyslological study of the species. No further references were available on the rearing oftheIndigenous species.

For normal larvel development to occur, suitable dietary and envlronmentnl conditions arc nceded to allowforsurvival, moulting, growth and morphogenesis of larvae.accordina to literature referred to.

Larval development among planktotrophlc decapod larvae, p:artlcularly the Palaemonldat Is known to be vamble with respect to the number of instant moulting and morphology (section 2.6.2). Knowlton (1974) suggested differential control ofdevelopmentnl process .29

by environmental factors and a semi-independent functioning of developmental processes. Although the underlying causes for this variation are poorly understood, various environ­ mental factors have been implicated in laboratory studies as contributing factors ofvarying degrees ofimportance (Sandifer and Smith, 1979). Diet, temperature, salinity, parental population, photoperiod, time of the year and pollutants were Iisted by Sandifer and Smith (1979) who suggested that in addition to environmental influences, the variation in de­ velopmental processes may be inheritable, serving to ensure dispersal oflarvae and possibly increasing the probability ofrecruitment to existing parental populations and subsequent successful colonisation offreshwater environments.

2.7.2 Nutrition and diet

Nutrition. Food was considered by Knowlton (1974) to be the main factor determining how developmental processes in P. vulgaris are carried out. Knowlton (1974) suggested the arrangement ofdevelopmental processes into hierarchy based on the utilisation of food­ energy, rust for maintenance activities at the expense ofmoulting processes, which in tum have priority over growth and morphological development. In addition, Knowlton (1974) suggested that the direction ofenergy flow may change underthe influence ofenviron- . menta] factors, possibly acting differentially on hormones which are thought to control moulting, growth and morphological development.

Although Ling and Merican (1961) claimed that M. rosenbergii larvae commenced feeding a few hours after hatching, results ofother workers suggest that the form I larvae of M. rosenbergii as well asM. acanthurus do not feed at this stage, (Choudhury, 1971b; Dugan and Frakes, 1972; Goodwin and Hanson, 1975; Moller, 1978), and survive on the yolk-sac for the rust two to three stages (Goodwill and Hanson, 1975).

Diet. Macrobrachium larvae have been reared on a wide variety of foods (Ling and Costello, 1976), both prepared and live, ranging from snake-head meat (Suharto, et al.• 1980) and egg custard (Ling and Costello, 1976) among prepared foods to live Artemia nauplii, com­ monly considered to be the food of choice for the early stages ofdevelopment (Goodwin and Hanson, 1975). Sick and Beaty (1974) and Ong et al.• (1977) reported that M. rosen­ bergii larvae could not complete larval development without the inclusion ofArtemia in the diet. Artemia have become a staple food for the Macrobrachium industry (Smith et aI.• 1978) and are, in fact, considered a fundamental, near essential dietary component for successful rearing ofM. rosenbergii larvae, with survival and growth being poor where 30

ArltmJo is absent from the diet (Thompson, 1980). Allhough Arttmlo are the food of choice for the early stages, the quantlty fed isusually reduced with development through thelater stnges (Sandifer and Smith, 1976, 1978; Lingand Costello, 1976), thus minhnlsina the cost of u51nB Arttmlo (Ling and Costello, 1976).

Steamed egg custard and soya bean curd are prepared foodsgaining popularity because of Iheir generalavailability, economy and ettractiveness to larvae (Ling and Costello, 1976). The eggcustard can be mixed with other nutrients including yeast and vJtamins (Lfng, 1962, 1969b).

QualUnlivc aspects offeeds. Food particle size has been an importanl consideration In the feelling of prepnred food to Macrobracl,/lIm larvae, Prepared food is graded Into approp­ riate sizes for differenl sized larvae by forcing it through a range of mesh sizes, usually commencing wilh 20-25 meshes per centimetre and up to 7-8 meshes per centimetre for the later larval stages (Ling, 1962, 1969b;Choudhury, 1971 b;Goodwin and Hanson, 1975; Mennsveta and Piyatlratltlvokul, 1980). Stnge X and Xl larvae of M. TOstnbtrgllcan be fed particlesof I mm in size (Thompsen, 1980). Dugan et al.•(1975) recommend uslna the width of the thoracic region of the larvae asa gauge for theappropriate particle size. However, Moller (1978) In an Jnvestigation of the feeding behaviour ofM. rosenbergl! larvae, concluded that particle size was notcritical and considered that particles of0,2-1 mm In diameter could readily be manipulated by Inrvne. Food capture was unselectlve with re­ spect to shapeand size but ingestion of theparticle depended upon chemical cues (Moller, 1978).

Quantity. and concentration of food. Although moulting frequency is not appreciably affected by differences in food concentrnlioninP. vulgaris (Knowlton, 1974) and P. strra- tus (Reeve, 1969), growth and developmentof these species depend directly on the amount ofavallable food. At reduced feeding levels orconcentrations, where growth and subse­ quently morphological development had ceased, moulting in these species still continued, resulting in an increase in the number ofstages tometamorphosis inP. serratus, or a repe­ tition of forms, in P. vulgarlJ.

Moller (1978) found that food capture by M. TOstnberglllarvac occurred principally by chance encounter, and that food which did not make contact with thefeeding appendages was Ignored. This appean to contradict the finding ofChoullhury (1971 b) Ihal M. acant­ "ufUsJarvae were capable of following and calchlng a sinking particle orprepared food. 31 "

Nevertheless, it Is known that for efficient feeding ofMaerobraeJrlllltl larvae it Is necessary to maintain food p:utlclcs in suspension inthe reuing water, either by means ofalrstone ngilation (Ling, 1969b; Goodwin and Hanson, 1975: Dugan etal., 1975: Aniello and Singh, 1980:Chlneah, 1980) or by means of water now in properlydesigned tanks (Sandifer lind Smith, 1974). S3ndifer lind Smith (1974) were able to accelerate the developmental rateof M. roJenhugllin closed recirculation systems, so that larvae developed as rapidly as those reared in greenwater culture systems, by malntalnlng food In suspension.

It is therefore clear that the density of food particles suspended Inrearing water could be a critical factor IntheIIvall3bility of food to thelarvae, particularly with respect to prepared foods. While there IIppears to be agreement that a density of 5-1 5 Artemta nauplil/ml of rearing water is asatisfactory density for the feeding of larvae (Reeve, 1969; Sick and Deaty, 1974~ Duganet ai" 1975; Sandifer and Smith, 1976; Goodwin and lIanson, 1975; Sandifer and Smith, 1978), there is far less information available on thequantity and/or density of prepared food to be fed to larvae. Ling (1962) :1I1d Choudhury (l97Ib) concentrated the larvae in one area of the rearing tank before fceding prepared food, then fed the prepared food until all larvae were seen carrying food, after which the procedure was repented. A quantity equivalent to 30% of the estimated biomass of larvae per day has also been recom­ mended (Ling, 1962; Mcnasveta, 1980; Mcnasvctn and Piyatirntitivokul, 1980). Reeve(1969) reported that consumption of Artemla nauplli by P. serratus larvae was 20/larva two days after hatching, increasing to I50/larva after24days, when 50% of the larvae had meta­ morphosed to post-larvae.

Frequency of feeding. E:nly techniques used by Ling (1962) involved a gradunl increase in the frequency offeeding prepared food from twice per day initially, up to five timesper day for the later forms, with Artemla being fed once in the afternoon asan overnight feed. Ling (1969b) recommended that A rlcmla naupli i should be present atall times to reduce thechances of cannibalism. Dugan et al., (1975) fed larvaein rearing experiments three times per day on prepared food and once per day on Artemlanauplli. Larvaeof M. rosen­ bagU reared under mass culture conditions In Mauritius are fed hourly on prepared foods, with Artemla being fed once in the afternoon(Thompson, 1980). The practice of feeding prepared food during the tinyand Artemla once in the late aftemoon or evening has been adopted by varlous workers (Sincholb andSukapunl,1980; Anlello and Singh, 1980; Menasvctn 1980;Menasveta and Piyatlmtltlvokul, 1980; Suharto et al; 1980). 32·

2.7.3 SAUn1ty and temperature

Most A/ocrobrac1llwn species are inhabitants of tropical regions, although some species ere lib Ie to tolerate lower temperatures than others (Hanson and Goodwin, 1977). Although the genus includes entirely freshwater forms (Panlkkar, 1968), according to literature referred to,most of Ihe species (such as M. rosetlbergll, M. OCOIII"UruS. M.core/nus, Itt. ohtone, Macrobrac!IIum americanum (Bate, 1868) lind M. rude] already investigated for theircul­ ture potential, require saline conditions for part of their development (Panikkar, 1968; Goodwin lind Hanson, 1975). Seawaterwas used by Ung(I969b) for the preparation ora saline culturemedium, as he found thatcrude salt WllS unsallsfaclory for this purpose, although reconstituted sea $:Ills worked quite well.

Rend (1982) considered the presence ofspecies ofMacrobrac"lum in both fresh and estu..rine environments to be indicative of II genus in the process of colonising freshwater. The osmoregulatory capacity of prawns isrela ted to theirdistribution (Denne, 1968; Rend, 1982). That temperature playsnrole can be deduced from the example, cited by Panikkar (1968), of temperate marine species ofNatantia which tend to become inhllbl­ tants of brackish or freshwater under tropical climatic condltions, Temperature can modify the effects of salinity (Read, 1982). Panikkar (1968) suggested that in addition to investi­ gations of the individual effects of temperature and salinity, it isnecessary to include the combined effects ofsalinity and temperature on survival lind development of larvae, Al­ though thisapproach has been adopted by research workers such asSandifer (1973), Knowlton (1974) lind Read (1982), most studiesof the effects ofthese parameters on larvae have involved the investigation ofindividual effectsfirst, with the purpose of establishing tolerance limits and optimal values for survival, lind later the rearing of larvae at the oprlmum values obtained individually. It should also be remembered that conditions which favour survival do not necessarily promotedevelopment (Read, 1982).

Temperature isknown to effect the moulting frequency of P. senatus and P. v"lgarlJ directly (Reeve, 1969; Knowlton, 1974) and the number of lnstars to metamorphosis to post-larval stage (Sandifer, 1973). In addition, growth of P. vlllgaris larvae is directly re­ lated to temperature, with an increase In moulling rate and growth at increased tempera­ tun: (Knowlton, 1974). However, growth decreased with respect 10moulting in this case. Knowlton (1974) found Ihat morphologkal development In P. ~"Igarls followed that of growth, proceeding at approximatc:ly the same rnte. 33

It has been suggested that the growth ofanorganlsm maybeexpected to be maximal In lin Isosmotic environment where a minimum amount of energy isexpended on osmoregulation (Panlkkar, 1968; Singh, 1980). Singh (1980) has pointed out that this may not necessarily be the case,citing theexample ofM. rom,bergll post-larvae which grow best in hyposmotlc medi... At the isosmotic point of 17,SS0/00 growth Isretarded. Crustacea are known to absorb water at moulting to enable increase in volume before hardening of the new cuticle (Megli:sch, 1967; Darrington, 1969; Singh, 1980). Singh (1980) suggested that this mllY be the reason for theretarded growth ofM. rosenbergll post-larvae at snlinities equivalent to the isosmotic point.

However, salinity appears to have little effect on the growth rateof the larvae ofM. aeanthu­ rus (Dugan u 01., 1975) or on the rate ofdevelopment of Maerobrac/rl"m amazon/cum (Heller, 1862),(Guest and Durocher, 1979). Yet Sick and Deaty(l974) found that dlffe· rent larval stnges ofM. rosenbergll had rather narrow Slllinity preferences for optimum sur­ vival rates, Read (1982) demonstrated thatlntruspcclflc variatlon in osmoregulatory ability occurred among different larval stagesofM. petersll, suggesting possible s"linity preferences. In considering thedevelopment ofM. pnm/l. which exhibits wide salinity and temperature tolerances, Read (1982) concluded that larval development ofM. petersit WIlS independent ofsalillity. Only in the low salinity range (0,S--6 SO/oo) which was below that required for development (8 S ~/oo) was there II significant effect of salinity, while at higher salinities temperature was thedominant factor influencing development.

It has been suggested that temper..turc is more critical for larval development than salinity (Knowlton, 1974; Goodwin and 1[anson, 1975;nod Read, 1982). Maintenance of haemo­ lymph osmoticandionic concentrations byCrustacea involves active transport processes (Darrington, 1969: Schmidt-Nielsen, 1977). The role of (Na-K)ATP ase in these processes has been established (Donat et al..• 1979) and the importanceof temperature in develop­ mental processes possibly lies in its effect on enzyme systems.

Optimal temperatures for rearing most Macrobrachlum specks range from 2S-300C with the lethal range being 10-1SoC (Guest and Durocher, 1979). Forbest growth ofAI. rosen­ bergll there is wide agreement tllat 28°C Is preferred (Goodwin and Hanson, 1975).

Salinity requirements for larval development vary among MucmbracltlulII species. In addl­ tlon, there Is varlallon.Intraspcclflcally.fn this regard, between thestages of larval develop­ mcnt (Dugan and Frakes, 1972). Alacrobrl1chlllm roscnbugll for instance requires S3l1nlties 34

(rom 10 - ISS0100 (Sick and 8C:3ty. 1974). For optimal survival and development. Itt. care/IIIIS and M. acan thurus require salinities of 14 - 17,5 S 0100 and 15 - 20 S 0100 respectively (Choudhury. 1970b; 1971 b). However, Choudhury (1970b; 1971 b) did not p~y much attention to temperature Inarriving at these values,

2.7.4 1"11

The question of a sultable pll range (ortherearingoflttacrobraclllllm larvae and the effect o( pll on developmental processes hasreceived scant attenlion. Guidelines for pH canbe obtained (rom references to I'll in experimental studies.

Ling (I 969b) recommended 11 pH mnge of7-8 and later (Ling and Costello. 1976) 7,5-8 for rearing of M. rosmbrrglliurvae. In summarising the work of Fujimura (1966) and Mlnuml1.:lwa and Morlzane (1970), Sick and Deaty (1974) reported that water used In experiments to determine adequate condltlons for mass culture had n pH of 8. I and con­ talncd a heavy concentration of CMorella.

In mass culturing experiments with M. carctnus and AI. acanthunu Choudhury (1970b, 1971 b) recorded Iluctuatlons in pll from pll 7-8.5 for M. carclnus and pll 6.5-8 for M. acanthurus.

111c latter results suggest that these species could tolerate awide range of pll levels.

In investigations of suitable mass rcuring systems for larvae, by Dugan et 01.• (1975). the pH range in the most successful of the rearing systems was between 8.1 and 8.2 lit tempe­ ratures of26 to 30°C and salinities II,S - 14 S 0/00 for M. rosenbergll uuve«:

Suwannatous and Sukapunt (1980) recommended a 1"11 of7 to 8 for freshwater and 7jS to 8.S for seawater used in providing brackish water condllions for rearing ftf. rosmbtrgll larvae.

High pll (above 9) W:lS Identified as a probable culprit In c.1using deposition of calcium, carbonate Intheprawn carapace (Go()~lwin and Hanson, 1975). 35

2.7.5 WaterJfardness

Sick And Beaty (1974) reported highermortalities amongAI. row,btrg/llarvae reared in I; culture medla using well water of hardness 50and 100 mgJ2 than in groups reared incui- hire media using distilled water. At a hardness of 100 mgJ2 mass mortality occurred after 5 to 8 days of growth lind none of the larvae reached stage four. Larvae reared in a culture medium using water of 50 mg/2 hardness showed a 97% mortality after 5 to 10 days.How- ever, larvaereared through stage four In adistilled water culture medium and then inwell water culture medium, metamorphosed through all developmental stages with a relatively good survival (Sick and Beaty, 1974).

2.7.6 Photoperiod and Ugh t Intensity

TIle onset of metamorphosis to post-larvae may not be exclusively controlled by mechanisms associated with larval growth and differentiation (Knowlton, 1974). Evidence from investi­ gations by Knowlton (1974) with 1'. vulgaris larvae indicates thatdaylcngth may be a con­ trolling factor;short days or no light promoting postponement of metamorphosis and re­ sulting in large-sized late stage larvae and post-larvae. Under longer daylength periods Knowlton (1974) found that there were fewer instars and smaller post-larvae. Continuous lighting was found to be detrimental to thc development of Itt. acanthurus larvae and con­ tinuous darkness detrimental to M. acanthurus and AI. carcinus larvae (Dugan et 01., 1975).

Afourteen hourlight, ten hour dark photoperiod regime has been used by some workers In experimentalsituations (Sandifer. 1973; Hubschman, 1975; Dugan et 01•• 1975) while Moreira et al.• (1982)employed a twelve hour Iight/twelvchour darkness regime in a study of thc effect of sa lin ityon metabolism ofcertain Palaemonld larvae.

According to Liao and Liu (1982), larvae should not be reared in direct sunlight or bright. light orintensities higher thun 4 000 lux, neither should the light intensity be lower than 200 lux.

2.7.7 Stocking dcn~Uy

Referencesto the effccts ofstocking dcnsity on developmental processes arc scant and recommended stocking dcn\iUcs for the rearing of larvaeare related to the culture system employed. 36'

Sick :1Od Beaty (1974) reported differences Ingrowth and survival ofAt. TOs~nbt,,1I brvllC reared under"green water" conditions at different stocking densities. with a maximum moulting rate and survival at an initialstocking density of 20-40 larvae/2 •

Goodwin and Hanson (1975) report that results obtained bySmith, Sandifer and Trimble (1974), although inconclusive, suggested that survival and larval development rates may be Inversely related to stocking density. However, these authors also reported that Hagood (no data given) had found that the early I:lrval stages were unaffected by crowding.

Ling and Costello (1976), in a reviewof freshwater prawn culture, reported that stocklns densities of larvae were maintained at 100 to 150 per litre In theInUinl stages, belllS re­ duced by losses to 20to 60 per litre toward the end of the larval period.

In Ilawalln culture, Initial larval stocking densities lire usually higher th:1I1 in later stages - 130 per litre Initially, compared wilh43 per litre In the later stages (Malecha, 1979).

For clear water rearing systems where water Is routinely exchanged, densities of 100to 200 larvae per litre are acceptable initially (Chlncah, 1980). Menasveta (1980) stocked a mean of 38 larvae perlitre In rearing trials employing closed recirculation systems. In their most satlsfactory rearing trial, using closed recirculation systems, Dugan tl 01., (1975) stocked 115 larvae perlitre Initially.

TIle early larval stages ofM. rosenbergii u» gregarious, tending to swim together in large groups until about ten days of age. (Goodwin and Hanson, 1975; Dugan et 01., 1975)and high initial stocking densities are recommended by Chao and Liao (1977), as larvae feed and grow better at high densities duringthis phase. However, once the tolerance to crowd­ ing Is lost, they should be given more space (Dugan ~I ClI., 1975) oralternatlvely periodically thinned out (Chao and L1:l0, 1977).

2.8 POST·LARVAL nEARING

2.8.1 Dnckground

As mentioned In section 2.4 of this chapter, the renrinc of posl·larvae was not intended lIS part of the present project. TIle literature reviewed in thisc:hailicr Is presented however, to enablediscussion of some results arising from the holdingof post-larvae produced during 37

the course of Ihe rearing programme, During the early development ofMacrobrathlum cul­

ture techniques, theusual practice (or there3ringof posl·l3lVlle WllS to stock them in nursery tanks forI short period prior to transfer to earthen ponds, or to transfer posl-l31VILe directly to these ponds (Plyatiratitivokul and Mcnasvela, 1980). According to the bUer ..uthors, both praclices were found to be un$;llls(uctory, and the nursery phase was re­ introduced. Kneale lind W..ng (1979) reported on the introduction o( a nursery phasefor the Hawalanprawn industry. lJigh densily nursery techniques using closed intensive culture systems were inveslig:lted by Sandifer lind Smith (1974, 1976, 1978), Willis et al; (1976) lind Kneale and Wllng (1979). These investigations dealt mainly with the effects of age, temperature, slocking density, nutrition lind artificial h:lbilllis on survival and growth.

2.8.2 Temperature and snllnlly

Kneale and Wans (1979) obtained higher growth rates of juveniles lit 28°C compared 10 24°C, but at theexpense of survival which was found to be higher at the lower temperature,

Ling (1969b) reported that young M. rosmbt'rgll thrive in varied types of fresh and brackish waters at temperatures between 22 and 32°C. Sandlfcr and Smith (1974) found that M. roscllhl!rgll may grow more rapidly in sliHhtlysaline water (2,5 - 5 S 0/0 0) than in freshwater or III higher salinitlcs (10 - ISS %o),during the Ilrst two to three months, with considerable growth being possible at salinities as high as 10- 15 S 0/0 0• Growth is however retarded al between 17 and 18S% 0 (Singh, 1980) while II salinlty of 2S SO/00 has been found to belethal to juveniles (Sandifer and Smith, 1974).

Ling (1962 and 1969b) recommended that newly metamorphosed post-larvae be accllmi­ tlscd graduallyto freshwater over a 7-8 hour period. S:lIIdifer and Smith (1974) however reported that direct transfer of post-larvae frorn higher salinities used during rearing to lower salinities or freshwater, had no apparent effect on IheS\llVival of the post-larvae.

2.8.3 Nutrition

Acomprehensive knowledge of the nutritional requlrements of prawns is considered a basic necessityforthesccccssfu! culturingof any specles under Intensive culture contlltions (MeSwceney, 1977). Natural food Isconsldercd to be Ihe IHinclpal food for rearing of M. rostllbtrgl/ juveniles by Ling (1969b) anti ling and Costello (1976). Although Ihey 38

found these foods to be satisfactory foruse Inlaboratory culture systems, Forster and " Wickins (1972) list certain disadvantages Inthe usc of fresh foods, namely:

• rapid decomposltlon • l/mNonslIm/"g dally preparation • vlIlnerabllJly 01supplies • variation /n quality

Willis et al.• (1976) considered natural diets to be uneconomical for use due to the lengthy preparation, cost of Ingredients and short storage life as well as the possibility of intro­ ducing pathogens Into the system.

Inextensive culture, food for growing prawns would be provided mainly by natural pro­ ductivity, while In fully intensive culture systems juvenile prawns would be fed on prepared diets, as natural productivity would be negligible (Forster and WickIns, 1972). UnderInten­ sive culture conditions It Is necessary to provide a more nutritious and well balanced diet (Sandifer and Smith, 1976; Sandifer and Joseph, 1976; Iliddle, 1977).

External mastication, exposure to water currents, aeration systems nnd physical grasping by an animal can hasten pellet disintegration and result In nutrient loss (Farmanfarmalan, et al., 1982). In addition, rapid disintegration of feedstuffs in water makes them unsuitable for shrimps (New, 1976). Water stability is a prime consideration In the formulation of diets for aquatic animals (Meyers and Brand, 1975; Diddle, 1977).

Protein requirements ofjuveniles reared in closed culture systems were found to be relative­ ly high by Millikin et al..• (1980) and Willis et 01.• (1976). From a review of investigations into the protein rcqulrcmcnts of cultured shrimps, New (1976) concluded that levels from 27 - 35% protein were required for rearing shrimps. Balaz and Ross (1976) ob­ talned results which suggest that dietary protein levels inexcess of35% may be required for the pond rearing ofjuvenile M. rosenbcrglL Willis et 01••(1976) obtained most rapid growth rates using a 40% protein trout chow. Doth Dugan tl 01.• (1975) and Kneale and \Vllng, (1979) obtalncd satisfactory results using trout chow in post-larval rearing cxperi­ ments In closcd systems, while other workers have used commercial shrimp feeds (S:mdifer and Smith, 1974, 1978; Mancebo, 1978; Piyatiratltivokul nnd Menllsvet:l, 1980).

Feeding ratesemployed by sorne workers range from 3% to 20% of biomass per day, usually with hlBher rates In the early developmental stages (Willlsel01., 1976; Mancebo, 1978; 39

Piyatir:ttitivokuland Menasveta, 1980) with I rate of 3% per d3Y being the most widely used rate reported ina survey conducted by McSweeney (1917). Willis et al.• (1976) re­ commend a feeding rate of 20% of biomass perday following metamorpbosis into post­ larvae. In theirrespective investigations Into nutrition andgrowth ofjuveniles. B:1I31 and Ross (1976) and Mancebo (1978) selected feeding rates of S% and IS% at the beginnlna of their investigations. Both sets ofinvestig3tors modifiedthe ntes Inter and gauged the quantity to be fed by the quantity consumed between feeding times.

In determining feeding frequency and time of feeding, twofactors related to the feed Ina habits of/tI. rosenbergli juveniles need tobeconsidered:

• Juvenile M. rosenbergll are active IIlgllt/ceders (LIII" 1962).

• M. rosenbergli ts 0" intermlttent feeder and better utilisation offood couldbe obtained byproviding several portions per day, while maintainlng great care to avoid over!ct:dlllg (McSwceucy. 1977).

The latter recommendation was made originally by Ling (1962) inan early reference to the rearing of juveniles. Dalal and Ross (1976) fed juvenileAt. rosenbergit once per day, in the afternoon, adjusting the quantity bytheamount eaten the previous day. Knealeand Wang (1979) fedjuvcnilc M. roscnbergli in rearing tanks twice perday on trout chowat levels such that there was always some excess food present. Kneale and Wang (1979) siphoned food particles from the tank bottoms once per day to maintain water qualily.

Plyatiratltlvokul and Mcnasvcta (1980) fed juvenile M. rosenbergil a 30% protein com­ pound diet at a rateof 5% of prawn biomass per day. in two feeds per day, over a three month rearing period. Stern et 01., (1976) investigated survival and growth ofM. rosen­ bergll juveniles fed on single species plant dietsand found these to be unsatisfactory, al­ though all the test diets were ingested. Plant diets investigated bySternet al.• (1976) In­ cluded Azalia/li/Cliloides, Cladophorasp., Elodea sp. and Lemna tp.

2.8.4 Stocking density

Adirect comparison of results obtained In investigations of the effects ofstocking density on survivalandgrowth ofjuveniles is handicapped by the fact th:lt different workers mea­ sure density In different ways, will' a common fault ofquoting stocking fates whichapply 40

'. to particular configurations used (McSweeney, 1977). McSweeney (1977) drew attention to the need for astandardised formula which should consider prawn biomass, available area, system volume and perhaps the turnover rate of water.

Mancebo(1978) found that growth of M. rosenberglljuveniles In a six month rearing trial was influenced bystocking density, animal size and tank size. Sandifer and Smith (1976) found the growth ofM. rosenbergll, juveniles was inversely proportional to stocki.lg density at levels between 150-600 juveniles/m2•

The density at which juvenile prawns are stocked affects survival as well as growth (S:mdlfer and Smith, 1976; Kneale and W:lI18, 1979), withjuvenile /tf. roscllbtrgll showing poor sur­ vivlli and growth,ln spite of added shelters, at stocking densities higher than 400/m2 (Sandifer and Smith, 1976). The latter authors however, subsequently suggested that crowd· Ing may not directly affect survival rates, which may depend on the container size and shelter provided (Sandifer and Smith, 1978). According to McSweeney (1977) optimum juvenile stocking density is Inversely proportional to the individual sizesand therefore It is necessary to make periodic reductions innumbers for best use of tank space, McSweeney (1977) and co-workers employ a six-month grow out programme in which prawn density is reduced by culling at two and four month intervals. Mancebo (1978) conducted trials on M. rosenberglljuvcnilcs in closed recirculation systems, using different initial stocking densl­ tics. Mancebo (1978) made periodic reductions in the dcnsity of the juveniles by culling the population for small and weak prawns every two months. For thelast two months of the rearing period t'he density was reduced to 14/m2 in all the tanks. Mancebo (1978) reported good survival rates ofjuveniles for all initial stocking densities. The stocking densities referred to by Mancebo (1978) were for the total available surface area, which included tank sides, bottoms and substrates.

2.8.5 Juvenile age and "breakpoint"

Asudden increase in mortalities among ftI. rosenbcrgli juveniles of acertain age is known to occur(Sandiferand Smith, 1976; Willistlal., 1976; Kneale and Wang, 1979). Kneale and Wang (1979) refer to this rise in mortalities asa "breakpoint" and reported that it occured at approximately 8-9 weeks in their experiments, where inilialjuvenilc stocking densities were higher than 900juvenilcs/m2 (surface area of tank bottom), Breakpoint was not in­ Ilucnccd by temperature, habitats, specific density, mean mass or total biomass. (Kneale 41

'. and W:mg, 1979). Although the specificcause was not readily Ilpparent at the time, Kneale and Wang(1979) implicated age and density, which may influence the incidence of break­ point.

2.9 DISEASES AND OTHER FACTORS AFFECfING PRAWN SU RVIVAL IN CULTVRESYSTEMS

2.9.1 Diseases

As late as 1975 it WIlS still possible for Goodwin and Hanson to report that disease was not considered II major obstacle to commercial prawn culture, with freshwater pmwn cullurists experiencing fewer disease problems than culturists of other crustaceans (Goodwin lind Hanson, 1975). These authors, however, added a note of caution thnt the history ofanimal husbandry did not permit complacency. Johnson (1977) predicted that diseases would be­ come more prominent obstacles to the successful production of freshwater crustaceans, IlS they become cultured nt higher densities under unnaturalcondllions. Indeed, losses of Macrobractuum prawns are experienced Jlllrticul:nly in the hatchery phase where they are cultured most intensively (Johnson, 1980).

Outbreaks of diseases In closed recirculation systems, where II large degree of environmental control is possible, ispredominantly the result of poor husbandry (Delves-Broughton and Poupard, 1976; Wickins and Beard, 1978) and once established, infections can be spread verY rapidly (Delves-Broughton and Poupard, 1976).

Attention to husbandry techniques and routine monitoring of water quality arc therefore of paramount importance in the prevention ofdisease outbreaks. According to Delves­ Broughton andPoupard (1976) who noted that in most cases of crustacean disease reports there was a paucity of information, diagnosis was inconclusive, and details of infectivity and pathogenesis were absent. Wickins and Beard (1978) observed that a major difficulty in the study ofprawn diseases, had been 10 correlate specific recognisable signs with the losses which occurred In laboratory tanks, asm:,"y mortalities occurred during moulting nt night and corpses were frequently cannlbattsed, By 1980 the causes orsome: disease conditions of prnwns wen: s~i11 unknown (Johnson, 1980). Abrier review ofsome of the marc Importan: dlsensc conditions recorded Is presented here: 42

Epibionts. Filamentous bacterin, most often designated Leucothnx species are frequently a problem in 13rval culture ofMacrobraclllum species (Johnson, 1980), attaching to gills and external body parts, and interfering with normal body movements (Johnson, 1980; Lewis et 01., 1982.)

Sessile protozoans such as Ep/sty/ls. Zoothamn/urn and COlllrnla may present problems in both hatchery and grow-out phases ofMacrobracll/um culture (Johnson, 1980). Fuuling of larvae or post-larvae byepibionts is related to high nutrient loads in culture water (Johnson, 1980). According to Lewis et 01•• (1982),filamentousbacteria such as Leucothrtx mucor, and eplcommensal protozoans such as Zoothamnium, utilise thesurface of the anlmalonly liSa substrate, relying on dissolved nutrients in the surrounding water for nourishment.

According to Johnson (1980) management schemes for epibionts have met with various degrees of success. Inhatchery rearing, chemical control of bacteria with antibiotics and copper sulphatehave been partially successful (Johnson, 1980). Reduction of stockingdensily, and nutrient load, anti maintaining clean tanks, have helped as well (Johnson, 1980). EctOo commensal protozoans have been successfully treated chemically wilh formalin, sodium chloride and quinine derivatives, and the feeding of hlgh protein diets, to encourage moult- lng, can sometimes assist the prawns to outgrow an epibiont problem.

Delves-Broughton and Poupard (1976) conducted an investigation into disease problems en­ countered in closed recirculation systems, inuscat laboratories in the United Kingdom. Some of the results arc presented below, including related work byother investigators. .

She." Disease. Symptoms arc brown to black spots on the exoskeleton (Delves-Broughton and Poupard, 1976; Wickins and Beard, 1978). Delves-Broughton and Poupard (1976) Isolated chitinoclastic bacteria from lesions on M. rosellbergll. identifying Beneckea species asthe predominant form, although Aeromonas and Pseudomonas were also present. These authors considered this type ofshell disease to be the resultofasecondary infeelion, following mechanlcalinjury, whieh could occur as the result ofdifficulties In moulting, aggressive behaviour, and handling, and which Is enhanced by high stockingdensities. Witkins and Deard (1978) did not consider thecondition lethal, while Delves-Broughton and Poupard (1976) noted that the condition did nut lend itself to prOI)hylaetic or chemo­ therapeutie control. 43

'; Black Nodule. Symptoms are black nodules in the epidennls beneath the exoskeleton of M. rosenbtrg/l. with no breach in the eplcutlcle as in the above condition (Delves-Broughton and Poupard, 1976). These authors considered that the condition wasprobably systemic, resulting from bacterial infection, and found that they could correct the disease witha constant treatment of Furanace at 0,09 mg/2. Johnson (1977) recorded the occurrenceof brown spotting of the exoskeleton of /tI. rosenbergli, which differed from the lesions pro­ duced by chitlnlverous bacteria, but hedid not record disease agents present in the tissue. Johnson (1977) considered systemic Infections to be rare,compared with bactenal In­ fectlons on the body surface.

White Syndrome. Symptomsare that the prawns tum an opaque white colour accompanied by muscle necrosis (Delves-Broughton lind Poupard, 1976;Wick Ins lind Beared, 1978; Johnson, 1977,1980). The exoskeleton Is softer than usual, and slow growth with high mortalities occurs (Delves-Broughton and Poupard, 1976). These authors cau tion that the condition should notbe confused with prawn microsporldlosls, which has been recorded forMacrobrachium species by Johnson (1977). These symptoms are often noticed In prawns after they have been stressed (Delves-Broughton and Poupard, 1976; Wickins and Beard, 1978;Johnson, 1980), as a result of changes In salinity and temperature, anoxia, or other stress factors, such as the presence of foreign bodies in the tissues (Delves-Broughton and POUPOlru, 1976). Delves-Broughton lind Poupard (1976) considered that no method of con­ trol for this condition waspossible. Johnson (1980) recorded a similar condition in M. ohione adults collected from the wild in Texasand transferred to the laboratory.

FungalInfectlons. One of the earliest records of prawn disease was a fungal infection of M. rosenbergillarvae reported by LingIn 1969 (Ling, I969b) inwhich he described small opaque white patches, starting at the tail and at the bases of appendages, which eventually spread over the whole body of the larva. Although fungal infections have been recorded In freshwater prawn culture, they have not attained the notoriety that they have in the culture of marine prawns (Johnson, 1980).

Pllmsitie infections. Infections by nukes, and the occurrence ofexternally attached orga­ nisms such asbranchiobddlids, isopods, ostracods lind tcmnoccphalid worms, have been recorded on MacrobracMIl11l species (Johnson. 1977). These lire not considered relevant to the present study. Johnson (1977) recorded a rusty coloured condition, of unknown aetiologyt which occurred on freshwater crustaceans, 2.9.2 Prt

In indoor culture systems, sufficient control ls maintained over the system to exclude pre­ dators such liS birds. However, adult Macrobrochlunr species are known to be aggressive (Dugan et al: 1975).lIlthough varying In degree (Goodwin lind Hanson, 1975), and canni­ balistic behaviour hils been referred to widely In the literature. Attllcks by other prawns

present II disease problem, as surface dllmllge can become a focus for infection (Delves­ Broughton lind Poupard, 1976). Prawnbeh4vlour is D critical fllctor in intensive rearins systems (McSweeney. 1977) as they are malntalned in crowded conditions (Forster and Wickins, 1972), Nutrillon has been Implicated in cnnnlballsm, Ling lind Merlcan (1961) noted that adult AI. rosmbtrgll rnny become clInnibalistic when surflciently hungry. Forster and Wick Ins (1972) found that cannlballstlc behaviour occurred even when the prllwns were well fed. ond sUll8ested that further researchInto Ihe nutrition of prawns in intensive culture. liS well as the effects ofstocking density oncanniballsm was needed. Johnson (1980), suggesled that nutrition must inOuence cannibalism as a result of'Inade­ quate intake of food or lock of specific required nutrients. McSweeney (1977) reported an example where prawn survival was increased from 40% tobetween 80and 90% by improve­ ment of the diet,

Peebles (1977) found that aggressive behaviour at the time of moulling was responsible for damage to prawn sensory and locomotor systems. and not cannibalism. According to this author this moult related intraspcciflc allgrcssive behaviour (M RAB) played 3 primary role incausing mortalities in confined populations of M. Toscnbcrgil, and cannibalism was probablynot a major cause of mortality In well-fed, uncrowded populations,

2.9.3 Tolerance to toxic substances

According to Spotte (1979),most of the toxicity problems encountered in aquarium systems can be traced to nitrogenous compounds. of which ammonia is the most toxlc, followed by nitrite. Toxins, precipitants and Jow oxygen concentrations in prnwn culture systems:lfC Important causes of disease ns well as predisposing facton (Johnson, 1980). According to Johnson (1980) carbon dioxide Is an importanttoxic 3gent In rearing units with low pll and Iimitcflaer.nion.

Ammonia. Free ammonll1 (Nil) is SCfler3IJy considered to be more toxic than ammonium Ions (N1I4+) by many fish culturists (Wkklns and Beard, 1978; 80)'d, 1979; Spotte, 1979; 45

'. Stickney, 1979). The fonn that ammonia tues is related mainly to the pH but alsoto temperature and salinity (ionic strength). Increasing pH results In a higher proportion of NIIJ-N to NII4+-N in water (Boyd, 1979; Spotte, 1979; Stickney, 1979), with anIncrease of one pH unitC3uslng the percentage ofNtl3-N to increase about tenfold (Spotte, 1979). Stickney (1979) presented the following example:

In {mhwater at 26°Cand1'117 ther« Islesst/.an J% NlIJ-N to over 99% NII4+- N. whereasat p1/8.S and 26°C, more than 15% 01 total NJl4-N Is present as NllrN.

Rising temperature and decreasing salinity result in muchsmaller Increases in NII3-N (Spotte, 1979). An example from Doyd (1979) serves to illustrate the effect of temperature on the NIIJ-N : NII4 +-N ratio:

At 1'/18 th« percentage NllrN Is /.2% at fJCcompar~d with .5.2% at 2SOC

At Identical temperature and pll values, and the same amount of total NH4- N in solution, seawater contains slightly less NIIJ-N than freshwater (Spotte, 1979).

Ammonia toxicity Is exacerbated by low dissolved oxygen levels (Spottc, 1979; Doyd. 1979) although the mechanism is obscure (Spotte. 1979). According to Stickney (1979) there is still little information available on the toxicity of ammonia to invertebrates of Interest to aquaculturists, Wickins and Beard (1978) reported that a NHJ-Hlevel of 0.45 mg/2 re­ duced the growth of penacid prawns inculture by 50% afterthree weeks of exposure. In discussing the occurrence of a "breakpoint" in the rearing ofM. rosenbergli post-larvae, Kneale and Wang t 1979) appear to consider levelsof total NII4-N below 0,42 mgJ2 as satisfactory. as this was not implicated intheoccurrence of thebreakpoint. Wickins and Beard (1978) listed 0,1 mg/2 NHJ-N as the maximum acceptable concentration in their culture systems containing M. roscllbergll

Nitrite. Nitrite toxicity is affected by both water pH (Armstrollg ct al., 1978) and salinity (Spotte, 1979). The unionlsed fonn in which nitrite occurs Is considered more toxic tlun the ionised form. and Increased levels of the un-lonised fonn occur at lower pII levels (Amlstrong tt al; 1976). Spottc (1979)considers it unlikely that nitrite poses a serious threat to aquarium animals maintained In brackish or seawater conditions. However. inan 46

"

lnvestlgatlon of the toxiceffects of nitrile levels on Itt. rosenbfrglll:uvne (reared in brackish water), Annstrong eta1., (1976) obtained results which showed that the 96 hr LeSO value for the larvae was 6-12 O1g N02-N/.2. In addition, sublethal effects on growth, in the form of growth retardation, occurred at levels aslow as 1,8 mg N02-N/2. Spottc (1979) reeom­ mendsan upper limit of 0,1 O1g N02-N/.2 foraquarium waters. Wickins and Beard (1978) chose 1 mgN02-N/.2 as the maximumacceptable limit for their culture systems. These authors reported three week LCSO values of IS O1g N02-N/2 for M. rosenberglt: Kneale and Wang (1979) reported levels of nitrite below 0,24 mg N02-N/.2, which appear to be satisfactory, for rearing.

Nitrate. Although Itisnot acutely toxic, even at relatively high concentrations, the lona term effectsof nltr:lte have not been determined (Spotte, 1979). Spotte (1979) reeom­ mended that the concentration of nitrate inaquaria should not be allowed to exceed 20 mg N03-N/2. Wickins and Beard(1977) reported a three week LCSO value of 160 rna N03-N/2 forM. rosenbergll and chose II maximum acceptable limlt of SO rng N03-N/2 for their culture systems.

Carbondioxide and precipitants. Free CO2 Is very soluble inwater (Boyd, 1979; Spollc, 1979) and the complex equilibrium reactlons of the various forms in which it occurs in water areextremelysensitive to pH and alkalinity of the water(Spottc, 1979). The composite re­ action shiftsto the right as pH increases:

CO2 +Jl2°*'//2coJ"'lr JlCoi"lr + COl· > Increasing pH

Thismay be represented figuratively asfollows (From Wetzcl, 1975): oN100_~-----=-----.,...., o w 80 Ii.o ... 60 ~ Z ..0 o i= ~ ~ "" .. 5 6 7 8 9 pH Figure / Relation between ,,11andtile relative proportlom ull/wrganlccarbon sl/celes 01C02 In sotutton. 47

TI,e pH at which C~ eoneentratlon decreases to analytically undetectable levcls. and at which C032. appears In measurable coneentrartons isat pll8,34(Boyd, 1979). In well buffcred waters the carbonic acid - bicarbonate equilibrium reaction is dominant, IS can be seen from the preponderance of bicarbonate ions wilhin the pll rangc 7,0-8,0 in Flgu,~ J.

In most fresh waters, calclum ion is associAted with bicarbonate and carbonate ion (Boyd, 1979). Photosynthesis causes a rise in the pll of water, by the removal ofC02' which can cause carbonate to prectpltate as calcium carbonate(Johnson, 1980). Accordina to Johnson (1980), if this occurs, the calcium carbonate formed may become attached to the larVllc, weighing them down and causing mortalilles.

TI,c dangerof C02 toxic:lty exists only Dt pllievels below 8,34 where free C02 Is present (Figure I.) TIle toxicity ofC02 Is most detrimental to nsh when the dissolved oxygen In the water Is low, as carbondioxide Interferes with respiration (Boyd, 1979). Toxicityof C02 to prawns Is DJlJlarently not considered u serious problem as no reference to toxic levels was encountered in literature surveyed. Boyd (1979) reported that fish will tolerate levels of 10 mg/i C02 In culture waler,lfthedissolved oxygen is high, lind that free C02 levels in Intensive Ilsh culture water typically Iluctuate between 0 - 10 mg/i with no ob­ vious ill effects on fish.

With respect tosuitable I'll values for aquarium waters, Spotte (1979) recommended pH values of7,1 -7,8 Iorfrcshwater, lind 8 - 8,3 for brackish and seawater aquaria. Boyd (1979) recommended pl] values of 6,5 - 9 llS suitable for fish culture.

2.9.4 Chemical treatment ofdiseases inclosed recirculation systems

Chemical compounds used for trentlng disease conditions mllY llffect the functioning of bloflltersby the Inhibition of nltrlflcatlon.Ieadlng to accumulation ofammonia and/or nitrite (Spotte, 1979). Spotte (1979) presented evidence from Investigations by other workers Into the effects ofchemlcnb onnitrification, at therapeutlc doses, which showed that erythromycin, chlortetracycline and methylene blue had Inhibiting effects on nltrin· calion in fn:shwater sy~tems. Resultsoblalned In these inv~ligatlons however, were In· conchnlvewilh reSllect to the erfects ofchlcramphenlcol and potassium pcrmanganate, 48

Bower and Turner (1982) investigated the effects ofseven chemotherapeutic agentson nitrification In closed seawater culture systems. and found that methylene blue, neomycin sulphate, chloramphenicol and cupric sulphate inhibited ammonla oxidation. and that neomycin sulphate and cupric sulphate Inhibited nitrite oxldatlon. TIle effects of cupric sulphate and methylene blue in this study showed inconsistencies, with discordant results being obtained In some of the replicates. Gentamycln, nlfurpyrlnoland quinacrine hydro­ chloride did not produce biologically slgnifkant effects on nitrification in seawater aquaria, at therapeutic doses. Results obtained by Dower and Turner (1982) for nlfurpyrinol were consistent with the results of work conducted in freshwater aquaria, and the usc ofchlo­ ramphenicol,cupric sulphate, methylene blue and neomycin sulphate was not recommended for seawatersystems.

2.10 SUMMARY

A review of the literature relevant to the establishment of hatchery facilities for the cul­ turing of Macrobraelllum species Is presented. From this review the following polntsare considered important:

• The culture systems 01 choice fortire development 01an Inlandhatchery, under temperate cllmattc condltlons, would Involve tire lise of aclosed water recircula­ tion system, where water purlflcatfon Is by means 01biofllters. In addltlon, tilt use ofarttflcla! sea saltslor tile preparation ofa bracklsll water larval CUItIl~ medium would make such a IIatcllery lndependant olllatural supplies 01seawater.

• In order tooptimise production In such systems, Mglr stocking densities, In a comf'etely controlled Indoor environment are necessary.

• Brecdlng, larval rearingandpost-larval nursery rearing ofMncrobrnchium spceles ar« possible throughout tire year, undersuitably controlled condltlons oftemperature, salllllly, water quality, pl,otoperlod and stocking dcnslly, provided ,lrata properly balanced diet Isled to th« prawns at tire different stagrs ofdevelopment. 49

CHAPTER 3

MATERIALS AND MEllIODS Page INFRAS'J'RUCI1JRE . 52 Glasshowe ...... 52 Water supply ...... 53 Temperature control 53 AlrsUPI)ly ...... 53 Photoperiod and Ughl Intensity ...... 53 Culture vessels ...... 55 INVESTIGATION INTO TIlE USE OFSOLAR ENERGY AS A SOURCEOF IIEAT FOR CULTUitE WATER ...... 55 ANALYSIS AND MANAGEMENT OF WATER QUALITY . 56 pH ...... 56 Conductivity ...... 51 Preparatlon ofartificial seawater and the measurementof SlIllnlty 51 Wotcr hnroncss os a factor In lorvnl rearing ...... 58 Totnl ammonia nitrogen (NII4-N) 58 p02 and pCOl ...... S9 LARVAL REARING FAC,ILlTIES ...... S9 Initial facilities ...... S9 Bloflltratlon systems 61 HOLDING FACILITIES FOR BREEDING STOCK 6S HOLDING FACILITIES FOR POST·LARVAE 69 COLLECTION AND MANAGEMENT OF ADULTPRAWNS FOR BREEDING PURPOSES 69 Collection and transportation of ndultprawns 69 Wntcr conditions ...... 10 Communal and observation tnnks 10 ~fllting ...... • 11 Incubatlon llnd hatching ...... 11 Nutrition ...... 12 PRELIMINARY STUDY OF LARVALDEVELOPMENTAL FORMS •••.•••.••• 12 Prcpnrntion of matcrl:l1 ...... n Identlflcatlonof14MI forms ...... 13 LARVAL REARING PROCEDURES ANO MANAGE\fENT PRACrICES ••.• 1S Temperature ...... 76 so

Page SaUnlty ...... 76 lIatching of larvae and transfer to renrlng containers 76 Salinity levels (or retiring brvac 76 Salinity levels (or metamorphosis to post·larvae 77 Stocking denslty 77 Nutrition 77 EIB eustanl 78 Artemill nauplll ...... 78 Mlcrowonns ...... 79 Fish nesh ...... 80 Uquifry ...... 80 Frequency of feedlng, quantity of food anllporticle sizes . 80 Management practices ...... 81 INVESTIGATION OFTilE SALINITYPREFERENCES orrue EARLY LAHVALFORMS ...... 82 Background ...... 82 ApllnratusIIIllI general procedures ...... 83 Design of Individual experiments ...... 86 Prcliminnry experiment with M. petersitlarvae 86 Experiment 1 Salinity preferences of the earlylarval Iorms of M. pctcrsit 86 Experiment 2 Salinity preferences of the later larval forms or M. petmit ...... 87 Salinity preferences of the larvae of coastal and Inland populations of M. lepidactylus 87 Experiment 3 : Salinity preferences of larvae from theLake Cubhu population of M. lepidactylus 87 Experiment 4 : Salinity preferences of larvae from theLimpopo river population ofM. /epldactY/lIs 87 Experiment 5 : Salinity preferences of the earlylarval Iorms of M. tud« 88 Experiment 6 : Salinity preferences u( the earlylarval forms of M. alls/rale 88 Experiment 7 : Salinity preferences of the carly I:lrvnl fomu o( M. tcabtlculum ...... 88 POST-LARVAL REARING 88 sa

PIlP Separation of tune and post·brvllO ...... 88 Slocking density ...... 89 Nutrition ...... 90 Mllnllgement ofwiller quality ...... 90 TREATMENT OF DISEASE ...... 91 Larne ...... 91 Adults ...... 91 SUMMARY ...... 92 52

ClimER THREE

MATERIALS AND METHODS

Johannesburg Is situated approximately 700 km inland from the ellSt coast of South Africa, at an ultitudeofapproximately 2 000 metres. The climate istemperate with wet summen and dry wlnters, and is not suitable for the outdoor cultivation ofwarmwater prawns for most of the summer season. As no prawn holding facilities were llvllilnble at the inltlalion of this project, the development of pilot-scale hatchery facHilles WIIS begun from ground level. It was thus necessary to provide the following Indoor fucillties:

• lIoldltll: laclllll~s for brut/llIgstock for extendal periods under laboratory con­ dtttom, as IlIdlgellous species ar~ not c/lslTlbut~d ncar enougll 10 Johannesburg to enable rcxulcIf collectton01adult specimens lor breeding purposes:

• RtilTltlg laclllticos for larvae, ",hlcll might require saUnt condlttons, at a ellstatlct 01approxlma/dy 700 km from thenearest suppUcs ofsca water.

• Facilities lor lireaccommodation 01post-larvae produced during tnvcstlzattons Into larval rearing and at a later stage, facilities lor tlte grow-out ofpost-larvae 10luvenlles lor lirestocking ofponds.

3.1 INFRASTRUCTURE

The physicalstructures and facilities used during the course of this projcct nrc listed under thissection.

Glasshouse. The project was conducted in a glasshouse of dimensions 30,4 x 7,6 m, equipped wilh the following facilities:

• Louvres Oil lire rooffor III~ rezulatlon ofsImllglll penettation

• Alrcondltlmllng equll'm"nl/or tire control 0/temperature Inside lire glQ.fJlrollst

• Single and J.pllQs~ electricity slIp"ly 1'0lnU

• Co",pressed airslll'I'I,· polnts allel a RcavellmodelR7IONFM mark 2 atrblowe« of26,9 ",J/mcapacity 53

• Municipal aswel!as ""d~rgr()""d watersupplies. the latter from Q borellole. • Flourescent IIglrtlng with photoperiod control

Wnter supply. Although the municipaltap water wasused for a period of time, It was re­ placed by borehole water for use In culture tanks. A permanent supply of borehole water wascompleted towards the end of theproject.

Temperaturecontrol. Although the glasshouse was equipped with an olrconditlonlng system for maintaining constant temperature, this was found to be Inadequate for Increasing the temperature ofculture water. As 11 result, culture water was heated by means of 100 W­ 300 Waquarium Immersion heaters. An investigation Into the use of solar energy forheating culture water was conducted during the course of the project and Is dealt with In section 3.2 of thischapter.

Airsupply. For the first 8 months of the project an aircomprcssor fittcd with lin oilflltra­

tlon system was used. However this airsupply proved to beInadequate IlS the project pro­ gressed and there was the risk of fluctuating pressures occurring whcn thc compressor was serviced. Sudden decreases in pressure resulted in poor or no performance of airlift pumps while increases caused the aquarium airsupply pipe fittings to tear loose.

Once the supply from the compressor had been replaced byan airblower, a more constant and increased volume of air became available. The supply ofair toculture tanks was by meansof a closed circuit of 20 mm polyethylene pipingwhich had supply points along its entire length. From the supply points standard sizes of plastic aquarium fittings completed the air supply system. Dispersion of theair foraeration and for airlift pumps was by means of aquarium airstcnes or short lengthsofporous aerator tubing ofdiameter 6 mm. TIle blower, witha maximum capacity of 26,9 m3/hr, wasadequate for operating nearly ISO airdiffuser outlets in 27 holding tanks, 12 single containerlarval rearing units, 3 multlcon­ tainer rearing units and two Artemla hatching units.

Photoperiod and light intcnslty. Control of photoperiod was initiated towards the end of the 7th month of the project in mldwlntcr of 1982. Daylength was set at 14,5 hrs light ami 9,S hrs darkness. This wasdiscontinued after five months, and no control was exercised during the summer months. _B

Fi urc ~ II InsulatcJ olar pIIIII 11 Four . olar panels and thrc(' 11I1,Wf" m/l IIH'd /1/ lola' " II" xpcrimcn! C Water 1"11"1' and insulated dcllvcr v pifll'l 55

Louvreson the roofof the glasshouse were kept closed so th:!t a minimum amount ofdirect sunlight fell onthelarval rearing containers. Measuretllight intensilies in the vicinity of the larval rearing containers ranged from2,000-2.800 lux.

Culture ~Is. Avariety of containers were used for the holding and rellring of prawns. These included thefollowing:

472 (46 x 32 x 32 em) sUfi rimmed aquarium tanks Jl52 (91 x 33 x 38 em} allglassaquarltlm tanlcJ 2502 (122 x 45 x 45 em) allgloss aquarium tanks 3682 (182x 45 x 45 em) allglass aquarium tallks 6/82 (305 x 45 x 45 em) allglass aquarium tank: 6502 (J27x /27 x 40 em) retnforced /lbrfglass tanks 102and 202 whtte !'Q!ytt"ylme buckets

Containers were usually filled to between 60 and 80% of their maximum capacity wllh water.

3.2 INVESTIGATION INTO TilE USE OF SOLAR ENERGY AS A SOURCE OF IIEAT FOR CULTURE WATER

TIle experimental design of this systemcomprised three plasllc portapools, each of 3 m diameter and 0,75 In depth. and maintained at :I volumeof approximately 5.000 t Two of the portapocls were insulated on the outside as well as on the water surface (Fig. 2A). The third pool W:lS not insulated. One of the two insulated portapools was equipped witha heat exchanger connected to solar panels (Fig. 28). lnsulation of the pools was by meansof bubble plastic sheets, normally used (orinsulating swimming pools. Initially :1 singlesheet or plastic was placed on the water surface with a double layer around the sides (Fig. 2A). At a later stage a double layer was placed on the surface of the water. during the winter months. In addition, the water pipes connecting the panels and the heat exchanger were also Insulated with bubble plastic (rig. 2C).

Vnn Leer solar p:lIlels were used In the Invesligatlon, and these measured 1.17 x 1,8 m. Initially 2 Il3ncls and later 4 panels were connected to the heat exchanger, Thls consisted of the stainless steel envelope removed from 11 solar panel and placed at an angle in the portapool. 111e p:tnels were mounted onasteel frame, lit n35°lingle, foclng approxlrnatety north, for maximum sunlight. Circulationof the water between the p:melsand heat 56

exchanger was by means of It small motor-driven water pump. A1,2 Kw electric motor was used to drive Ihe pump.

TI,e pump was switched on anti off manually and ran for 6 - 7 hn per tiny when suitable climatic conditions prevailed. Each portapool was provided with a maximum/minimum thermometer,and temperatures were taken on a 24 hourly bnsis. Air temperature was measured simultaneously. TIle investlgallon covered both summer anti winter climatic conditions.

3.3 ANALYSIS AND MANAGEMENT OF WATER QUALITY

On two occaslons during the course of the project, water s:lmples from holding and rearing containers, natural habitats of the prawns, and water supplies were sent to the NlItlolIlll Institute for Waler Research of the C.S.l.R., for n detailed onlllysis. In addition, certain water quality parameters were analysed at the University. Excepl where otherwise mentioned, methods followed In the analysis of samples were accordlng 10standard methods for the exnmlnnllon of WOller and waste water (APIIA, 1976).

3.3. t pll

This was determined using a T & C 500 portable pll meter. pl] was monitored dnily in larval rearing containers, but not in holding tanks, until the latter part of the project pll was only checked occasionally in breeding stock tanks during the first stage of the project, but later, once all breeding stock had been transferred to tanks equipped with undcrgrnvcl filtcn, a routine check was kept on pll, as this could be expected to decrease as a re- sult of nitrification processes. Duringinvestigations of salinilY preferences of the c3J'ly larval forms, pll was monitored on n daily basis in eachof the larval containers. Although usc was made of a Radiometer model DMS3 mark 2 at times during the latter experiments, recurring problems with the IIII probewere experienced and theT & C 500 was used for determining pll,directly in each container.

During the carly st:lgcs of the project, Ircshwatcr In holding facilities, where blofilterswere ill lise, was not buffered against fluctuations in I'll. Later, whe" pll had been found 10 be decreasing, crushed lea slll~lls were added to these tanks, 10 buffer the pll. TIle SCa shells were nol mixed In with the filter gravel, to avoid disturbing the litterbed, but were sprinkled on the$urfacc of the filter bcd. 57

3.3.2 Conductivity CPS/COl)

Conductivity was not routinely monitored, but was determined (orthe two sourcesof fresh­ water by the N.I.W.R. (Table 1J. In addition, the conductivity in t:mkscontaining adult and post-larval prawns was monitored when problems were experienced with a deellne in pH and n riseIn the conductivity. Once regular partlaI water changes were introduced this was discontinued.Conductivity was also lowered In tanks containing Indlvidu.lI M. rude females In an attempt at stimulating ovary development.

Conductivity was determlned with either 8 T & C 20001 conductivity meter or a TOA ElectronicsCM-2A meter,

3.3.3 Preparation of ortmelnl seawater and measurement ofsalinity

Artificial sea water was made up withTropic Marine sea StIlls, normally used for aquarium purposes. The StIlts were dissolved in freshwater lind kept under constant neration. Fresh­ water used was from the borehole supply for 1110st of the project, except for the first two 1110nth~, when municipal water was used. No attempt was made to keep the salinity of the sell water In the stock tank lit a given level, Instead, the salinity was determined withen

ATAGOsalt refractometer, before saline water W&lS to be nudeup, and the required propor­ tions of fresh and sea water, for a given volume of water, were calculated by meansofthe following formula:

Vol ofsea water required os l1{(w.../rrJJ..sallll.ilJ!. x volumex for volume x Sllll"ily ofseawater

Salinity was determined to within O,S S0/00• The refractometer was set on zero with distilled water during salinity preference studiesand withborehole water for most of the project. As the instrument was vcry hcat sensitive, it often had to be cooled down before setting in thesummer months.

During the first 3 monthsof the project, determlnatlonof S3linilles was by means of con­ ductivity. The conductivity of frc5hly made up artificial sea water WBS determined and then sallnlties wen: made UI' on :l proportlonnl basis, nssumin~ that the sea water was

33 S 0/00• Thus, for example, In a 3 S°/00 s,111nity mixture, the conductivity should be t ~ full strength $(3 water. The proportions were determlned using the abovementioned formula, with conductivity values substituted for s.,Unily values. S8

During this period replacement water was Introduced It the same ulinity as that of culture medium, but this resulted in rising salinity. Thereafter only freshwater WIS used to replace lossesdue to evaporation. \Vater of thesame salinity as theculture medium was added when there was lin accidental loss ofculture water.

3.3.4 Water hardness as A Iactor ill larval Jenrins

During the nrsttwo months of the project, only municipal tepwater was used in holding and rearing containers. However, Sick and Deaty (1974) found thut water of hardness SO m8/2, used to prepare a saline culture medium, caused 97% mortality of1tI. rosenbtrgfl larvae reared In themedium, after S - 10 days. The tapwater slipply for this stully has I hardness of 38 mgJ2 compared with 12 mg/e for the borehole water {Table JJ. Due to U's softer quality,the borehole supply was therefore used forthe rcmalnder of the project.

Routine monitoring of water hardness was necessary during thecourse of the projectas munlclpal and borehole supplies were linked, and the water was occaslonally mixed.Once the borehole and munlclpal supplies were separated, routine tests for hardness were dis­ continued. Determination of water hardness was by means ofstandard methods (APHA, 1976).

3.3.5 Totnl ammonia nitrogen (Nff4-N)

Two methodsof NII4-N determination were followed during the course of the project. In the earlystages standard techniques, using Nessler's reagent, were employed for the determination of total NII4-N (APHA, 1976). Although this technique was satisfactory for freshwater,lt was unsatisfactory for saline water, due to salt precipitation. During the latter stageof the project the phenol-hypochlorite method was used 35 this \V3S satisfactory for both fresh and brackish waters. The technique followcd was that of Spotte (1979), with the rcplacernent of sodium nltrofcrrlcyanlde by sodium nitroprusside. Analysisof samples began within I hr of collection. Collection of samples was by means of 3601111 plastic syringe, theInlet of which was covered with :I piece of cloth held In position bya short sectionof plastic tubing, pressed over It. TIle doth prevented larvae and/or Artrmta naupll] being removed with the sample. Aliquot!! of 401111 cadI were collected and trans­ ferretl to SO nil volumetric flasks, which were Immediately stoppered. S9

Routine monitoring of NJl4-N levels was not conducted over most of the project.However, during the thiN and fourth months of the project ammonia was routinely monitored In Illrval rearing contalners. During this Inlllni phase of Luval rearing the sulinitics were relative­ ly low (0 - 8S0/00) lind the detennlnntions were made according standard methods (APIIA, 1976).

During the course of investigations of SlIlini:y preferencesof larvae, total NII4-N was deter­ mined dally for larvnl containers, as well asin stock tanksof fresh and sea water, before the mixing of experimental saJlnlties. Asmentioned, the phenol·hypochlorite method was used for these determinations. Samples from holding tanks as well asrearing containers were included with these when time permitted,

3.3.6 p02 and pC02

These par:lmcters were only monitored during investigations of s.,llnity preferences ofthe larvae. Determlnatlons were made using II Radiometer model OMS3 mark 2. Samples were collected in I ml plastic syringes! modified In the same manner as for the s.1Inpling of NII4-N aliquots. As soon as each sample had been collected, the syringc was scaled wUh a plastic cap, which was supplied by the manufacturers.

3.4 LARVAL REARING FACILITIES

3.4.1 Initial faeiUlie!

These include facilities used during thefirst four months of theproject. During the prcpara­ tlon of holding facilities for adults, several batches of larvae were hatched and these werc held in 47 2 aquarium tanks equipped only with an aer-Ier and immersion heater. Later, 10 2 plastic buckets, also equipped only with an aerator and immersion heater, were used. No flltratlon ofwater took place and water quality wasmaintained by siphoning out detritus which collected on the bottom.

A rearing container comprising a built In mechanical liIterofaquarium filter wool and activated charcoal was then developed (Fig. 4A). This consisted of II bucket Into whleh a layer each of filter wool lind a bas ofactivated charcoal were placed. A second bucket, with half the bottom covered by a sieve,w3s placed Inside the rarst so that the sieve screenlay above the filter wool TIllsbucket Wl1S also equipped with an airlift, which drew water frorn o o M

.5 ~ ..; i! '- ... ::s ~ -to.;. \j .:;:.- s § u ~ ::s ~ ~ - to.;. lU \j ~ ~ ~ , s... ·5 ~ C'1 i:~ -e ...... 'r 'E $ \j '- .9- ]-'~- ...~ l::I~ :::4: ... 0 .:: -::s "'l .. ClC ~ .==-~ lU .5 ... ~ $ lU ,S- C) .:::.., -::s 61

above the charcoal Into the inner bucket, thus circulatlng water through the filter wool and charcoal. These fllter buckets were found to be unsatisfactory for larval rearing as problems with theconstruction wereexperlenced, and larvae tended to be pulled onto the filter woolasa result of sieve: screens tearing loose. In add ilion, Artemia were drawn through the screen Into the filter wool.

3.4.2 Dlofiltrnllon systems

During the fourth month of the project blofiltration systems were developed for therear­ lng of larvae. Separate filler units were employed for larval rearing to avoid the risk of larvae being drawn onto the filter, when moulting. Two basic designs were employed for most of the duration of the project.

The firsl design consisted of It 102 white plastic bucket into which gravel and an alrllrt pipe were placed (J.7gs. 3A and 41/). The gravel used was approximately 2 mm in diameter, lind the airlirt was made from 20 nun polyethylene piping. An elbow and foot piece were attached to the base of the lift pipe, 10 anchor the nirlirt in the gravel. A layer of coarser I em gravel covered the foot piece to prevent finer gravel particles from entering thelin pipe. Al the lop of the lift pipe n 20 mm nylon tee-piece was filled, to allow for 3 delivery pipe to be connected, 35 well 3S an entry for the airstone or porous tubing. Doth alrstones and short lengths ofplastic tubing were used in airlifts with satisfactory results.

The second design, originally intended for comparative mass rearing experiments, was a largerscalemulticonulncrsystem (Fig. 30), in which fibrcglass tanks ofdimensions

1,27 m x 1,27 III x 0,4 m were used ascontainers for the biofiltcrs. Gravel of I cm diameter provided the filter medium for the full depth of thc filter;airlift pipes wcre buried in the

gravel, and provided with 20 mm elbows as foot pieces.These airlifts were of the S3I11C de­

sign 3S for the previous filters described.The airlifts returned thewater from the filter tank to 5 or 6 rcnring containers arranged mound one sidc of thefilter tank(Fig. .38). Watcr re­ turning to the filter from the larval rearing contnincrs was collected on the far side of the filler, behind II barrier consisting of a row of bricks placed on thesurface of the filterbed (Fig. 30). In later modlflcatlons, where attempts were made 10 improve water circulation throuch the filter beds, lengths of 20 nun polyethylene pilling wen: attached to airlift foot pieces(Fig. 4CJ. These pipes extended the lengthof the filtcr tank, and were laid on the bottom of the tllnk. 62

FIGURE4 A-D LEGEND A Larval rcaring unit with n builtin mechanical fllter system.

J• Filterwool 2. Activatedcharcoal 3. Sievc plate 4. Alnton" S. Porous aerator tubing In airliftpipe 6. lind 7. Air supply pipes

n llloOller unit constructed in a 102 plastic bucket.

I• Coarse gravel layer 2. Fine gravd layer 3. Alrstone 4. Foot piece 5. Airlift pipe 6. Delivery pipe 7. Plastic tubing 8. Air supply pipe 9. W:ller level above gravel

C Section through a multlcontalner rearing unit.

I. Gravel filter medium 2. Extended foot piece 3. Airlift 4. Delivery pipe 5. Return pipe 6. Rearing container 7. Immersion heater 8. Ilcat exchange compartment 9. Heat exchange airlift 10. Orick barrier

D Section through a larval rearing container with outlet pipe in position.

I. Outlet pipe 2. Outlet screen 3. Porous aerator tubing 4. nyp~s.s outletscreen S. Air supply pipe 6. Return pipe 63

lot) -----

N

.-

N The distal ends were sealed lind two rows of holes,increasing in size with distance from the lift pipe, were cut into each pipe. Returning water W3S collected over heat exchange com­ partments constructed in the filter tanks with bricks (Figs. JB and lie). four immersion heaters, each of 300 W capacity, were placed in three exchange compartments. Airlifts, lifted water from the bottom of these compartments over thebrick barrier placed on the surface of thefilter bed. One of the larger scale filter units was modified during the testing ofa transistor controlled heating system by the Instrument Development Unitof the University. In this filter tank nomodifications were made toairlifts supplying larval con­ miners, but four airlifts with foot pieces nanningthe length of the filter tank were installed. These drew water through the niter bedand deposited it into 11 J0 2 plastic heat exchange box. A single 2 Kw heater wasinstalled in the box aswell as aeration for c1rcul3ting water around the heater in the box (Flg.3O). An overflow from the heat exchange boxemptied into n narrow brick divided compartment around the airlifts to the larval ccntnlners. Gravel used was washed and disinfected withformalin at 200 m2/2.

Containers coupled to these blofllters were modified 10 2and 20 2 white plastic buckets. The design of the containers wasthe same, except for thesizes of the fittings used, IS mm for the 102containers and 20 mm for the 20 2 containers, TI,eappropriate sized outlets werecutintothe plastic containers and fitted with modified nylon nipples and PVC coup­ lings.whlch allowed for the water return pipes to the biofiller to be connected and dis­ connected :It will (Fig. 40). Outlets were fitted with 75 mm PVC pipes with attached sieve screens(1'7g.4D). Aeration and water circulation inthelarval containers wasbymeans of 6 mm porous plastic tubing in the form of a ring. When first designed, these rings were made the same diameter as the bottom of thc larval container, nnd held in position with a ringof galvanised wire scaled in plastic aquarium tubing. Alater modification involved attaching n smaller ring of this porous tubing around the base oft!'eoutlet pipe (Fig. JA). TIlls gave an improved pattern of water circulation, and was used inall larval rearing containers.

Regulation of the flow of waterto the larval buckets was achieved in two ways. Coarse adjustment was made by clamping airsupply pipes to each airlift with plastic aquarium clamps. Finer adjustment wasachieved by roising or lowering the air diffuser inthe lift pipe itself. When the correct ndjustment was achieved nmodified piece of plastlc piping was Inserted adlongsidc the air supply pipe to keep this pipe l,\ position (Hg. 40). 6S

Airlifts not inuse at 3 particular time were disconnected from the larval container lind allowed to empty directly backontothe filter bed, thus ensuring continual clrculatlon of the water through the filter bed.

Flow rates from the filter bed to larval containers was between I lind 2 ~/min during the initial phase ofblofilter usage. TIlls was reduced laterto now rates bet ween 0,5 - I 2min (approximately SO tank overturns/day) in order to minimise the nushing ofA,Umla Into the filter bed.

3.5 1I0LDlNG FACILITIES FOR BREEDING STOCK

nreeding stock was held in rectangular aquarium tanks of various sizes (Fig. SA), In the early stnges of the project maintenance of water quality was by means ofeither mechanlcal or biological filters. Mechanical filters consisted of 5 2glass containers or small clay Ilower pots. A single airlift pipe was used to circulate water through a layer of filter wool and then over a bag of nctivatcd charcoal (Hg. 6A).

Biological fillers were constructed In some aquarium tanks by means of standardaquarium uudcrgravcl filtcr liltings, In other tanks, lengths of polythcne piping were laid along the bottom of thetanks, and connected to airlifts (Fig. 6/JJ. These pipes had the ends sealed, and holes of increasing size: with distance from the airlift pipe, were cut in two rows. A layer of 1-2 CIII gravel chips covered these pipes, anda layer of finer gravel (2 mm diameter) was placed over Ihis(Figs. 5801/(/68). The depth of the filter bed was not constant indif­ ferent tanks. Single nirlirts wereused in the 250 2 tanks, and double airlifts in the 368 2 tanks. The 3 mlongglass tanks were filled with 2 rows of 20 mm pipes on the bottom, each with airlift pipes connected along Iheir length (Fig. SM Airlift pipes were open ended, al­ though asecuring piece of plastic pipe was inserted to hold the alr supply pipe and aerator in position in the Jift pipe•.

Acratlonresulling from circulation through the airlift was considered adequate inmost tanks and additional aeration was only provided where stocking density was consldcred to be sufficiently high, for example IS adults/m2.

The mechanical filters were found 10 beinefficient. Although these acted as detritus lraps, they did not deloxify the water, Routlnc maintenance, involving the clcanlng of filter wool nnd charcoal, :15 well as clclJnil1g t:lI1ks 10 reduce nlgac growth, W:1S considered a distlnct dls­ ndv3nt3ge and the use of these filters was grnduallyeliminated. 66

FIGURE6 A-O LEGEND A Mechanical filtration unit usedIn breeding stock holding tanks.

I. Activated charcoal 2. Filler wool 3. Alrstone 4. Airlift pipe o Section through an aquarium tank fitted with an undergravel filler

I. Course gJ'llvellayer 2. Fine gravel layer 3. Airlift wilh two foot pieces C Arum/a hatching apparatus,

I. Hatching container 2. Temperature controlled Willer bath D Larval container used in the salinity preference studies.

I. Container 2. Cork seal 3. Plastlc funnel 4. Porous acntor tubing 67

...

3 Q 58

Fleur«5 A II 69 e ,

Short sections of 32 - SO mm PVC l>iJl(s were placed in holding tanks as shelters for the adults. Clay dishes were placed in holding tanks toward the end of project for the feed ins of commercial diets, Uneaten food inthese dishes could then be easily removed bysiphoning.

It was necessary to provide cover over tanks in which adults were held, espectally AI. rosen­ bergllildulls, whlch lumped out of tanks during the night if uncovered. Pieces ofshade cloth and other netting material were used for this purpose,

3.6 HOLDING FACILITIES FOR POST-LARVAE

Post-larvae were held In the same type of facility as the adults, with respect to filter design. The tanks were equipped with sheetsof shadeclothor anchovy netting which were hung ln the tnnks to Increase available surface area. Feeding dishes were used in the early stages of post-larval rearing insmall 47 ~ tanks. When post-larvae orjuveniles were transferred 10 larger containers, sheetsofglass, supported above the filler bed, served as feeding platforms on which food was deposited. Uneaten food was scraped to one side and siphoned out.

M. rosenbergi! post-larvae were divided up Into 3 fibreglass tanks, as used in the larval bio­ filter systems, which were equipped with umlergravel fillers. Course 1-2 em gravel was used. Airlifts with foot piece running the length of the tank below the gravel circulated thewater through the filter bed.

3.7 COLLECTION AND MANAGEMENT OF ADULT PIMWNS FOR DHEEDING PURPOSES

3.7.1 Collection and trnnsportatlon of adult pm\ms

Elcctrofishing techniques were used for thecollection of prawns from the Limpopo river system, Messina, Transvaal, TI,e appantusused consisted ofa small portable powergenerator, lin electroflshlng unit and two electrodes, TI,e apparatus was specially designed for the R. A.U. and made usc of bot It AC nnd DC current. A shnil3t electroflshlng system, belongin8 to the University ofZululnnd, was employed for the collection of prnwns in LakeCubhu, Northern ZululJnd. As MQcmbrod,llIIn prowns lire restricted to Ihe shore line (Forbes and Dlckerton, 1977), collection was confined to the lilloralarens.

Captured prawns were held at the siteof collection in aented containers. Prawns were then transferred to tou&h nylon bags. contnining water from the source of collection. These h;ll~" 70

"

were then filled with oxygen and se:lled. Thls tough nature of the bags llS well III the smalter sized rostra of the Indigenous prawns, made It unnecessary to break off rostral tips to pre­ vent puncturing ofthe bags. Adult prawns. especiallyberried females, wen: packed separate­ Iy at densities between I and 5 per b:lg. TIlls WllS determined by the sizes of the prawns.

It took approxlmately 7 hrs to-transport the prawns to the Univ~rslty from both s:unpllns locnlltles. On arrival the bags were first noat..d in the water surface of the hoMingtanks for approxlmately I hr In order to allow temperatures to equalise. Dags were then opened lind water from the holding tanks was mixed in smallportions with the water In thebas. after which the prawns were released.

Identification lind sepnratlon of the species Into their own specific tanks was based ondes­ criptions and keys In Barnard (1950), Kensley (1972), and Holthuis (1950). Exnmples of the species were sent for positive identification to Professor L.B. lIolthuis at the Natural History Museum, Lelden, Net hcrlands,

3.7.2 Water conditions

As previously mentioned adult prawns were maintained In freshwater. either from munici­ pal or borehole supplies. An exception was the holdingofM. nICJ~ stock at between3- 14 S 0/00 for II period of roughly 7 months. This species was originally identified as Macrobrachium equldens (Dana), n brackish water species. After six weeks in the laboratory no berried females had appeared and the salinity wastherefore raised in an attempt to stimulate reproduction.

3.7.3 Communal lind observntlon tanks

Adults of each species were malntalnedseparately in either communal or observationtanks. with the exception of the holding of M. peterstt and M. scabtkutum ndulr!i In the same tanks. Difnculty was experienced In scpnr:lling theadults of the two species, parflcularly the fe­ meles, Incommunal tanks. moles and females of a species were stocked according to the numbers nvallablc, so that the usual sex rnUos of I male to S female! suitable for M. rosen­ bC'f'glJ was notpossible. Inltinll)' adullswere housed 1n3lnly In observation mnks, but I:lter only In ccmmunal tanks. during the final phn~ of the larval rt3rlng programme. 7. .

TIlesmlllkrobsenatlon lanks were introduced alan early Slll£( o( the projeciln order 10 monitor reproduction of Ihe indisefl(ous species more closely. These lanks were Inilblly provided wilh m«hllnkal filh:n, whkh we,,: replaced bl(r by hHAnk under~l1vel mien.

3.7.4 Mall",

Malins or pfllwflsln communal tanks Will flO! manipulated. Tanks were checked dally (or

moult Cllsil and berried (enualel. Where possible, one nule Will stocked (or every rcnllie In

the observatlcn 11Inks. Where ahis WIIS not posslble, 1lI1IIes were Inlroduced 10 (em3les on the mornlnll or a moult c4I1 beln; observed. In lome cases, where mllies were held loselher In lep:unlo lankl, the female would be transferred 10the male 11Inke (or rnnlln, purposes.

3.7.5 Incuballon nnd hatchln.

nerrled remales In communal I..nks were removed nfler mallna 10 separate contalnen No

provision was made (or lncubatlon l'IOks AS such. The conllliners used were 011 her mrln, contalners or theobservation lanks prevlou~ly described. Obscrvatlon tanks were dlvkJed and used for housing 2 berried (emllies per lank for a period of lime. When these IlInks were converted (oraccommodating posl·larvae, III.: 10 2 bloflltcr syslem, orlgln311y intended (or 10 IVai rc:uin8, was used for both incubatlon and halchlng. Prawns held individually In obscrvatlon Innks were kepi in IheseI:lnks during the incubation period. TIley were only

removed for halchlng OI1<.'C the ellf,s become eyed.

Originally,re;lrins ecntslners were used (or halchlng and the berried females were lrans­ fcrred (rom(ruh 10 saline conditions, Once the halchln; h;ltl been completed, Ihe)' were ng:lln transferred 10 freshwater, Asre3rina SlIlinillc:s durlnlt thec:lfly phase of the projeci

were low Ihls presented no problem wllh respect 10 the transfer o( femAles. When rmlna s:allnlllcs were raised, the procedure was altered, "Ilhollsh r.ob:abl)' 1'101 detrimental to Ihe (CIl1:IICI, the h"lchlna of larvae In brackish water was dlsconttnued. II w:a, fell IlIlll Ihl' llppro:lI:h would also be: more sull:able (orlI;ah:hlnR the brv3e or the Intlisenolll specles. (or the 1"lfJ)OSC of the1.1llnll)' preference siudies. J)urina the bller rut o( the l\rojccl, f.'C~h· wlIh:r blomler.y.lellll were used (or Ihe h:lIchlnao( the Luv,1c. Eilher the 10 eblorillcr 1)'Slcm.or oneof the brl,tCr ,.::arilll unll. \u. used for lilt. r"tl'01C. Tr;1n~(c:r o( the 1.1fv:lC

(rom Ihe bller hlllchina cent..ln"rs 10 fClIrlll' c:onl.alnc.'I is dlscuned under I4rvlll rellrln. (Ch:lplcr 3 Kcllon 3.9.2.) 72

3.7.6 NutritJon

After the first balch of Indigenous prawns wascollected, a variety of foods were used including; trout pellets. gelatin diets and fresh food such as raw fish and mosquito l3fvac. However, feeding was standardised after four months when a commercial prawn diet from Meadow Feeds was fed daily at a rate of roughly I pellet per prawn. Various commercially formulated food pellets were used during the course of the project.

Feedingdishes were Introduced for the feeding of commercial diets so that food which was not eatencould be e:15ily removed by siphoning.

TIle prepared diet W:lS supplemented with finely chopped raw fish. usual Tilapta spp, , once or twiceper week.

Originally adults were fed once In the morning. However this was changed to an afternoon feed to accommodate the nocturnal feeding habits of theprawns,

3.8 PRELIMINARY STUDY OF LARVAL DEVELOPMENTAL FORMS

A prcllmlnary study of the morphological development of larvae of the indigenous species was madefor the following reasons:

• To beable to dtstlngulsh the larvae oftile IIlfjew;t species from each other. • Asan aid to assessing the morphological development and therefore growtll oj thelarvae, under 'he rearing conditions employed,

Material used for the study of morphological development was obtained from rearing containersand the moulting history was therefore not followed.

3.8.1 Preparation of rnalerilll

Originally a study of 13rvl11 morphologv using II sc.1nnillG electron microscope was planned. Larvae were fixed accenJing 10 procedures for osmium Ictro'ide studies, TIlis studywas not completedlind Insle3d it was decided 11I3t larvae would be phOlOU:lphed under magniliCJllon. The following procedure WIlS adOI)led: "

• Larva« wert collected and[lmm'cclill nilalcohol gl)'urille mixture (70 " JO "/.',) 73

e .

• Onc« sulflC'lent numbers "ad bun preserved, larvae were cleaned In Q 50 : 50vlv lactic acid: pheno! mlxture,

• Lan'ae lI'ere then placed 01/ microscope slides al/d dissected In a drop o/lacto- plltllol mixture. •A coverslip was then pressed down on tilt dlsstCted larvae.

• Photograp"s were taken using 01/ Olympus VallI/ox camera-microscope. • Larvae wer« I'Jwtograp"ed on tile same day lnorder toavoid tire Iormatton 0/ crystals 11/ tile matertal wt!II time.

3.8.2 Identification of brvnl fonns

Knowlton (1974) has found that larval morphogenesis of P. l'u/garls closely paralleled growth but not moultlng history, and that there was a cessation of morphogeuesls with cessntlon of growth. TIllis evidence of morphological change was taken as an indication that the larvae were growing. For the purpose of this study II was necessary to estl~bllsh whether development was taking place alllong the larvae being reared. As specimens were taken from communal rearing containers, no moulting history was available and it was necessary to lise changes in morphologyasa yardstick of development. A comparative survey of the morphological development of M. rosenbergll (Ling, 1962, 1969a, Uno and Kwon, 1969),M. acauthurus (Choudhury, 1910a). Macrobruchlum carcinus (Choudhury, 1911a), M. idella (Pilla! and Mohamed) and M. petersi! (Read, 1982) was under- taken. From results obtained during the present survey,a set of morphological features were selected, which were considered suitable for usc in following the progress of larval development.

There isagreement on the appearance and development of certain features, in the species listed, for the first (our morphological forms. Thereafter descriptions of the morphology of the laterforms differed until the last larval form was reached.Here there was agreement among thevarious authors, whether this last form wasdescribed asstage VIII or stnge XII.

In order to follow thedevelopment of theflvc speciesselected for this study, it was con­ sldered necewry todivide the process ofdevelopment into /I v.ril! of seven morphologlcal fonns based on the1I1)pear:II1CC and devclonment ofselected features, TIll: first (our Imlll fonns in the grid correspond closely to thenrst four brYn! st:lgcs described for Macfl1b,.,· chlum larvac In theliterature referred to. (Ung. 1962, 1969:1; Uno and Kwon, 1969; 74

Choudhury In03, 1971n; Pillal and Moh:uned, 1973; Read. 1982.) Morphologk31characters developing during later larval development have been grouped into three larval formL

During development, the larvae undergo the following changes In selected morphological characters:

Fonn I byes Sessile Peretopod: land 2 present ns blr3lnoUI butts Tehan rusett with abdominal sesment 6

Form II byes Stlliked Carapace Appcnmncc of spines oncarapace Perlopods land 2 complete, appearance of additional perelopod buds

Fomllll

Carapace AI'pcarance of dorsal (eplgnstrlc) spines or teeth anteriorly Antenna Al,pcarnnce of scgmentatlon Perelopods Unequal degree of development, usually numbers 3 and S arc more

developed than 4, which may be present 3S a bud TeLson Artlculation with segment 6 present Uropod Appearnnce of biramous uropod, the exopod being setose, the endopod bare

FonnlV

Perelopods 3 and S most developed, 4 stili present as a bud Teboll Posterior margin narrewer, npl,roxlrn:ucly twlee the width of the anterlor mllf&ln Uml'od E:copods lind endopods setose

Form V

Carapouoml Teeth mil)' nppc:u domlly on resuum rostrum 7S

Peretopods All developed and, except for S which remains uniramous, consisting of exopod and endopod Telson Lateral margins parallel, shape rectangufer Antenna Flagellum and scale approximately the same length

Fonn VI

Carapace and Appenranceofsetae associated with rostrnlalld/or epigastric teeth or rostrum spines, as well as serra lions or small teethon the margins of the teeth In some species Peretopod« Commencement of rudimentary chel:1 development on perelopods land 2 Pleopods Appearance of unlramous buds Inltllllly lind biramous plcopods later. The exopods of the latter may show setauon on 2, 3 or 4. Telson Shape begins to taper posteriously

Fonn VII

The degree ofmorphological development at this stageofdevelopment Is similar in the species surveyed and the characters selected approach functional development.

Carapace and Associated setae and teeth have increased In numbers rostrum Peretopods Chelae developed on I and 2 Pleopods All plcopods with cxopods and cndopods, All exopods and most endopods wilh setae, paddlclikc, Appendices lntcrna on most cndopods, Tc/soll. Long, very narrow posteriorly, setae reduced In number Uropod Elongated, narrower, many setae present

3.9 LARVAL REARING PROCEDUUES AND MANAGEMENT PRACfJCES

TIle development of!:lIval re:Jring procedures and management Pr:Jctlccs for rearing the larvne maybeconvculcntly divided intoIwo phases. The nrst phase lncorpornted earlier ntrcmpts nt larval rC;trins. during the development of holding fllcilltl~ for adults, and 76

'i rearing facilities for the larvae of the selected indigenous /tIQcrobrachlum specieL This phase terminated wllh the successful test rearingof M. rosellbtfglllarvae to the post·larval stage In the blofilter systems which well: eventuallydeveloped, IS well as the productlon of I small number ofM. rude post-larvae, The second phase oftheItudy comprised therearlns of the Inrvae of the Indigenous species In the biofiltratlon units, as well as the Invesligatlons into the sallnlly preferences of the early lunal forms of these species. During this phase, material fora prellmlnary comparative morphological study of the larvai forms WI'S collected and preserved.

3.9.1 TempernhlfC

Water temperatures were controlled In all larval rearing units at atemperature range suitable for tho rearing ofM. rosmbcrg/llarvae. Although 2SoC was thelevel chosen, in praetlee temperaturesranged from 25 - 300e. • Towards the end ofthe second phase of the larval rearing programme, the temperature In one of the longer blofiltration units was adjusted to fluctuate around 250C.Marrobraclll/lm lepldactylus and M. rude larvae were being reared in thisunit at the time. The survival of the larvae of these two species to post-larval stage was low, and thus the temperature was lowered to ascertain whether the higher temperature range WM responsible for the poor survival rate. 3.9.2 Salinity

IIntchlng of larvae and transfer to rearing containers. Most batches of larvae were hatched under. freshwater conditions, although several batches were hatched under saline conditions. During the first phase of the rcarlng programme, brvae were hatched in freshwater and the S3linity was gr:\(Ju3I1y increased In the rearing container over a period of several days. later this procedure was discontinued lind the larvae were hatched only in freshwater and then transferred to II blcflltration unit in half the volume of water of the h:ttching container. The airlift pump was then set at a low delivery rate and the bucket nllowcd 10 fill UI' gradually with saline water over II period of 20-30 min. When necessary, the S3l1nity in tho blofilter system was adjusted to 1I11ow for the addition or freshwater to the system.

Salinity levels for!torin, l:uvac. During the nnt phaseof the l:t/v31 re;trinB programme, the S3linlty in the rearing ccntalners seldom exceeded 8 S °/00, with the exception ofM rosen­ bcrr// larvne, for which the prerencd So1linlty levelswere known, and M. rude larvae which 77

were Identified lIS AI. equtdens at the time. These two species were reared at a sallnlly of at least lOS 0/00• In the second phase ofthe !larval feuing programme salinity levels were acJjusted on the bilsls of results obtained In the salinity preference investigntions. InaU cases the sallnlly levels for the rearing of larvae were increased above 8 S 0/0 0• and usually ranged from 10-17S 0/0 0•

Salinity levels for metamorphesls to post·lafVnc. Sllllnily was not reduced with th"approach of metamorphosis, except when attempting to induce metlllnorphosis, where thisappeared to be delayed.

3.9.3 Stocking density

During the Initial phase when the survival time of larvae was short, no reduction In numbers of larvae WIISnCCCSs.1ry. Later, with Improved survival of larvae II was considered necesS:lry to subdivide large batches into more than one rcarlng container. The hatching of a single female was normally accommodated In B single 10 or 20erearing container. The resulting stockingdensities ranged from SO - 500 larvae/2 initially, but more commonly these ranged around 100/2. Usually no further reductions were made, and In fact individual batches of the same species were often combined when numbers were seen to decline. Small batches , of larvae were also combined after hatching Into single rearing containers. No specific in­ vcstigation ofsuitable stocking densities was conducted during the present study.

Estimating of the numberof larvae In any container was done by means of subsampling. For this purpose the aeration to a larval container was increased to ensure even distribution of I.arvne throughout the container. TIle numberoflarvae in cad' of either S or 10 100 101 samples was then counted and the number perunit volume was established by averaging,

3.9.4 Nutrition

Variousdiets were fed to the larvae during the initial phase of the proiect. These included Iiquifry (0 commercial preparation for nCJunrium fish fry), gratled boiled fish Ilesh, micro­ wonns (rQl/ag~lIus sp.J,nn tItS custard mixture anti Arumla nauplil. The latter twofeeds became thestaplc diet for huval rearing for most of the project. 78

Ess Custard. The e~ custard mixture consisted of two heaped teaspoons of 5kimmed milk powder mixed with one eJU;. This was well beaten to remove lumps and then steamed In a closed pot until firm. Eggcustard was refrigerated when not In use. TIle egg custard WIS forced through nylon meshes of varying sizes to produce a range of particles suitable for varyingsizes of larvae. Meshes employed were 26,14 and 12 per centimetre.

Artemlll nauplll, During the secondmonth of the project ArUmla nauplll were Introduced forthe feeding oflarvae and these became a regulardully feed for the duration of the project.

When nrst Introduced, the cysts were hatched in diluted artificial sea water, of salinities between 8-17 S0/0 0 , In one litre conlcal glnss containers. 11\1: contalners were placed In a water bethconverted from a 47 2 aquarium tank lind provided with aerations lind Immersion heaters, During this period tap water was used for the preparation of saline mixtures.

Later, when the hatching units had to be increased Inscale, larcer2S0 2 aquarium tanks were used liS wnter baths and S 2 detergentbottles were used liS hatching containers. These bottles, with their bases removed were placed In an inverted position ina polystyrene foam frame­ work noating on the water surface of the water bath (Fig. 6C, page 66). Temperatures were maintained between 2S and 300C. Moderate aeration was supplied to ench hatching contalner,

Either taporborehole water was used with equal success. Originally the cysts lind salt were added simultaneously to the water inthe containers. Later theprocedure W:lS varied and the.waterwas first aerated with the required amount of cysts ill suspension. After20-30 min the appropriate quantity ofsnit was added, Coarse salt proved IlS effective as the more expensive nrrificial sea salts. One to two heaped tablespoons ofcoarse salt (8-16 s) and one level tablespoon ofcysts (:t 8 g) was added to each S2 container. This gave salinities of between 8 lind 16 S % 0 • In the final phase of the larval n::Iring programme thesalinity was kept at ±7 S0/0 0 for h:ltchillg, with satlsf:lctory results.

Nauplii were originally harvested after 24 houn of Incubation. Unhatched cysts were chen returned for another 24 hrs'of Incubation. A partlal water change W3S conducted before the unhatched cysts were returned for Incubntion. Later, n:luplil were incubated ror 48hn before harvesllns. Unhatched cystslit that St3~ weredlscanlcd liS very low numbers of 79

nauplli were obtained after 72 hn oflncubatlon, NlIuplll were separated from hatched cysts by removing the hatching containers and allowing the nauplii tosettle to the bottom of the conical container from where they could then be siphoned off. Once nauplil had been harvested from all the hatching containers they were poured through a piece of linen In order to concentrate them in 8 small volume of water before being used for feeding. Ibtchln. containers were properly cleaned before usc.

The above procedure (or separating nlluplll from cystswas not entirely satisfactory as un­ hatched cysts were genernlly siphoned offwith the nauplll, Hutchins tanks with bulll·ln light traps were constructed and tested during the course of the project but results were unsatisfactory lind the procedure was considered timeconsumllllJ.

Decapsulation of Cyst5 prior to hatching was also Investigated, but the procedure was con­ sidered to be time consuming and o( lillie practical usc. erpeclally at a time when batches ofArtemia cysts were prepared for halchlng In the morning aswell as in the afternoon, In order to have both 24 hr and 48 hr Incubated batches available (or the afternoon feed.

Mlcroworms. Allhough microworms were fed to larvae onoccasions. they were notincluded in the regular dle], Stock cultures were maintained throughout the project with the lnten­ tion of using this diet in larval nutrition studies to follow at 3 later stage.

Oatmeal was used to culture the microworrns using the following procedure:

• Two hundred m! 01oatmeal by volume Is placed In aplastic soup dis" andQn equal volume 0/boiling water added to tlll1.

, • Tne mixture Is allowed to cool andsoIMI/y.

•A quarter 0/a teaspoon 0/tlrltel actlvuted yeast Is thm sprlllklcci Oil th« sllr/ace 01tire oats.

• TI,t culture was I,,,,oelliauelwltl, worms.from Q stock (flIIIiTC. ",1,leI, "ad bUll \\IQJ/red 0",1 litell concentrated tn 15-25",1o/watt"

• Dis/Iu wer« covered wit" acircular ptec« 0/plastic to kup III lite moisture QJ,d floofed 011 culture tanks In apolystyren« foam fram«, to maintain a tr"'ptTlltIlI't %bout 2j°C. 80

Wonns may be harvested either by scraping them off thesides of the dish or by rinsing them off the surface with water, if the oatmeal is finn enough. Harvested worms were washed in 11 1 2 conical container by allowing the worms to settle,after which the cloudy water was siphoned off. This procedure was repeated to remove Ilne particles of culture-medium. A given volume of sedlmented wormscould then be fed.

Fish flesh. Flesh from Tllapla spp. was generally used forfeeding larvae. but this was not included in the routine diet employed during later renring attempts. Flesh was immersed in hollins water unlil it turned while. It was then graded through the same range of meshes as for the egg custard mixture.

Liquifry. Originally this W:1S fed to larvae on :1 few occesions but was considered unsati,. factory (Schoonbee, pus. comm.).

Frequency of feeding, quantity of feed and particle sizes. Larvae were fed 3-4 times per day throughout the developmental period. Once the egg custard lind Anemia were intro­ duccd as thestaple diet. the cgg custard was fed two to three times during the day lind Artemla once in the lute afternoon, as nn overnight feed.

Although nauplil which were hatched after 24 hrs were fed to earty larval forms formuch of the rearing programme, this became hnpractlcal later and larvae of all sizes were fed on nauplil harvested aftcr 48 hrs of incubation. The range of nauplii sizes after 48 Ius was not considered too large for the newly hatched Macrobrachium larvae investigated here.

Egg.custard and fish Ilesh were graded to appropriate sizes by being passed through the re- quircd mesh slze twice. Newly hatched larvae were fed on 26 meshes/em particle size. As the larvae of the different species varied insize at the same level of morphological develop­ ment. particle sizes were adjusted at different limes for respective species. Graded egg custard W:lS shaken innclosed container with nsmall volume of water. to loosen and separate :I~greS3ti()IlS of panicles, before feeding. Alacmbracl,llIm fUICl/bugll. M. petersi! andM. austra!« Iarvae were fed 14mcsh/cm graded p:arlicks until forms lv ancl Vwere reached nllll 12 meshes! em for forms VI and VII. Mac:robraclli/im rude, M. Itpidactyills and /tI. scabrtculum larvnc were fed on 26 mesh/em graded partlctcs for 11\0st of the larvae period, with 14 mesh/ern graded pnrticles being fed only to the sixth and seventh larval Iorms, 81

Two considerations were taken into account in determlning the qUlIntity of rood to be fed:

• Encounter rate. Where water ctrculatlon ensures suspmston ofparticleso//ood or Artemln naupllt, tire rat« at which th« larvae collide wit" food particles become3 an Important factor In /udlng.

• Continuous fcedlt,g habit o/Ianoae - a continuous pmence offood Is ,,~ctssary.

Difficulties were experienced in maintaining n balancebetween these two consldcratlons, without causing a build up of food material on the bottomof the rearing containers.It was therefore decided to reduce the frequency of feeding while malntulnlng n high enough density of particles or nauplll at each feeding. For the feeding ofearly larval forms,a half a teaspoonofconcentrated egg custard particlesper feeding was considered sufficient for a single 102 rearing container while 1-2 teaspoons of concentrate was provided In 20 2 containers, depending on the larval density In the container. For the larger larval forms. sufficient particles were fed to ensure that each larva had grasped a food particle. Asmall quantity of additional egg custard was then added.

Originally. Artemla nauplii were exclusively fed to larvae during the first two week 1ofde­ velopment but this was later changed to once per day. throughout the rearing period. Originally rates of 5-10 nauplii/ml were fed to larvae, Towards the end of the first phase of the larval rearing programme. the initial density ofArtemla provided was increased to 20-30 nauplii/ml and the water circulation through the rearing container turned off for 20-30 min. This allowed feeding at a higher encounter rate. Airlifts were engaged again. During this period delivery rates from the airlifts ranged between 1-2 2/min and this re­ sultcd in a decrease in the Artemla density in the rearing container. Circulation of the nnuplii through the filter medium and back to the: rearing container was possible. but losses ofArtemla also occurred in the filterunit. The deliveryrate from the airlifts was therefore decreased to between O,S and I 2/min during the second phase of the larval rearingpro­ gramme, in an attempt at malntalnlng a higher density of n:lllplilin the rearing containers. Densitiesof between 5-10 nauplii/ml after a 12 hr overnlght feed were considered 53tis­ factory for fcedlng.

3.9.5 Manngement practlces

Throughout the larval rcarlng programme Itwas nCCCS$3ry to ensure th,ll excess OrFJnlc matter was removed from rearing containers to prevent pollution of the water. In ;IIIJllion 82

the screens on the busesof outlet pipes tended to block after prepared food WIIS fed. Outlet screens were cleaned by siphoning adhering particles through the screen and by removlna and washing outlet pipes when necessary, Excess food material was siphoned Intoacontainer so that any larvae which were accidentally removed could bereturned to the rearIns con­ talner,

The sides of rearing containers developed a growth of organic matter with time, and al­ though not detrimental to the larvae, the containers were cleaned when this Impllired visibi­

lity of larvae in the container. The volume In the container WIlS decreased by siphoning water from the outlet pipe and the outlet pipe was then removed, cleaned, and replaced In II clean container. The larvae were then transferred to the clean container which wns then replaced In position next to the fllter,

Shading, in the form of sheets of plastic or pieces of shndecloth, was originally placed over the rearing containers, to reduce light penetration. However, this was only employed at a later stage.Jfdirect sunlight fell on a renring container during the day,

As previously mentioned, the biofiltratlon systems were not buffered against I'll fluctuations where saline conditions prevailed. Accidental lossesof water from the system did occur lind in these cases water was replaced by water of the samesalinity.

3.10 INVESTIGATION OF TlJESALlNITY PREfERENCES OFTIJE EARLY LARVAL FORMS

3.10.1 Background

During the initial period of this project attempts at re:tr:ilC larvae were unsuccessful due to inadequate lnformatlon on the natural distribution of thespecies Involved, with respect to fresh and brackish water. Salinities used In rearinc attempts were either too low (± SS0/00) or marginal (1: 8 S0/0 0 ). Later n series ofexperiments were conducted to establish the sollinity requirements for survival and morphologlcal development of the e.trly larval forms (I~1I1) at a temperature suitable for reJring of M. rosell/mgll larvae. This approach of maint:tlnIIlS:t constant temperature W:lS adopted as n comparative rearing trial was envisag· ed, including M. rnscllbrrglL The aim ofthese cxpcrlmcnts was toestablish a slmtil1B point for the rearing of thc larvae, 83

TIle basis for the experiments was the hypothesis of Knowlton (1974) that 4 hierarchy of developmental processes existed, based on the: utillsatlon of food energy (Chapter2, section 2.7.2). The design of the experiments w..s based on the experiments conducted by Choudhury (1970b, 1971b) on M. care/nils and M. acanthurus larvae. Choudhury fint investigated the effect ofsalinity onthe survival of form I larvae of the Iatter species. If. according to the hypothesis of Knowlton. food energy Is utilised for matntenance activities at the expense ofmoulting processes, then form Llarvae, surviving on the contents of the yolk SlIC.would be unable to moult toform II in unfavourable salinities. as nil the food energy from the yolk would be channelled into survival. Larvae able to moult. but not sup­ plied with food would survive vnrying amounts of time,accordlng to the sultabllltyof the experimental snlinlty. possibly moulting to form III under otplmum conditions. Choudhury (I 970b. 1971b) then investigated thcsuilabillty of'varlous dlets. For the purposesofthis study. Artemia nauplii were considered satisfactory. being an important component ofany diet (Chapter 2.section 2.7.2). TIle third stnge in Choudhury's investigations involved the rearing of Imneatvarious salinities fed the appropriate food.

3.10.2 Apparatus and general procedures

As the experiments were conducted during the summerbreeding season in the glasshouse. no control ofdaylength was cxccrclscd, III order to prevent direct sunligh t in the afternoon from raising the temperature in the water buthJblack plastic sheeting was hung behind the apparatus used.

Experiments were conducted in a temperature controlled water bath which consisted of a glassaquarium tank measuring 182 x 4S x 45 em equipped wilh a 300 Waquarium heater (Fig. JC, page 66). Acratlon was provided in the vicinity of the heater to circulate the water and thus create uniform watertemperature conditions throughout the tank. The heater was set to range around 28°C.

Containersfor the laMlc were prepared from 7S0 mlgreen glass bottles from which the bases had been cut, TIle necks were scaled with new corks so that the bottles could be inverted forusc in the temperature bath(Fig. 6D, pa.flC 68). Green gl:1s~ containers were chosen IlS this colour is thought to nld larvae in their orlentatlcn toward food (Read, 1981).

TIIC contalners were held in posttlon bya frame of polystyrene foam containing 24 hlll"s for the bottles and noatlngon the water surface of the bath. rlasllc funnels of 7 em 84

diameter were positioned over the openings of the containers In order to minimise evapo­ ration. Aeration was provided to each container through the opening ofeach funnel by means of arigid piece of plastic polyethylene piping, to which ashort porous aerator tube was attached (Fig. 6D page 68). Initially the airstream was controlled by meansofplastic aquarium clamps. Later plastic aquarium valves were used as a finer control of the airstream could beobtained with these.

Two 47 2 aquarium tanks were used as stock tanks for freshwater and artificial se:l water. These were each equipped with 100 Waquarium heaters and were constantly aerated. Sea water was prepared according to the methods described under section 3.3.3 of this chapter, The freshwater t:lnk contnincd borehole water,aerated for at least 24 hrs.

Allotted sallnlties were randomly assigned to containersIn numbered positions in the frame of polysterene foam. The general expcrimcntnl design consisted of four levels of salinity, each in turn comprising six replicates.

Larvae used In each experiment 'yere all hatched from the same female, in hatchingcontainers describedin(Chapter 3. section 3.7.5). The following apparatus was set up for the accllmltl­ sation of larvae toallotted salinitles, at the start ofeach experiment. and for the cxamlna­ tion of larvae during the experiments:

•A 202 plastic bucket wos filled with water. This wos equipped wtth 0100 W aquarium heater to maintain atemperature appro:dmately tlte same as the hatching tank and water bath temperatures. ,III enamelled metal basin/looting on the water surface served as a containerlor accllmltLflllg larvae 10 sal/lie con­ tlitlon: as wellas for purposes ofexaminatton. The apparatus reduced tempera­ ture /1uctuatlolls during transfer and examIllation.

Once hatching was completed, the female was removed:and tho volume In the bucket re­ duced to about 2 2. Larvae were thentransferred to the enamelled metal basin described above. Larvae to be held in freshwater were first counted out Into the containers used, and then transferred to the water bath. A white plastic teaspoon was used for the handling of lavrae. TIlismethod of handling resulted in :I mun;!nal Increas~ of the water volume In the bottles after transfer, Larvae were stocked lit 30/500 011 of culture medium in mostof Ihe experfments, Afler tho transfer of the larvae to freshwater, an aerator W:lS placed in the basin and sea water was allowed to drip Into the water.Jn the vicinity of the airstouc, The 8S

s:llinlty was checked at regular Intervals until the lowest of the allotted salinities was reached. Addition of ICOl waterwas stopped, the aerator removed.and the required numberoflarvae were thencounted out into bottles containing 500 mlofthe allotted salinity. Sell water was agaln added and the procedure repented until the larvae at all the salinities had been trans­ ferred to the water bath. The process ofeccllmltlsatlon usually extended over a period of 2-3 Ius.

Daily examlnatlon of larvae was conducted using the same metal basin, once waterquality parametershad been monitored. Larvae were transferred to the metal basin by pouring the water directly out of the container Into the basin. Each container was rinsed to ensure that all the larvae were transferred to thebasin. Dead and dying larvae were counted and removed and morphological development was checked.

The criterion selected for survival was the ability of the larvae to perform whole body movements, as opposed to movements of the heart, gills orpcrelopods alone. Where this was uncertain larvae were drawn up into a pipette, which was then rotated to stimulate movementof the larvae, Larvae able to right themselves In thepipette were countedamong the survivors and returned to the basin,

Survivors were checked for morphological developmentunder adissecting microscope, and then counted and returned to the containers, in which freshly mixed culture medium had been placed. Experiments were conducted for a period of6-8 days, which allowed for the appearanceof form III larvae.

At each salinity level, 3 out of the 6 replicates were provided with freshly hatched Artemla nauplil, while food was withheld from the other3 replicates. The provision of Artemla nauplii was initiated on the second day of the experiment, at a rate ofnpproxl· mately 5 n:luplii/ml. Although the form I larvae do not feed on the Artemla (Chapter 2, section 2.7.2), references to fed and starved larvae will imply the provision ofArremla or the withholding ofAnemia: Artemia nauplli were provided daily to allotted containers, after cxamlnatlon am) transfer of larv:lc to a fresh culture medium. The provision of Artemla nauplll tocontainers at eachsalinity level W3S by mC311S of randomlsatlon.

Water quality parameters were monitored on a dnily b:lsis. Temperature was usunlly monl­ tored twicc per day, In the morning and In the afternoon,Temperature was read In only 86

one container asno variation in temperature was observed between individual contalnen,at

:1 given time. Temperatures were determined to within O,50C.

The S3Unity of seawater in the stock tank waschecked before the mixing of culture media and the correct volumes required forallotted salinities were calculated. Salinity of the medium in each container was monitored before transfer of the larvae to the metal basin forobser­ vation.

Total NII4-N was monitored in the freshwater and seawater stock tanks before theculture media were mixed, samples being taken in the morning. TIle total NII4-N in eachofthe larval containers was monitored daily, with samples being taken in the morning.

3.10.3 Design ofIndividunl experiments

Preliminary experiment with M. petersll1:Jrvnc

A prclimlnary experiment using the apparatus and procedures described above was conducted' using M. fJt!lerslllarvae. Although theidentifications of the female, from which the larvae were hatched, was uncertain at the time of the experiment Ihis was later confirmed as M. petersu.

The experiment was divided into twostages, The first was an investigation of the salinity requirementsof the early larval forms, and the second, of the salinity requirements orthe later larval forms.

Experiment 1:Salinity preferences of the early larval forms of M. petenit, Salinities of

0, 4, 8 and 12 S % 0 were chosen forthe experiment wlth the early larval forms. larvae were stocked at 20/500 ml of medium. The duration of the experiment was 8 days. During the course of the experiment problems wen: experienced wilh the adlustrnent of the 2 x 100 Whealers Installed in the water bath, and these were replaced by n new 300W heater on thepenultimate dAY of the experiment, In addition, problems were experienced with the salinometer, which had to be repaired during thecourse of the experiment.as well as the Radiometer, on which pl], p02 and "C02 were to be measured. As mentioned, control of aeration to the containers W:lS by means of plastic aquarium plpc clamps and WAS also problemnlc, Due to technical problems encountered, the use of the phenol­ hypochlorite method for NI14- N determination wasonly Introduced 011 the last dayof the experiment. 87

Salinity was not monitored on days6 Dnd 7 as the salinometer was being repaired.

Experiment 2 : AI. peterst!later larval (onns. Form 5and 6 larvae, obtained (rom Ihe same hatchingasthe firsl stage of the experiment, but reared sepllf'lliely to forms Sand 6,were used in this experiment.

Based on resulls obtained in the Orst slage of the experiment, snllnities of 8, 12, 16 and 20 S 0/0 0 were chosen. Larvae were again stocked at 20/500 ml and were fed on Artemla nauplll once per day. The duration of the experiment was again 8 dais. Temperature, salinity, pll, 1'02' peo2 and NII4-N were monitored asdeserlbed in section 3.10.2.

Salinity preferences of the larvae o( coastal lind inland IJOpulllllon ofM. IcpldactY/IIJ

TI1C salinity preferences ofAt. lepldaaylus obtained from Lake Cubhu, Empangenl, Zululand and M. lepldactylus obtained from the Limpopo river, near Messinn In the Northern Transvaal, were invcstlgated in Iwo separate experiments, TIle reason for invesli· gating bothpopulations was because Ihe population in the Limpopo occurred at :1distance of several hundred kilometres from the coast, and therefore the possibility existed that the populations differed in their salinity preferences.

Experiment3: Salinity preferences oflarvaa from the Lake Cubhu population of M. ICfJldaety/us. Salinities selected for this experiment were 0, 4, 8 and 12 S 0/00, thus coveringtherange from freshwater tosalinities suitable for rearing M. rosenbersu larvae, It was considered unlikely, at the time of theexperiment,that higher salinities would be required. Larvae were stocked at 30/500 ml accordingto procedures described in section 3.10.2. TIle duration of the experimentwas.? days.

Experiment4: Salinity preferences oflamle from the Limpopo river population of M. lepklactylut. Salinities of0,5, 10, IS S0/0 0 were chosen for this expertment.as re­ suits from the previous experiment suggested that the specles llIay have higher s:Il1nlty preferences than 12 S 0/00. Larvae were stockell at 30/500 ml according to the procedures described In section 3.10.2. The duration of the experiment was 8 days. No water changes were made ondllY 2 35 results from previous trials showed tlut NII4-N levels were low before the provision ofArtemla In the containers. For the same reason water was Ill>! changed In conlalners with starved larvae ondny 7 as these: were few in number. 88

Experiment 5 : Salinityprc(crenct's of theearly I4rVll (ornts ofM. rude. M. rude Imac used in this experiment were stocked :It 30/500 ml, accordlng 10 the procedures described in section 3.10.2. Salinities selected (orIhls experiment were 5, 10, ISand 20 S 0/00. Fresh­ water W:lS not Included as the larvae of this species had already been reared to post·lIIrval stage in saline conditions. TIle duration o( the experiment walSdays.

Experimenl6 : Snlinlty preferences oftheearly larval (onns ofM. austmlc. M. austral, larvae ~sed in this experiment werestocked at 301500 ml, according to the procedures described insectlon 3.10.2. S:lUnlties of 5, 10, 15 and 20S0100 were chosen for this experi­ ment. Freshwater was excluded as post-larvae had been recorded from l1 rearing container holding M. austral« larvae. These later proved to be /II. pctmll post-larvae however, lind no M. australe post-larvae were in fnci produced duringthe project.

Experiment? : Salinity preferences oftheearly larval fonns of M. scabrieulum, M. scabrl· culum larvae used In this experiment were stocked at 30/S00 011, according to the proce­ dures described insection 3.10.2. Posltlve identification of Ihe female as M. scabrlculllln was possible as a result of Information received by Prof 11.1. Schoon bee from Prof. lIolthuis on the scparatlon ofM. petcrsll and AI. scabrlculum adults, Salinilles of 0, 4, 8, 12S0/0 0 were chosen for this experiment and Ihe experiment was terminated after 7 days.

During the experiment, only temperature and 5.1linlty were monitored as results (rom previous trials showed that the techniques employed In those experimentsresulted insatis­ factory water quality for the survival and development of the early larval forms,

3.11 POST-LARVAL REARING

3.11.1 Separation of Inrvne and post·lame.

TIle technique used for separatlon of larv:le and post-larvae was manual as small numbers were involved. When post-larvae reached 50-100 011 a visual estimate, the rearing container was dralncd lind the larvae and post·larvac trnnsfcrred to the mel.1l basin, used in S3linity experiments (section 3.10.2). Here thelarvac were removed wilh:l small pL1stic cup and returned to the larval r"uing container. PoSt'!:UV3C were then counted out into II 500 011 beaker, They were accllmitlzed to freshwater by :ldding slII:1I1 tlUanlltics of water to the beaker, lifterwhich they were rdc:lscd.AccllmitiS3tion took appruxlm:llc1y S mlnures. 89

3.11.2 StockJnl density

The production ofincreasing numbers of post-larvae coincided wlth II peak in the numbers of breeding slock. Hence stocklng densiries were determined by the splice available durin. the later stages ofthe project Only among the first batch ofIt/. rosenbersit post-larvae pro­ duced WllS Itpossible to periodicallyreduce numbersin the tanks to accommodate lnerease

in size. Even among these prawns, II point was reached beyond which no further reduction in numbers was possible ns D result ofalllck of space. The procedure adopted for the re­ duction of numbers among the .,rnwns referred to above WIIS ns follows:

1000 post-larvae ofPL21 to PL40 were stocked nt 733/012. The populntlon consisted of a wide range ofsizes. After 12 dnys the longer specimens comprising about I of the population were transferred lind stocked first at 342/012• and after I month at 146/m2. Five weeks after initial stocking, about half the population in the original lank were transferredlind stocked at 187 m2. Again, after eight weeks. half the remaining population were transferred lind stocked al 100 m2• Due to a lack ofspace it was not possible to repeal these reductions in density :my further.

Duringa later stage, batches ofM. ptlmil and M. rosenbergfl post-larvae were stocked at high inlUnl densltlcs in 47 2 aquarium tanks, They were then transferred to 250 2 aquarium tanks after which no further reductions indensity were possible. Initialstocking densities in the 47 2 tanks were very high. rnnging from 2520 - 8306/m2of tank floor with a mean of 4413/012. Post-larvae were held in these tanks for periods of 2.5 - 13 weeks with a mean of 5.7weeks. Thereafter they were transferred to 2502 tanks (area of tank Iloor 0.55 m2). Records arc available: for only two of four tanks stocked in this way. In these tanks. posr-Iarvac transferred from 472 tanks were stocked at 3174/m2 for M. ptUrsll and 20 141m2 of noor space for M. rosmbtrglL In this case /tI. roStnbtrgli were transferred directly after metamorphosis to the latter tank. and nor from the 47 2 tank. After transfer to 250 2 tanks, no further thinning out of numbers WIlS posslble due to lack ofspace.

M. rude pcst-larvee were held only in 47 2 t:mks due to the low numbers of post-laMc produced. The first batch of post-larvae numbered 15and were stocked at :I

fllIbUlIts. ArliOchll substrates.In theform of sh3dl:cloth oranchovy neUing. were hun, vertlcally inIII the tanks containing post-larvae. These provided lIpproximOitely theurne surface lIre3 lIS the tOink bot tom.

3.11.3 Nutrition •

Newly metamorphosed post-larvae were fed on eggcustart.lllt a frequency of 2-3 feet.llnss per dllY. The quantity of food was based on the amountconsumed between feeds. Partlclo sizes were between 1-2 mrn in diameter, 1Ilthough egg custard "worms", prepared by p..ssing theerg custard only once through :I sieve, were also tried. Approximately two weeks after metamorphosls a commercially prepared diet was Introduced together with the egg custard, which was then reduced until only II prepared diet WllS fed. Four commercially formulateddiets were available overthe courseof the project. Ofthese, one fonnulallon was found tobeunpalatable to all the specle~, due to theinsolubility and strong blndin. properties of the pellet. M. roscnbergl! post-larvae were fed on two commercial prawn diets. TIle first batch was originally Icd a formul..tion from Meadow Feeds and later, together with the second batch, an Epol prawn grower, which was provided In 3crumbled form. M. p~tt,sll were fed ona 48% Epol trout starter 3Sthis was preferred by the prawns.

Pcllcted prawn formulatlons were crushed and made intoa ball with a small amount of water so that small softened particles would be available to the post-larvae. Whenformula­ tions such as the 48% trout starter wen: introduced, these were just crushed, strained, and the whole particles fed, as these readily softened in water. Formula feeds were fed twice per day - once In the morning and once In the afternoon - thequantities being based on the amount consumed. Uneaten food was siphoned off the feeding platforms on dishes before each feelllnl!.

3.J 1.4 M:m38ement of water qunUty

Due to the tl3l1y siphoning or uneaten food out or the tanks, lind the replacement of this water resul:uly, It wns considered unnecessary to conduct resulJr pllrtl:al ch:IIl~'CS of wllter. TI1CSC were necewry only when mort:lllt1es occurred or when 11 deterioration In wliter qU:lllty \VIIS antlclpatcd, Crushed SCI shells were added to the: niterbeds of tOlnks, as WIIS tho case for adults, to buffer the system :I&4ll1lt nuctuations In I'll. 91

3.12 TREATMENT OF DISEASES

Larvae, Larvae were only treated for disease on one occasion}when mortalities occurred in one of thelarger bloflltration unlts, Although the larvae showed no obvious disease symp­

toms when examined, a 2 hr treatment with 40 ml/i of formaJin W:lS conducted, before transferring the larvae to another biofiltration unit. Forthe purposes of the treatment, tho airlifts were stopped, the buckets drained to half their volumes and the formalin admInis­ tered, TIle larv:le were then transferred in the same rearing contalners to the other filtration unit,.and water clrculntlon started.

Adults. Adults were treated for exoskeletal conditions on II few occasions. Blackening of gills andlordark brown patches on other regions of the exoskeleton occurred among edu]t M. /~plclaclyills lit one stage of the project. Affected prawns were removed to separate tanks for treatment. Aformalin-malachite green mixture W:lS used to treat the prawns with blackened Sills. The concentrations were 2S m~/2 formalin plus 0,I mg/2 of malachite green. W3ter was changed and fresh formalin-rnalachlte green added on evcry alternate day. Temperature was controlled at approximately 280C. Prawns were held in the treatment tank until the gills showed signs of lightening.

The brown spot condition was thought to be due to I'll values below 7, with resulting

corrosionof the exoskeleton. Affected prawns were transferred to II treatment tank con­ taining 0,3 mg/2 ofa chlorarnphcnlcol-furazolidonc mixture from Israel. In addition crushed seashells were added to the tanks in order to buffer them against a decrease in pH level. Prawns were returned to the holding tanks once the brown spots had been lost with moulting. From this point in the projcct regular 50% water changes were made in holding tanks and thepH level monitored.

Opaque while shrimps occurred occasionally and thesewere treated for 11 period of7-10 days witheither the lsrncli compound or furanace at 0,3and 0,1 mgl2 respectively.

AlthoughnotII disease condition, damage to adults resulting from aggressive behaviour Wilt common, lind damaged prawns were tnnsferrcd to individual tanks until damaged appendages reappeared after moulting. The prawns were then returned to their respective tanks. Chcmical treatment was found tobeunnecessary, • 92

3.13 SUMMARY

• Laboratory-scale indoor "atclluyfacilities wert developed, tested and ustd /0' tht brudlng and larval ,earlng ofth« /lve Indlgcnolls Macrobrachium specltl­ Closcd waterrecirculation systems, employlllg gravtl blo/llttn were usedlorthe "oldlllg 01adultsami post·lanoae, and lor tilertarlng oflarvae In a culture medium prcparcd from syntlutic sca salts. • Th« usc 01solar energy forheaUn, culture wat«was Im·tstlgated lor passlblt incorporatton I" a commemal productlon writ atQ lourstage: • IlIdlgcnolls Macrobrachlum spccles were cotlected from tile wild on several oceasion: to provide breedlllg stock willie M. rosenbergil adults were obtained from acommercial enterprlst. • Th« culture practices adopted.for tile IIoldlng 01adults and post-larvaeand the rearlllg oflarvae, were based Oil experienceofImocstlgators studying othn Macrobrachium species. • I" edduton to tile attemptedreartng ofth« kuva« o/Indlgenous species. aprt­ Ilmlnary study ollnterspecl/lc larval morpllology was conducted, In order to monitor development ollarvoe In rearing unttsand tomabie Iclentl/leallon of t/le lan'ae 01tile tndigenous species III tile laboratory. • Asaflnt step In establishing optimalconditions lor the rearing 01larvae. a senes 01salinity preference experiments ""OJ conducred to establish th«preler­ ences of the early larval forms: • A study ofpost-larval rearing techniques WOJnotundertaken, altlrollg" post­ larve« produced during the course 01tirelarval rearing programme. wereIIeld WIder similar conditions oflntmslve nursery grow-out for other Macrobrnchium species. • Dtseas« conditions which appeared were m'atedchemically or by Improvement 01tcclllllqlles lor water tllla/lty management. 93

CIIAPTER4

RESULTS PlIac PIlYSICO-eIiEMICAL PARAMETERS OFWATER QUALITY . 9S Wnter chemistry orthe sources Irom which indigenous prawns were collected 9S Waterchemistry orthe borehole and tllp·water ~uppUes . 91 Wnter chemistry orculture water InIlarval rearingunits . 98 Waterchemistry orculture water in adult and post·larval tanks . 101 COLLECTION AND IDENTIFICATION OFTilE INDIGENOUS SPECIES 103 Collection orwild pro,,·••s . 103 Identification or the Indigenous species .. lOS POTENTIAL OF SOLAR ENERGY AS ASOURCE OFIIEAT FOR CULTURE WATER . 106 SURVIVAL AND REPRODUCTION OF TilE ADULTS UNDER LA801{ATORV CONDITIONS . 106 Survivnl of the Indigenous prawns In the IlIb(.rntory . 106 Reproduction or the Indigenous prewns in the laborntory . 110 ~f:1ting . 110 I\toulling.sp:nvnlng and Incubatlon . 110 Fecundity ••..••••••••..••...... •...... •••••••••••..••...... •.••.•..•...••••••••••••• 112 BI"t!C(lins season . 112 LARVAL REARING PROGRAMME 113 Development lind prelimin3ry:testlng ofl:Jrval renringsystems . 113 Unfiltered culture water . 113 Theusc ofmechanical filtmtion units . 113 L,n'3C reared in biofiltrntJonunits . 114 Survival and morphologlcal development of larvae reared during phase two of the rearing programme . 114 It,. It/JltlQcl",u~ . 116 ~I. rude .. 116 }of. scabrkulul1l and ,'I. /Jtltrsll . 117 M. scabr/culum ...... 111 ~I. ptltrsll . 118 ",. tIIlslralt . 118 Snllnlty prdcrences of the enrly larval ronus .. 119 Salinity Ilreferences of the c:uly 13rval fonns ofM. pttmll . II? 94

Pago Salinity preferences of the bier Ianni fonns orAt. p(tersll •••••••••• 121 S:lIinlty preferences of the clirly Innal fomlS oC AI. lepldactylus,

Lake Cubhu populatlon ...... II 121 Salinity preferences of the clirly IDrvnJ fonns oC M. lepldactyills. L1mpOI)O river population ...... 121 SaUnlty preferences of the clirly InnDI fonns oC M. rude . 122 SaUnlly preferences of thecarly IlInnl fonns oC M. austral« . 122 SaUnlty preferences of thecarly larval forms oC M. scabricu/llm 123 Developmental morphology ...... 131 Form I ...... 131 Form II ...... 131 Form III ...... 134 Fonn IV ...... 134 FonnV ...... 137 Form VI ...... 138 Form VII 139 POST-LARVAL REARING ...... 140 /If. rosellbergll ...... 141 /If. rude ...... 141 M. petmll ...... 141 M. lepldactylu$ ...... 142 TREATMENTOFDISEASES ...... 142 SUMMARY ...... 144 95

CIIAPTER FOUR

RESULTS

4.1 PIIYSICO-CUEMICAL PARAMETERS OF WATER QUAUTY

Results of the detailed analyses of physlco-chemical conditions of the water bodies from which the prawns were collected, as well asthose of the borehole and tap-water supplies in the laboratory, arc presented in Table J. Detailed analyses were also made of the three multi-container larval renring units and one post-larval holding tank. TIle results arc presented in Table 2.

4. t.l Water chemistry of the sources from which Indigenous prown! were collected

Samples from theLimpopo river, near Messina, were taken lit the end of the dry season when waterflow in the river had virtually ceased aboveground. A comparison of thewater chemistry of Lake Cubhu with that of the Limpopo river at Messina showed that there were marked differences between these sources (Table 1).

Dissolved solids In the Limpopo river, liS reflected by electrical conductivity. are consider­ ably higher than those in Lake Cubhu, 11l1s also applies to all the cations analysed. Even so. a calcium value ofonly 32 mg/2 was recorded for the Limpopo river.

As could be expected chloride levels in theLimpopo were higher. due to the severe drought prevailing in the Northern Transvaal, although the chloride levels in Lake Cubhu are higher than could be expected for normal freshwater. This can beexplained by its proximity to the sea.

Values for ammonia, nitrate, nltritc.as welllls phosphateswere In both cases not high and of the same order.

Results forC.O.D. indicate that orS.1llic loads in the Limpopo were the highest. Thls iscon­ firmed by thevalues obtained fer organic carbon.

Heavy metals Cu, Fe, Zn and Mn were present in low concentratlons at both localities. · 96

TABLE I: Analysis or laboratory (resh\Vllt~rSUI)pl~llIld o(the natura! sources(rom whkh tho indibocnous prawns WOI'C collected.

Waler source

Water quality Lake Cubhu Limpopo River Laboratory Laboratory parameter Zulu land Messina borehole tap-water sampted sampled supply supply 14/6/82 28/8/82 sampled sampled 15/9/82 15/9/82

Turbidily (N.T.U.) 2,3 2,4 2,6 1,8 Colour (Cobalt Platlnum 20 37 10 10 Colour Unil) Electricalconductlvlty cpS/ern) 348 1286 91,6 335

Sodium (No) 44 188 4 26 Potasslum (K) 2 4,5 < I 5 C:llcium (ea) 9 32 7 26 Magnesiurn (M~) 9 71 < 5 12 Total AlkalinilY liSCaC03 24 490 48 118 Chloride ten 72 190 < 5 19 Silicon (Si) 6,4 2,8 9,0 3,0 Kjeldal-ultrogcn (N) <0,2 0,7 <0,2 <0,2 Ammonla-nltrogcn (N) Of <0,2 <0,3 <0,2 <0,2 ~ Nitrate- Nitrite e 0,2 <0,2 0,7 0,4 nitrogen (N) III Nitrite-nitrogen (N) < <0,01 <0,1 <0,1 <0,1 Sulphate (5°4:) 29 50 < 5 41 Total phosphate (P) <0,2 <0,2 <0,2 <0,2 Orthophosphate (P) <0,2 <0,2 <0,2 <0,2 Chemical oxygen demand 24 37 < 10 15 (COD) Organic carbon (C) 5,0 12,4 2,0 3,8 Doron (D) <100 375 <100

Copper (Cu) Of < 25 < 25 < 25 < 25 Iron (Fe) ~ 56 105 185 65 Manganese (Mn) ::l < 25 < 25 35 < 25 Lead (Pb) .:( < 25 < 2S < 2S < 25 Zinc (Zn) < 25 < 25 2970 95 97

4. J.2 Water eJI~mistry of the borehole and tllPwllter suppUes

Borehole water WilS used exclusivelyduring the latter slilse of the project for larvlll rearina units, holding lanks, and (or replacement purposes, Judging byIhe results o( the lalVll rearing programme, the use of IhisWiller supply appeared to be utis(actory for the prepara­

tion of artlI1claI seawaterand its ulilisation in rearingunln, Once II permanent borehole IUppl)' lind. reservoir had been completed, the presenceofrusl from the supply pipes and originlllreservolr lank was ellmlnated.

Dissolved solids In the borehole water were much lowerIhan inthe tap water(Tablt JJ. O( the Iwosources of freshwater in the laboratory, the mlnerallolldsin the borehole water were found 10beconsiderably lower than those in Lake Cubhu and the Limpopo river, while the minerai content of the tliP water appeared 10 be similar to that of LakeCubhu. The highervalues obtained for Na and Clln the tap water mlghl have originated from Ihe treated sewage effluent which enters the Vllal river above the Intake of the source of potable water supplies (Schoon bee, pers. comm.),

TIle combined calcium and magnesium hardness of 38 mrJ2 forthe tap-water supply approached the SO mv./2 level found 10 be detrimental for rearing the early larval forms, by

Sick and Bealy (1974). However the hardness of the borehole supply \VlIS only 12 mgJ2 and thereforemore suitable for the preparation ofn saline 11Irvai culture medium.

Exceptionally high values for Fe (185 nl,y2) and Zn (2970 1lg/2) recorded in Tabl« J can be ascribed to rust in the supply pipes and reservoir, which were in both cases replaced by a PVC system,

4.1.3 Wllter chemistry of culture water in IlIrnl rearing unUs

S3tisfaclory temperature control WIIS achieved using aquarium immersion heaters Inlarval, post-larval and adull tanks.•Iowever, the temperature lit IIliven selling fluctuated lIS much 0C 115 2 fromthedesired temperature. An exception WIlS lin electronle temperature conlrol unit developed at the University by lisInslrument Development Unit. and used for heIlU". one of the muill-conl:alner I:uval rearing II nils. TIlls pruvlded constant temperature levels In the blom'cr, bUI not In the attached re.1rina contlllneN, due to Y3rL1ble innow nIcs of heated Wiler10these. 98

TABLE 2: Mt'M Yllues obtalned from adetliled onlllysJs of water quoUty conditions In three multleontnincr ItllrinS units, with analysis of thc wotcr from a post· Iarv:d holdins tnnk

Multlcontalner rearina unit. Water quality Post-larval parameter 2 3 hold Ins tank

·Snllnily S 0100 10,S 14,5 IS,S 2

TurblJily (N.T.U.) 0.8 1,25 1,0 1,4 Colour (Cobalt Platinum ) 500 > 500 ) SOO >300 Colour Unit) "pH 7,58-7,70 7,60-7,59 8,17-8,09 E1eeti'ical conductivity (pSI em) 1255 1510 165S 1950 Sodium (Nn) 3200 4250 4900 230 Potassium (K) 135 173 183 29,S Calcium (Ca) 120 ISO 220 61 Magnesium (Mg) 350 47S 478 49 Total Alkalinity as CaC03 < 5 < 5 37 200 Chloride(CI') 5605 7373 8643 310 Silicon (51) 1,2 3.2 0,2 < 0,2 Kjcklal-nitrogen (N) 0,55 0.6 0,65 1,3 Ammonia-nitrogen (N) at 0.2 0.35 0,4 0.2 Nitrate-s nitrile C"A 26.0 29,0 17,0 18,6 nitrogen (N) E Nitrltc-nitrogcn (N) .< < 0.1 <0,1 <0,1 0.3 1 Sulphate (5°4 ) 780 1095 940 190 Totlll phosphate (1') 4.5 4,2 3,3 1,9 Orthophosphate (1') 3,7 3.4 2,5 1,8 Chemical oxygen demand 60 (COD) Organic carbon 18,4

Boron (8) 1325 1725 1700 300 Copper (Cu) at 45 SO 45 < 25 Iron (Fe) C"A 58 200 110 110 ~l3nJ;anesc (Mn) ::l < 25 < 25 < 25 < 25 Lend (Pb) .< 43 68 110 35 Zinc (Zn) 453 405 148 110 Approximate period in usc 4 months 3 months 2months 10months

·ltefrnctometer 1't3dlngs on the «by sampled_ "Vulues for the Inlet and ourlet of a single reuing container. 99

TI,e snit refractometer used during the project proved to be a useful tool in the manaaement of salinity as long as the influence of temperature on the rC:ldings was taken into account. TI,e syntheucsea salts used provided asuitable salineculture medium for the rearin.of larvae.

Evaporlltion in larval rearing units was higher than that inholding containers and water had to be replaced regulnrly. During the Initial phaseof the rearing programme, replacement water in therearing units was first adjusted to the salinity level In the larval containerbefore addition. TIlls resulted in gradunlly Increasing snlinities. When borehole water was used to­ wards the end of this phase. to replace losses due to evaporation, the levels becamemore constant. Fluctuations in salinity occurred In the multl-contalner rearing units, liS the volume of replacement water was not directly measured and excess freshwater was occasionally lidded when replacing water to the marker level. For a given salinity level the extreme values varied as much ns 5 S 0/00 asa result of occasional overfilling of the units.

TI,e levels at which salinity was maintained In the three multl-contalner units is presented in Table 3. Salinity in the three rearing units was altered occasionally, although the mean levels gene roily ranged from 8,2 - lOS0/00 for one unit and from J3.5 - 17,2 S 0100 for the other two units (Table 3).

The pH of the water In the multi-container rearingunits varied between 7,3 and 8,5 (Table J). Due to the failure to buffer the bioflltratlon systems ofthese units. there was a gradual decline of the pHover the second phase of the rearing programme. To­ ward the end of the project pH had however not declined below 7,5 in these units and did not rise above 8.5. (Table 2).

Differences in the electrical conductivity recorded.colncide with the actual salinltles selected for the three multlcontalner rearing unils {Table 2). As can be expected Na and C2·concen· trotions were high. Similarly the values obtained for both cations and nnions were nlso much higher than would be the case for freshwater.

In both multlcontnlncr and single container rearing units ammonia levels were generally satisfactory, with the execptlon of the single container mechanlcal Illtratlon units used briefly in phase one. The eoncentratlen of ammonnla in the mcchanl~1 Illtratlon units was found to be high, with levels above I mgl2 In some cases. In the nnt single container hio­ filtration units developed, ammonla levels were more acceptable. usually below 0.1 mdi lind seldom above 0,2 rngl2 (sec: table below). TABLE 3: Ranseand mean monthly nlues for temperature (0C) salinity (S°/00) and pHin three multicontai.ner rearin& units

Rearing Water . October November December January February March unit quality 1982 1982 1982 1983 1983 1983 . parameter

Temperature 16/10 to 31/10 1/11 to 30/11 1/12 to 30/12 3/1 to 31/1 2/2 to 25/2 R3ngc . 25-27,5 24-29 27-29 23-29 23,5-27 Mem 26,6 27,2 28,1 27,1 26,3 Salinity 0-406/10-23/10 ) Rmge 7-1 U24/10-31/10} 7-10 8-11 9,5-12 8-11 Mean 8,2..<24/10-31/10) 10,1 9,7 10,7 9,9 pH ranJ;C 8,2-8.4 7.8-8.3 7.6-8 7.6-8 Temperature 17/11 to 30/11 8112 to 30/12 311 to 31/1 2/2 to 25/2 1/3 to 31/3 Ran;c - 20-27 27-28,5 23-29.5 23-28 22-28,5 Mean 25,6 27,2 26,3 25,8 2 Salinity Range - 8-11 16-18.5 14-19 ll-IS.S 11-15.5 Mean 9.3 17,2 16.1 13,5 13,5 pH range - 8.0-8.S 7.3-8.3 7,5-8.1 Tempcnhlre 18/12 to 30/12 311 to 31/1 25-28) 2/2 12 1/3 to 17/3 Range - - 27,5-29 23-29,5 27,2) -11 24-26 Mem 28,1 27,1 22-25) 14/2-28/2 24,7 24,3 ). 3 Salinity - - Range 13-1,7 14-18 13-18 13-15.5 Mean IS 16.6 15.2 14,2 pH range - - 8,1-8,2 8,0-8,2 -8 101

Rearins Nfl4-N rnsl2 NJl4-N ms/2 UnU Rlln,c Mean I 0-0,13 0,078 2 0-0,12 0,055 3 0-0,22 . 0,107

Tabl« lor va/lltl/or NII4-N.1or tile /lr" 17 days ofus«. In tht /1m single contalner bto­ filtration ftorln,"n/ll.

Spot checks (or lImmonia concentrationswere made InI:unl re:ulns unlts at the endo(the eleventh lind durin, the 12th month o( the project. In thesingle contniner rearina unUs. levels were below 0,1 mg/2 In some unUs but AS high ns 2 msl21n others. Levels of Ntl4-N in one of themulticontalner unlts ranged from O,OOS- 0,13 mg/2 for this period.

Arnmon13 concentrations were determined again for the three multlcontalner unUs, aspart of a detailedanalysis of water quality In the ISth month of the project (Table 2). Levels recorded at this stage were approximately twice the vlIlue oflevels during theeleventh and twelfth months. However, If one takes Intoaccount thefact thllt these muUicontainer units were inoperation for periods of 4-6 months when the samples were analysed, vlIlues for ammonia were not exceptionally high, Indicating rellltively sallsfactory functlonlnaof the biofllter lit that.tlme.

Abuild upof nitrates, varying between 16 and 29 msl2 can be expected, as no control of nitrate levels WlIS exercizcd in these unUs.

4.1.4 Wlller chemistry of culture water inadult andpost·!llrval tanks

Adults and post·larvae were malntalned In waterfrom the tap water supply In the euly stllges of the project and in borehole water for the remainder of the project. Temperatures in these tanks ronged from 25-30oC over thecourse of the project with mean values be­ tween 26-28oC during the second phase. Adetllilcd analysis of the water chemistry of one AI. mscnbtrgll post·larval rearing tank, in usc for almost ten months at that time, is pre­ sented in Table 2. The lln:lIysis Jllowed that the quality of wlIlc:r In this tank, aner '. a prolon,cd period ofUSo1se, was very much thesameliS thatrecorded for the larvAl rearina units.. wIth Ihe excepuon of the ~UnU)' levels. There was however Abuild-up ororn.lnil: u r­ bon which II also reflected In the COD 0(60 mali. MHLATUZE RIVER ~

/ l r~

RICHARDS BAY ~ t

~ 7: SllUllllon ofLaU Olbhu retattv« to RlcJuzrds Bay. with collection sites IndIca/ed.

o -N 103

Failure to buffer pH in the freshwater biofiltration units resulted in a decline of pll durin. the second phase ofthe breeding programme.

Although pH levels declined to close on pH 7. only two tanks declined to levelsbelow pH7. namely 6,4and 6,9. The addition of crushed seashells to buffer the pH had the effect of raising the plL which stabilised between pH 7 and 8.

Spot checks for ammonia concentrationwere conducted at the end of the II th and durin, the 12th months ofthe project. In holding tanks contalnlns adults and post-larvae the levels of NJl4-N It this time were below 0,1 mg/2. ranging from O,OOS - 0,06 mg/2 In tanks which had been used for varying periods of time.

4.2 COLLECTION AND IDENTIFICATION OF TilE INDIGENOUS SPECIES

4.2.1 Collection of wild prawns

Indigenous /tIacrobrac1l/um sp. werecollected on four occasions during the period December 1981 to October 1982. from Lake Cubhu, Zululandand once at the end of August 1982 from the Limpopo rlver:t 2S Km west ofMessina, Transvaal. Two collections slte~ were utilised at Lake Cubhu, site I being at the pumphouse on the southern perimeter, and site 2 at the overflow and along the raised wall on the northern seaward end of the Lake (Fig. 7). At the site one, prawns were found along a 100mstretch of shoreline In open shallows(:t: SO em) and water around the roots of mangroves. The detailed analysis ofwater from Lake Cubhu was conducted on samples collected from this site. At site two, prawns were again found In the shallows, amongst the rocks which lined the inner face of thewall Pmwns collected from the Limpopo were also found in shallow water, often where rocks or other cover were present.

Data relating to the collection of prawns during the course ofthe project is presented In Tabt« 4. Atotal of87adults and 116juveniles were collected over the course ofthe project, III of these being M. Icpidacl>,IIu adults and juvenilescollected In the Limpopo river.

Dcrried females of four specJes were collected from Lake Cubhu during the months of October, December lind January. These were Initially identified u M.scabriculum, M.ptltnll lind M. l~pldaCI)'''IS. No berried M. rud« females were collected lit this time. Only two M. l~pldaCI)lI'11 males were collected In early and late summer It Lake Cubhu. TABLE 4: Sources ofMacrobraclUum Breeding Stock

Locality M. scabrlculum M. lepldactylus M. rude M. rosenbergit M. austrak Assorted and date and Juveniles ofcollection /II. petersit Lake Cubhu S adults 2 females 2 Males - - Not counted Site I (2 berried 15/12/81 females) LakeCubhu 4 Males I Male 4 Females" - - 11 larger juveniles Site 1 9 females 2 females and assorted 11-12/1/82 (6 berried) (1 berried) smaller ones Lisbon atates - - - 30 adults Hazyview, Transvaal. 20/3/82 Lisbon atates - - - ± 30 adults 3/6/82 (lost in transport)

Lake Cubhu S juveniles, 13 females Possibly 3 adults and - 6 , 19 assorted sites I and 2 10juvenUes provisionally (2 berried) juveniles juveniles 15/6/82 identified as 1 male /II. lepidactylus Limpopo river - 21 adults 26-29/8/82 90juvenUes

Lake Cubhu 4 Males 1Male 4 females - 5 Males sites 1and 2 11 Females S Females I juvenile II females (1 berried) (4 berried) 8/10 (7 berried 10juvenUes females)

2- lOS

llurinJ: the winter months, when thewater rcmpcruturv ill a depth o( 30 em W.1S IS"e. two berried M. rud« (email's were collected at site I but no berried (email's were recorded (or the otherspecies. Macmbrad,Ium uustrale was cottected predominantly at site two of lake Cubhu,

M. 1t>!'/daCI}'ltls collected in the limpopo river did not include any berried (emales or males showing secondary sexual characters. These prawns were collected during early sprin, when measured water temperatures ranged between .7oC In themorning and 27°C In the after· noon.

losses of prawns during transportation to the laboratory were low and were commonly a result of dalllage duringcollection.

4.2.2 IdentlOcatlon of 'he Indlaenaus species

Species collected from lake Cubhu were first tentatively ldentllled as M. leptdactytus, M. equldens and AI. scabrlculum. This was possible as the males ofthese species arc not difficult to separate. Females and juveniles can then be separated on the basis of the similaritiesof thefemales and juvenilesto the adult males. However, these identifications later proved to beInaccurate. Examples of the species, as Identified, sent to Professor Holthuis.Il.eiden, Netherlandsl.wcre found to differ from the ldentlflcatlons applied. M. rude had been mistakenly identified asM. equidens, and prawns grouped under M. scabrlculum were in fact a mixture ofM. scabriculum and M. peterst! adults. In addition, mature specimens of certain juveniles previously grouped with M. rude, proved to be /II. australe. According to Professor lIolthllis. these records ofM. sustrat« one probably the first recordsof this speciesfrom the African continent.

Thus, while theproject began wilh the Identification of onlythree species, I total of five Indigenousspecies were eotlected. The five species under consideration Ire:

AI. '''I'/(/O'')'''IS AI. nltle M, .,callr/ml"", M. I'tltrsll M. ollStralr 106

4.3 POTENTIAL OF SOLAR ENERGY AS A SOURCE OFJlEAT FOR CULTURE WATER

Temperatures for the period 27th of January to August the 25th 1982 are presented In figures8 and 9. Figures 8A and 88 represent results usin,two solar panels connected to the heat exchanger in one insulated pool. Figures 9A and9B represent results obtained using 4 panels connected to the heat exchanger. durins mid winter. In the case offisure 98 results were obtained after most ofthe external pipin, had been insulated and thewater surface insulated with a double royer ofbubble plastJc.

Results Indicate that water can be more than adequately heated in mid-summer (between 240C and 35 0C), using two panels to heat the pool (Fia. 8A).

However this was not adequate for heating the pool in winter, asthe water temperature did not rise above 200C(Fig. 88). When four panels were connected toheat the pool in the following trial, the mean temperature did not rise above 230C(FIg. 9A). When the water surface was insulated with a double layer of bubble plastic lind the piping taking heated water to the exchanger was Insulated, temperatures were obtained which arc suitable for maintaining prawns during winter at temperatures ranging between 240C and 290C (Fig. 98).

4.4 SURVIVAL AND REPRODUcnON OF TIlE ADULTS UNDER LABORATORY CONDITIONS

4.4.1 Sun/val ofthe indigenous prawns in the bboratory

Out ofa total of 2C3 adult and juvenilewild prawns collected over the course of the project, 107 survived to the end of the project. Most of the breeding stock used during the fint aix months of the project were lost accldentallY,lIs a result of drastic water changes. These prawns had been held in tanks equipped with mechanical filten lind had been showing• loss of appetite forsome lime prior to the accidental loss.

The surviv:1J of wild pmwns held in communal tanks for approximately 6 months durinatho second phasennsed between 30%and S4,8J [Tabl« J). Temperatures at which the " prawns were held ranged between 2S-30oC with mean temperatures between 26,6oCand 0C 28 (Tobl~ J). After the holding tank blonJten had been burrered against I decline of 107

Flg.SA Mean 24 hourly tompetaturOl 2 Panels Inlul,t.d lolor pool Inlul,t.d pool Open pool Ambl.nt

28

20

27 28 20 30 31 1 2 3 4 5 January 1982 February 1982

Flg.SS Mean 24 hourly temperatures 2 Panels ~ Inlulat.d lol,r pool '/////tl Inlul,t.d pool 20 1111111111111 Open pool Ambl.nt 18

a

8 10 12 14 18 18 20 22 24 28 28 30 2 .. • June 1982 Jul, 1882 Flg.9A Meen 24 hourly tempereture. 4 pinel. connected 108 ~ Inlulaltd lolar pool 24 '/",,/, Inlul.ltd pool 1II111/1I1111 Optn pool Ambltnt 22

20

11 'C 10

14

15 10 17 18 18 20 22 23 24 25 20 27 28 28 30 31 July 1882

Flg.98 Mean 24 hourly temperature, 4 Panels connectod addltlonsllnsulatlon

Inlulaltd lolar pool -oOllIIlf~'v 28 ';'"",/, Inlulaltd pool f~. IIUIIIIIIIII Optn pool A.. •• Amb'en' I -r 'l::; 24 .~ 22 ,/ 20 'C I

10

12 13 14 15 10 17 18 19 20 2t 22 23 • AuguI' 1882 109

TABLES: Selected data relatins toenvironmental conditions II weD IS pnwn aumYil In communal holdinS tAnu

DATA M.ltpldaclylus M. rudt M. scabrlculum M. peterstt" M. austral'

Mean tempe- 27.6 28,0 27,4 26,6 27,9 raturc °c pH ransc 7,3 - 7,8 7,1 - 7,9 7,4 - 8,5 7,3 - 7,9 7,4- 8,0 after buf· fcring of biofilten

Initial 231m2 23,31m2 73,3/m2" 36,7/m2 25,6/m2 stockins 2'" density 301m of prawns

Approxl· 6 6 6 4 6 mate dura- months months months months months tion in holding tanks

Survival 54,8% 40,6% 30,0% 45,5% 47,6.

, Data for the period after M. ptttnil adults were removed to aseparate tank from the tank containing"'. scabriculum adults.

,. IncludesM. ptttnll. initially stocked togetherwithM. scabr/culum.

"·M. scabr/culum density after transfer ofM. pettnll adults from the tank. 110

pll, the plileveis ringed between 7,1 and 8,S in these t:mks (Table j). Although theexact causesof mortalities were not monitored indetail, avatlable dal3 suggests that IlWCSSlve behaviour III moultlng,lInd possibly inadequate diet, eontributed to increasing morlalitlel lind decreasing reproductive potential with time in the laborltory.

4.4.2 Reproduction or the indigenous pnlwns in tho IllborAtory

The five Indigenous species collected for this study survived and reproduced in the labOrA­ tory. although there wnsn tendency for reproductlve potentlal todecline in some species.

Mntlng. Mllies and femnks readily mated In holding tanks and observed muling behaviour resembled that described for M. rost'nbtrgll. Mnting behaviour was often observed shortly after the lntroduetlon of newly moulted females to males during the early stngel of the project. In communal tanks in the later stages, mutingW:lS observed in territories excavated by males of the species, in the filter gravel. This was particularly common among males of M. lepldactylut bUI to a lesser extent among M. australe, M. {Jcttrsll and M. scabrtculum males. MlIles of the latter two sp~clcs commonly excavated depressions under shelters pro­ vided. TIlls behaviour W:lS not noted for M. rude, the adults of which commonly moved about In theopen, making less use of the shelters provided.

Only a single male M. lepldactylus was avallablc for mating purposes for 3 months during the first phase, and no sexually mature males were available for the next six months.

Moultin8..~pll\Vnlng and incubation. for the period during which individual females were monitored,M. pmnll and M. scabriculum adults were held together in the same tank and the data available for these two species has been combined. Among the adults of these prawns,deviations In the colour of the felltales and esgs were observed. Females, and occasionally males, changed to n rustcolour and II perc~nlage of these females 13ycd orange ew at times which later became opaque, and were shed.

M. austrak fcm:tles spawned rcgularly but u~ulllly shed 1Il0st of their c;tgS before they hatched. M. rud« fem:tles also shed their cBS' but to II lesser deBree, while M. IcpldactyluJ females. of II smaller slze,shed few cggs.

AmongM.sC'abrlC'lIll1lt1. M. pCI~rsllnnd M. austral« femalcs OVllIY development was 1J',1I.1Ily complete by the time spawning occurred lind II pre-matins moult usually look place wilhin TABLE 6: Relevant data on spawning and incubation for individU3lJy monitored females

DATA M. lepidactylus M. rude M. scabriculum M. petersii M. austral« Fecundity SOO- 1,100- 1,800- 1,080- 2660" 10,500 3,340 3,280 2,940

Mean 5,502 (9)- 2,047 (7)- 2,365 (4)- 2,220 (3)-

Egg Dark green Grey to Olive green Olive green Olive green Colour to olive green grey-brown (Initial) Mean 26,6-- 26,5-. 26- 26- 25,4- incubation 29,1 27,8 28,2 28,2 27,9 temperature °C

Incubation 15-16 16 12-18 12-18 period days days days days

-Number of females from which mean was obtained "Single female

- 112

24 hrs, Thiswas not the case for M. lepldac/ylus and M. rude females, where the period between spawning and the following pre-mating moult took longer. Incubation of eggs took from IS-16 days for females of the latter specieswhile inM. scabrtculum lind M. peterst! the Incubation period was between 12 and 18days, usually In the region of 14 days [Tabl« 6). Mocrobrachtum ails/role females were only removed from the communal tank prior to hatching as the femllles easily shed their eggs, and therefore females could not be monitored. Macrobrachlum lepltlac/yhls and M. rud« females spawned every 4-6 weeks while M. scahr/cllilim and M. pttmlf females spawned every 2-3 weeks, In the laboratory. Hatching took place overnight usually, although this was often spread over two nights among M. l~p/daCI)'llIs and M. rude females.

Fecundity. The fecundity of the Indigenous species was not Investigllted in detail. Never­ theless there was evidence that the fecundity of the prawns deteriorated after a prolonged period in thelaboratory. This was In the fonn of eggsdropping offgradually, the appear­ ance of abnormally coloured eggs which also gradually dropped off,lind smaller slzed hatches. This was the case for M. am/ralc adults shortly after transfer to the laboratory, and for adults of the other species towards the end of the project. Records of the sizes of hatches for females that appeared to be carrying the full complement of eggs nrc presented in Table 6. Although the sizes of the females were not normally recorded, 10,500 larvae were hatched bya female M. lepidactylus collected from the Limpopo river and measuring 7 ern from rostral base to tclson (approximately 9 g mass), and 2940 larvae werc hatched from n female M. scabrlculum measuring 5,I em in length (approximately 3 g mass). This female M. scabrlculum was the largest in the tank,

Breedingseason, Berried females of the indigenous species were collected from the wild In the warmer months, with the exception of/tf. rude femnl,.s, whleh were collected In berry during mid·wlnterfTable 4).

Macrobrac!,I/lI1I l~pltlacl)'llIs adults collected from theUmpopo In early spring did not In­ elude any berried females or males showing secondary sexual characters, such :IS eharacrerls­ lically developed chcllpeds, After:l period of rime in the labor:llory under controlled condi­ lions, secondary sexual characters appeared among the m:lles at the time the fint berried females appeared, This occurred approxirllately two months after collection and colncldcd with the lI1'pe:uance of berried female, among M. I~I'/clac/)'I/I$ adults from Lake Cubhu approximately three weeks after collection. Males of the laller spccie5 began 10 shed their cheliped! towards the end of summer in the Inborn tory where photoperiod had been dis· continued at the onset ofsummer. 113

A peak in the appearance of berried females of all species, including M. rude, occurred during November toJanuary, which coincided with the period in which berried females of four of the species were collected from the wild. As a result of the increased numberof berried femnles, and limited facilities for larval rearing, notall the batches of larvac were reared. Nevertheless a total of 104 berried females of nil five indigenous species were reo corned over a six month period from communal tllnks used during the second phuc ofthe project. From these, ninety one batches of larvae were hatched.

4.5 LARVAL RF~IUNG PROGRAMME

4.5.1 Development nod I)rcllmlnllry testing of larval rearing systems

As previously mentioned (Chapter 3, section 3.4.1) the development lind preliminary testing of larval rearing systems took place during the first phase of therearing programme. Results for this phase lire presented in the order that development proceeded, from unfilteredculture water to theuse ofblofllters for water punrlcatlon. Data for "/II. scabrtculum" applies to M. scabrlculum and M. petersit, which had not yet been taxonomically separated. No M. australeadults were available during this phase, andM. rude females only came into berry toward the end of the first phase.

Unfiltered culture water. Aquarium tanks and 102 plastic buckets were only used tempo­ rarily from the 24th December 1981 to the 6th of February 1982, prior to the completion of the mechanical lilt ralion units. Seven batches of "M. scabtlculum" and :1 single batch of M. lep/dactylus larvae were held inaquarium tanks or buckets. Larvae were hatched In freshwater, with the exception ofa single batch of "M. scabr/culu","larvne, and the salinity was'gradually Increased from the first day to levels varying from 1-4 S 0/00, but not above 8 S 0/00. Survival of these larvae was from 4-1 S days, wilh morphological development, extending beyond fonn II. in only three batches of "M. scabric/llllln'~ Mortalities were generally high, sothat nlnnagement of the 13rv3c became difficult IlSweak lind dyinglarvae were inevilllbly siphoned out with debris from the culturecontainer. nil' usc of mechanical fillmtlon unirs. The rearing unitsdeveloped provided difflcultles from the points of design lind functlon,rciulUng In unnecessary losses of Inmu:. A total of four batches of,\t. lcp/t/actyilis and three of "M. scabriculum" were stocked in these units over a period of four weeks. Salinity In the units was gradually increased after hatching with the excepuon of three batches of M. ltp/dactylus, hatched lind held al 3 S 0/0 0. I:or 114

the other batches, thesalinity did nol exc~ed 4 S 0/00 at termlnatlon. Survival of Wvao in these units was poor, varying between 4 and 6 days,at temperatures between 25 and JOoe.

Larvae rellred In blonltratlon units. Larvae were reared In blofillratlon units for theJut few monthsofthe nrst phase. with varying degreesof success. Eighteen batches of larvae were reared In these units at mean temperatures of 26,20C-2SoC and s:tllnitles ranaln. from 6-15 S0/00. Of these batchesonly M. Tose"btrgll and AI. rude larvae reached post· , larvalstage, reared al S:lllnities from 10-15 S 0/00 (Tablt 7J. Only a single batch ofAI. l~piJacl>'llls larvae were hatched, as theonly male at that lime WDS lost when the blo­ filters were nrst used. Survival ofthese laIVae was only five doys at 3 S 0/00, and morpholo­ glc:1I development did not progress beyond form I. Survivalnnd morphological develop· ment among "M. scobrtcutum ,. larvae was found to be poor at the snlinltiesselected for these, which were below 8 S 0/00(Tobit 7). Generally survival In these units was favourable al 5:llinllies above lOS 0/00, with one batch of/II. rude larvaesurvivingfor 89 days, while larvae reared nt salinities below lOS 0/00 did not survive longer than 16days (Table 7J. ltIacmbracl'lwn TOsen~trglllarvae were reared for the first time In the project, using these blofiltratlon units.

A total of 2424 M. rosenberglt and flfteen M. rude post-larvae were produced duringphase one. A dcflnlte effect of daylcngth on the metamorphosis to post-larvae was noted among M. rosenbergi! larvae. (Table 8). The percentage of the Initial total completing develop­ ment to post-larvae was 19% and 40% respectively for two of the M. rosenbergli batches, while only 0,5% oftheM. rude batchcompleted development (Table 8).

4.5.2 Survlvallll1d morphologlcal development of larvae renrtd during phase two of the rearing programme

Out of 11 total of 94 batches of larvae hatched, during thJs phase ofthe ~arinB programme, 24 batches were discarded or accldentatly lost. Of the remaining total, 27 batches of larvae were reared for Icss than 21 days. Survival improved oncethe snlinilies in the ~arins unlit had been increased In accordance wlth results obtnined from the5:lJlnity preference studies, It was possible 10 resr 4J batches of Iarvne forperiods longer Itlan 21 days with the produc­ tion of S065 M. pctmll. I 14 M. rude and ISM. IcpldaCI)"US posl·larvae from among Ih~se batches. Macrobrllc1,11l1tI scabrlculum and M. nuslrolc were not reared through to 1l0sl·l.lI,\,ae. 115

TABLE 7: SUrvivll1lU1d morphological development of IzUVIC In blofiltration unitsduring phase one of the renring programme

Survival Macrobrnchium Initial and Developmental Survival period species final fonn time In days salinity S 0/00 reached days

0- 4 2xM."scabrlculum" 1 S - 10 /tI. leptdactylus 3 .... 3 1 S /tI. rude 6 .... 6 9 M. "scabrlculum" 6 .... 6 9 M,'scabrtculum: 6,S .... 6,S 10 I I- 20 /tI. rude IS .... IS 4 18 M,"scabrlculunf 6 .... 6 2 17 M. rosenbergtt 7 .... 7 16 30 - 40 M. rude 9 .... 9 36 M. rud« S .... 10 3S Over 40 AI. rude IS .... II 73 M. rude IS .... 10 4S M. rude 10 .... 10 PL 89 AI. rosenberztt 6 .... 10 SO M. rosenbergil 6 .... II IS PL 57 M. rosenberglt 6 .... 10 PL 61 2xM. rosenbergi; 7 .... 12 PL 61

TABLE 8: Effect ofphotopcriod control on the production of post-larvae for fourdlffe­ rent batches of larvae

Period from Age of larvae Initiation Total hatching to al initiation ofphoto­ post­ appearance of 14 hr period con­ larvae of the 1st photo- trol to first produced post-larvae period post-larva

M. rude 66 days 3S days 29 days IS M. rosenbergll SIdays 30days 21 days IS M. rosenbergll 44days 26 days 17 days 801 M. roscllbcrgll 36days 18 days 14 days 1608 116

Most batches of larvae that were reared beyond the Sth brval form, were combined wllh other batches of the same species andapproximate age, for practical purposes. In addition, larvae were removed from some batches for use in ullnlty preference studies.

Data presented here Is for both uncombined and combined batches. (Refer also to section 4.5.2 for morphological development In the salinity preference siudies.) Morphological development, for Ihe early larval fonns ofthe indigenous species was more rapid in the ulinlty preference uudles than in rearlns units at the same salinities.

M. Icpldactylus. Form I larvae moult tofonn II between 4 and 7days after hatchlnS, at 0C. mean S3llnitles between 10 and 17S 0/00 and mean temperatures ranging around 27 Two batches reared at mean tempcralures around 250C fell within this range. From III larvae appeared at 10- I I days under the same conditionsofullnity and temperature. although I batch took 16 days to reach fonn Ill. At mean ulinllies between 13 and 15 S 0/0 0 form IV brvae appeared 12-16 daysafter hatching. Form Vlarvae were noted 15-28 days after hatching at mean s:alinlties between 10and 16.6 S0/00• However at this stale the development become staggered, and three larval forms were present at one time. In addi· tion, survival decreased rapidly from Form Vto Form VII. At mean salinltles between 13 and 17S0/00 fonn VI larvae were recorded between 24 and 47 days after hatchln.. Survival to forms VI and VII ranged from 0,27% - 2,6% of Ihe initial total. nlthough 52,9% survival was recorded for a single batch reared to form VI. The longest survival recorded was 2,6% reaching forms VIand VII after 64 days.

Allhough form Vlllarvac and post-larvae were less than I%of the Initial totals, data from the.sc suggests that I3rvae could reach these forms between 40and 50 days. at s:alinities r:mslng around ISS0/0 0 and a temperature of 27OC.

Out ofa total of 17 batchesofM. /tpldo(lyllls IaI'Vl1C reared, 14batches were reared for periods ranging from 28 - 64 days at salinities In the region of 15 S0/00 and temperature around 270C.

M. rude. Form I larvac moulted to form II between4 llnd 7 days after hatchln" It ullnitIcsbetween 15 and 17 S 0/00• Two batches reared It mean ....linitiesor 9,3 and 10S 0/0 0 moulted after the same period or timc to form U. Form IIIlarvllc appearnl ~I 10-14 d3ysafler hatching, with one recant of 17 days,II mean I4llnltles or 14-17 S%0. At mean salinities nnglns between 14 and ISS 0 /00 ~/. rod' larvae reached form IV In 12 117

and 17days. Form V larvae appearedbetween 17 and 23 days after hatching. withone record o( 28 days where the larvae were initially reared ata mean salinity of9 S 0100. A relatively long period followed between the fifth and sixth larval forms, with the latter appearins between 38 and 4 I days after hatching. At mean salinities ranging around 16 S0/00, fonn VII larvae appeared 48 days after hatching in one batch o(M. rude larvae, Post·larvae were recorded between 42 and 6I days after hatchingat salinities ranging (rom 13-17 S 0/00 and temperatures nuctuating around 27°C.

Due to the (act that M. rude batches were all combinedat some stage, with other batches, survival ligures could not be calculated,

Of the 13batches o(M. rude larvae hatched during thisphase, three did not survive longer than 21 days, and the remainder survived at least to form VI, with some reac1ling post·larval stage. Rearing periods ranged from 27-61 days.

In the case o( five batches combined with each other over a period of time, with 17days difference between youngest and oldest, larvae were in thereulng container for 124days. During thisperiod post-larvae were produced from among these batches.

M. scabriculum and M. petersil larvae were reared separately only for the last two months of the project, once the adults had been taxonomically separated, TIle poor survival of M. scabriculum larvae, compared with the regular development through to post-larvae of the M. percrslllarvae, made it possibletoseparate batchesoflarvae of the two species, aU previously identified asM. scabriculum:

. . M. scabriculum larvae were reared at mean salinities ranging from 8,6- I5,2 S 0100 and at mean temperatures between 24,3 and 28,loC. Onlyone batch survived longcr than 21 days. Survival (or the remainder ranged (rom 9-19 days. Morphological development proceeded as(or as form Vin this time, nod n few of these fonn V larvae were reared to form VI in 500 mlcontalners, used in the snlinlty preference studies.

At mean salinities between 9,9 and 14S0/00 fonn II AI. scob,/nllum larvae appeared about 6 days after hatching, while fonn III larvae appeared 9 days after hatching 3t s:llInltics ranging around 15 S0/00• Al 9.9 S 0100101. scabrlculum 13rvae of I batch took IJ days to reach from III. Abatch of AI. scabriculum brvae reared at 15,2 S0/00 reached the 41h larval form II days after hatching. At salinities mnging around 10S0100 two batcher of 118

larvaereached from V at 20 and 21 days respectively. The few Lune reared (rom fonn V to (arm VI In a smaller container rellched form VI in 19daYL

M. petersii larvac were reared at mean salinities ranging around 10 S 0/00 and mean temperatures ungins (rom 2S,6-28,loC. Survival anddevelopment to post-larvae were the highest (or this indigenous species, with some pest-larvae belns produced (rom all 16 batches reared. Earlier batches identified IlS M. scabrlculum at the time, proved to be /of. petersti, once the juveniles reared besan to show development of species characteristlcs of the malc chelae.

Morphological development ofItt. petml/ was more regul3r than for the other indigenous species. Form ll larvae appeared at 4 days after hatching, form IIllllrvne at 7-8 days after hiltchlng, form IV larvae at 8-10 days and form V larvae at 10days. Form VI larvae appeared at 14-16days lind form VII13rv3e at 15-21 days. Post-larvae first appeared at 21 days in 6 outof 16batches reared. TIle longest period before the appearance of post· larvae was 30days and the shortest 18 days. Survival from hatching to forms VI and VII was 10,9% for one batch of larvae, while for another batch survival to post-larva I form was 32,3% of thenumber initially stocked,with 12 larvaerem3lnlng on the 54th dny. D3tl for other batches could not be calculated due to recombinations.

Soon after the appearance of the first post-larvae, a rapid Increase inmetamorphosis to post­ larvae occurred, with a peak between 10 and 14 days, afterwhich a rapid decrease occurred. Most batches were reared for 30-40 days, at which stnge most ofthesurviving larvae had metamorphosed to post-larvae and only a few larvaewere left. Inone batch ofItt. pttmll larvae, metamorphosis to post-larvae extended over a period of 22 days.

M. australe, Of the eight batches of M. sustral« reared,only I batch survived 27 d3Ys, the others being termlnated in under 21 d3YS. In the latter batches mean salinity levelsrnnged from 7,7-10,7 S0/00• In the batch which survived 27 days, the salinity had been incre3scd to range around ISS 0100 on day 9 when the larvae had reached forms lIond Ill. TIle latter larvae had reached form II between days 3 and 4, form III between days 9 and 13 and fonn VI between days 17 and 18. By the 27th dny no 13rvae were left.

M. roscnbergil. Out of the 3 botches hlltched during this ph3SC, one wasdiscarded aOer hatching, onewas terminated early, and one batch was reared through to post·brvnl form. 119

The latter batch was reared in 320 2rearing contains of amulticontalner unit Initially, but returned to one eontalner as the numbers began to decline. The first post-larvae were ob­

served on day 30, at which tlme the sun'ival from hatchin,was 29%. Arter Il period of66 days 22% of Ihe initial total had reached post-larval form, and 0,89% remained as lame. A total of 1323 post·larvae were produced from this batch.

4.5.3 Phase 2 :SaUnity preferences or theearly larval fonns

Salinity preferences or the early larval forms ofM. peteniL

Preva1l1ng water conditions arc presented inTable 9. Durin, this experiment only four larvae percontainer were examined for morphological development, in contrast to the examination of thewhole population In later experiments.

The survival time ofM. peterstt uavs» In freshwater (Figs. IDA alld 0) was short, with 50% mortalities occurring between days 2 and 3 and 100% mortality by day 4. TIlls applied to both fed and starved larvae, neither of which progressed beyond form J.

The survival time ofstarved larvae increased with increasing salinity, and 8 50% mortality occurred among these larvae between days 4 and 5 at 4 S 0/00 onday 5 at 8 S 0/00 and between days 6 and 7 at 12 S 0/00. (Figs. IDe, 1:: and GJ. Morphological developmentof starved larvae did not progress beyond fonn II at 4 S 0/00 (Fig. IOC), while larvae fed Anemia at this salinity were able to moult to form III, butsurvival decreased ropidlyas the larvae reached form III (Fig. JODJ. Starved larvae at s:lJinilies 8and 12S 0/00 were able to reach form III but subsequently died, although survival lime ofform III larvae appeared to be greater at 12 S0/0 0 than lit 8 S 0/00. (Not indicated inFigures). Survival and morpho­ logical dc:velopmenl of Illrvlle was bestats,1l1nities 8 S 0/00 and 12 S 0/00 where the larvae were fed Arttmla (Flgt JOF and II). Ofthese larvae, those held at8 S 0/00 exhibited • highersurvival rate thnn those at 12 S 0/00 (78,3% compared with 40%) on day 8, while morphological development was the most rapid lit 12S0/00 where all the surviving13n'ac had reached form IV by dlly 8 (Figs. IOF and /1).

Aneml« nlluplll fed at0 SO/00 were all dead lifter 24 hrs lind of those fed ot 4 S 0/00 the mlUority were delld ordyina ot 24 hn. TABLE 9: Summvy ofthe raa;e mel IDQD nlues for water. quality parameters Oftr the course of the individual salinity preferaace cxpaimeDb

Experiment Tnn;»crature Range of mean daily salinities pH NH4-N l'o"H4-N NH4-N andspecics lUnleand for six rcpliates at each salinity range larval freshwater sea water ~ ~ "QD~C) level containers stock UDk stock UDk mg/2 mll2 mtJ2

I n.o-31.0 0.036- 116-147.5 0.5-1.5 M.~tmiJ 0 3,8-4,0 .8,~,O 11.7-12,0 8,0-8,5 0,134- 29.1 0,093

2 26,0-30.5 0,16-1,05 125-160 0.5-{),8 M.pe/mil 7.7-9,0 11,3-12.5 15,0-16,0 19.3-20,3 7,8-8,4 0.310 28.0

3 27.0-28.5 0-0,239 0-0.102 O-o,D42 108-IS6 0,2-3.5 Ii Iq1G«tyAa 0 3,$-5.0 7,$-9,$ lJ,7-13,2 8,0-8,$ (UkcCubbu) 21..1 O,OJ2 0,03 O,()(J9

4 27,5-29,5 0-0,054 0-0.01 o-aoos M. kpidtlctylus 0-0.3 5.0-5.3 10.0-10.0 14.3-15,2 7.9-8,6 (Limpopo riwa) 27.9 0.024 O.OOS 0.0lU

S n.o-JO.5 0.001- 112-142 0,5-0.9 14. rudtl $.0-$.8 10.0-11,3 14.0-1$.7 19,7-20,2 8,3-8.$ 0-0.135 0-0.01 0.12 28.3 0.026 osas 0.007

6 25.0-29,0 0.003- 119-135 0,8-1.0 M. IIIJ1IIJt $,0-$.7 10.0-10) 14.8-1S,2 19,3-20,3 8.0~,4 0.127 0,008-· O,oos" 27) 0,030

7 n,5-31.D JL s=bric-..J:un 0 4.0-4,5 8.0-8,5 11.7-12,2 28,7

-VIJucs fOf fed larvae OQ the rmalcby oftbe experimc'nt. --Only one rcadinl N Q 121

Salinity preferences of the later larval rorms ofM. petenU. Prev~lIing water conditions arc presented in Table 9. In all the experimental salinities, survival of larvae declined to below 50% between days 4 and 5, except (or larvae 12 S 0/00. At 12 S 0100 survival approached 50% on the last day of the experiment. Although morphological development reached form VII, no post-larvae were obtained at the termination of the experiment.Thls experiment w~s termlnated as a result or the poor survival and raised NJl4-N levels (Table 9J and no fl,ures (or survival and development arepresented.

Salinity IJI-ererences of the early larvnl ronns otM. lepldactylus. Lake Cubhu popubtlon. Prevailing water condftiollSQrc presented In Table 9. Although survival time of larvae In freshwater was slightly longer among starved larvac then larvae red Anemia nnuplii, 50% mortalities occurred between days 4 and S for both fcd and starved larvae(Figs. JJA and B). Morphological development in freshwater did not progress beyond form I.

Survival time of larvac in 4 S °/00 was shorter than in rreshwater with 50% mortalities occurringllmong both starved and fed hrvae, between days 3 and 4(Figs. 11C and DJ. Morphological development of larvae at4 S0100 progressed to ronn II, but these larvae were eitherdead orin the process of dying when examined, and did not fulfil the criteria for survival.

Survival and morphological development were simil3r forstarved larvae at 8 and 12 S0/00 with 50% mortalitles occurring when the larvac had reached form Il,between days 6 and 7. (Figs. JIE and GJ. Morphological development did not progress beyond form II.

The highest survival rate and the most rapid morphological development occurred where the larvnc were fed Artemta nauplif at 12 S 0/00, with a 94% survlvlll rate by day 7 (Fig.! JlI). At this point approxlmlltely 50% of thesurvivon had reached form III.

S:dlnity preferences o( tile early lamal romu of M. lepldllclylus, Umpopo populnUon.

Prevailing water conditions lU'C presented In TobIe 9. Survival or IllI'Vac In (reshw01ter WIIS similar nmong both red and starved lame, with starved larvae surviving sllgluly longer than Iarvlle provided with Artt'mlo. (Figs. J2A and OJ. The 50% morfallty level WIIS reached be­ tween days6 and 7 where larvae were provided with Arum/a, and on day 7 for stllrved Iarvac, but 100% mortailly had not occurred In either of thefreshwafer treatments by day 8, the termlnauon of the experiment. Survival at this point was 10% ror starved IArvac lind 2,2% for fed larvae. Devclopment in frcihwater did not progress beyond Form I. 122

Survival and development of both fed and starved larvae were similar at S S 0/00 (Flgs.12C and D). Fifty percent mortalities occurred between days 6 and 7among both fed and starved larvae, when most of the larvae had progressed to form II.

At 10 S 0/00 and 15 S 0/0 0, survival and development were slmllar for starved larvae, with 50% mortality occurring between days 7 and 8 at lOS 0/00, while at 1S S 0/0 0 • survival rate of 54,9% was recorded on day 8. (Figs. 12£ and G). Morphological development did not progress beyond Form II in either case.

Larvae fed Artemia at 10 and 1S S 0/00 showed similar survival curves, with 82,4% and 83,4% survival byday 8 at 10 S 0/0 0 and 15 S 0/0 0 respectively (Figs. / 2Fand II). Morpho­ logical development was more rapid at 15 S0/0 0 , with 94,7% of the larvae reaching form III on" day 8, compared with 72% present as form III at 10S 0/00•

Salinity preferences of the early brvalfonns of M. rude. Prevailing water conditions are presentedin Table 9. Survival of starved larvae at S S 0/0 0 was poorer than for starved larvae at salinities of 10, 15 and 20 S 0/00 , with 50% mortalities occurring between days S and 6 at 5 S0/00 and between days 6and 7 at the higher salinities (F/gs. /3A, C,E and GJ. Morphological development ofstarved larvae did not progress beyond form II at anyofthe salinities investigated.

Survival of larvee fed Artemla at salinity 5 S0/00 decreased rapidly with the appearance of form II larvae, reaching 50% mortality between days 5 and6,aswas the case with starved larvae at thissnllnity (Figs. 13A and BJ. Survival of larvae fed Anemia at salinities 10, 15 and 20 S 0/00 did not drop below 50% by the terminationof theexperiment at 8 days. (FIgs. 13D, Fond11). Survival rate of the latter larvaewas highest at 20 S 0/00 where 85% survival was recorded at the termlnatlon of theexperiment at 8 days.

Morphological development ofall fed larvae (Figs. / 3D,D,F and II) progressed to form III, but with increasing proportions of form III larvae with Increasing salinity. At 20 S 0/00 66,7% of surviving larvae fed Artemie had progressed to form III,compared with 15,6% at S S 0/00•

Salinity preferences of the early larval fomu of M. a\lstrale. Prevailing water condltlons are presented inTobit 9. Surviv:d of starved larvae was similar It all salinities with SO~ mor- 123

talities occurring between days 4 and 5(Figs. 14A.C,Eand GJ. once all the SUrviVOR had reached form II.When 100% mortalities are considered forstarved larvae however. survival time was longest atsalinities 10, IS and 20 S 0/00•

Survivalof larvae fed Artemta was similar at 10, IS and 20 S0/00 but poorest at 5 S0/00, with 50% mortality occurring betweendays 5 and 6 (Figs. 14B,D,F and 1/). In addition, morphological development at 5 S 0/00 did not proceed beyond form II. Survival of fed larvae at 10,15 and 20 S 0/0 0 was between 80 and 90% byday 6.lIowcver, differences in morphological development werc evident, with the percentages ofform 111 larvae present at the termination of the experiment being lowest at lOS 0/00 (l,3%) and highcst at 20 S 0/00 (29,6%).

Salinity preferences of the early larval fonns of M. scabriculum. Prcvailing watcr conditions are presented in Table 9. Survival timeof starved larvacwas slightly longer than fed larvae in freshwater, with 50% mortalities occurring between days 5 snd6 for starved larvae and days 4 and 5 forlarvac provided withAnemia (Figs. 1SAand BJ. Morphological develop­ ment did not progress beyond form I infreshwater.

Larvae fed ArtemiD at 4 SO/00 survived longer than starved larvae at this salinity with 50% mortalities occurring between days 5 and 6 for starved larvae and between days 6 and7 for fed larrae (Figs. 1SCand DJ. Morphological development did not progress beyond form 11 ineither starved or fed larvae.

Survivaland development of starved larvae were similarat 8 and 12S 0/0 0, with 50% mor­ tality occurring between days 6 and 7 at 8 S 0/0 0 and on day 7 at 12 S 0/0 0 (Figs. 15£and GJ. Morphological development did not progress beyond fonn IIand was morc rapid at 12 S 0/00 than at 8 S0/00•

Survival of larvae fed Artemla at 8 and 12S 0/0 0 was similar, with 90,1 %and 86,7% sur­ vivalnt 8 S 0/00 and 12 S 0/0 0 respectively at the termination ofthe tri:ll(FIgs. lSf'and 1/). Morphological development was faster at 12S 0/00, where 48,7% of the surviving larvac reached fonn 111 lit the termlnation of theexperiment, compared with 14,6% at 8 S0/00• 124

Results of the Investigations Into the salinity preferences of the early larval forms (Figs. 10-15)

The following code has been used to Indicate the percentage of surviving larvae present as a given larval tonn:

~ Fonnl ~..",.~~ Form I ...... , Fonn • ...... •••••• •• ••••• 11.1.II ••••••. ••••••• ••••••II•••••••••• 111.'111111 Fonn IV ~ FonnV 125 126

• ::::::::::::::::::::;:::::::::;~~ ... ~ u v,.."..."..",.",.A • ~ ~l ~ Q ., ~ V",,"""''''''.Al! 11 ~~ ~ .. % J V~""''''''''''A J V,.,.,.~. " J ~ ~SSSlW_ N I • I ... J V,,.,,,.,,.~ • J V,..".."..".~ ~i ! ~ ~ ...~."..."..".~ .. *~ 0 ~ I ..."..AI8BB888S88 " N I I ~ • • ~,,.,,,.,,..,,,~ ... f ~,,.,..,,..,,..~ • :. ~ l7',,,,,,,,,,,,,,A Wlc! T ~ c ~ V,,,,.,..,,...ASt ., .. I&. J i1 .. ~ • " III 0 J • ~ N I t I • f ... , v,,..,,..,..,,.... • 1 V"AIrT""~ "1 ~ v,.,,.,."'~ ., &II • I ~S"SSM " I ~ ... i

.. /'Atlung .. 11110/ IIA1W'l ,,..~ .. 11110. ,.4,., 127

v"",~V,,,,,.,... V,,,,,.'A V""'~ ~

V"",,.'A V",,~~ V",,.'A ~~

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v,,.,~~ . v"",~ ... v""'''''''~ • V"",..... "'1 .SSS~ • "

\ WIG, ...,., 128

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4.5.4 Developmental morphology Results presented lire based on the examlnatlon of a Iimlred quantily of material and arc incomplete forM. scabrlculum and M. australe. Data was collected mainly from larvae in multicontalner rearing units, during the last two months of the project. Generally the larvae passed through developmental forms similar to those summarised from :available literature inChapter 3, seclion 3.8.2. Forms VI and VII of M. leptdactylus and /tI. rud« continued forextended periods. during which lime a greater variety of morphological changes occurred than for M. petersll. which reached post-larval forms in about half the time of the other two species.

In addition to the features of the morphological forms listed inChapter 3 section 3.8.2, the following data was collected for the indigenous species:

Form I Telson. In all five species there are 7 pairs of posterior marglna] spines. the inner pair being very small (Fig. 16AJ.

Antenna. Endopod unsegmented. exopod annulatcd, Almost unchanged in form II (Fig. 16DJ. Nodata available forM. scabriculum.

Chromatophores; TIle overall appearanceof M. leptdactylus, M. scabrlculum and H oustral« larvae is almost transparent. Chrornatophores arc poorly developed inM. scabrlculum and M. australe, and almost entirely absent in M. lepldactylus. Macrobrachium rude and AI.petersll larvae haveagrey brown appearance anteriorly and medially.This results from well developed chromatophoreson the third abdominal segment. a single dorsal and paired dorsolateral chrornatophores as well as well developed chrornatophores on theanterior region of the carapace. below therostrum and between thesupra-orbital spines. These chromatophores consist of red and blue pigments in AI. nul« andM. petersit. The dorsal chromatophore or ltI. rude is more prominent than the dorsolaterals, while the dorsolaterals ofM. pcursll arc more developed than the dorsal. Chromatophores ofM. lepldact)'llIs. M. scabriculum anll ltI. australe arered. In M. leptdactylus the red chromatopbore on the eye is the only proml­ nent one. Yellow colour was associated with the maxilliped bases ofM. nit/c. M. ptlersll and M. austrd«

Form II Telson. Eight pairs ofsplncs present on the posterior margln were recorded for. M. Ilelmll. M. rude and Itt. scabtlculum (Fig. 16CJ. No data is avail3blc for Itt. Irpldacl)'IIu :lnd M. australe. 132

FIGURE 16 A-I Morphology of the larvae of theindigenous Macrobrachium species.

LEGEND

A Telson ofItt. leptdactylus, form I

B Perdopods I nnd 2 buds, Itt. Irpldacl)'/IIS, form I

C Telson of Itt. rude, from II

D Anlenn:l of!tf. rude, form II

E Telson and uropods ofItt. Irl'ldacl>'IrIS, form III

F Antenna of ftI. lcpldactylus, form III

G Pcreiopods 3 and 5 buds, /II. peterstt, form III

II Tclson and uropods ofItt scabnculum, form IV

Tclson of /II. scabrlculum, form V 133 134

CI,romalOpllores. Larvae ofM. leptdactytus show onlyone well developed chromatophore on the ventral eye stalk. the chromatophores on abdominal segment 3 being poorly developed. Macrobrad'/IIIf1 australe larvae also have poorly dewloped chromatophores on abdominal segment 3. bUI one pair ofchronutophores on the antenor carapace below the rostrum is fairly prominent, These chromatophores are paired In M. scabriculum as well, whlle in M. petersllllndM. rude they appear fused.

Form III

No data available for M. scabrtculum.

Clrromatop/rom. M. It!plc/actyills larvae hove the chromatophores on abdominal segment 3 finely branched and red. but not prominent. wilh Ihe chromatophores on the ventral aspect of the eye and a pair on thelnterlorcarapace below the rostrum, being themost prominent. InM. rude and M. petmlllarvne the chromatophores on the 3rd abdominal segment and theanterior carapace are well developed and a mix tUN of red and blue piS· ments, Chromatophorcs of the Ihlrd abdominal segmcnl of M. rude have distinct centres, while in AI. peterstl they arc widely spread, but with small centres. Macrobraclr//lm australe has a poorly developed dorsal chromatophore on abdominal segment 3. while the laterals arc more developed. Abdominal segment 4 of M. rude, M. petenu and M. australe bears a red chromatophore ventrally.

In addition toM. lepldactylus, M. australe has prominent chromatophores associated with the eyes. These chromatophorcs arc less prominent in M. petersii and poorly developed in Itt. rude,

Antenna. Three segmented flagellum (folg. J6FJ.

Peretopod. Duds of 4 and S. (Fig. 16GJ.

Telson and Uropod. EXOIlOd of uropod wilh six setae in all five species (Fig. 16£).

Form IV

No datnls available for M. australe.

TclsOII. The spines on Ihe posterior margin are reduced 10 S,,,,irs (Pig. J6/IJ. This number remainsconstant until Form VII, when the number may be reduced to 4 pairs.

Clrromatnp/rorts. Abdominal Stgmenl 3 ofM. pt'tcrsll13rvac bc:us well developed, densely branchedchromatophores c:o,ubllns .,f red and bluecolman. The same c:hrOI1\.lIl1phorcs arc found inM. ltp!dllctyilis and M. srnbrlcul"", larvae but these are red in colour. These are small In M. scabriculum, while In M. '"plc/aetyills the chromatophores co...ct :tl:rrllc IIrea 135

FIGURE 17 A- J Morphology of the larvae of the indigenous Macrobrachium species.

A Telson ofM. /(l'lda(."I)'llls, form VI

D Antenna ofM. leptdactylus, form VI

C Rostrum ofM. petersit, form VI

D Pleopod buds of M. pelusll, from VI

E Tebon ofM. lel'lddcl)'llls, form VII

F Rostrum of M. rude, form VII

G Rostral tooth lind setae of M. rude, form VII

II Rostrum of M. lepldactylus, form VII

Plcopod of M. rude, form VII

J Pcrclopod I chela of M. rude, form VII 136

..,

.

w ..

CJ

Q 137

but centresare small and the dendrites poorly defined. Red chromatopborea arcpresent on the ventral surface of abdominal segment 4 inM. lepldaCI)'/IIS and M. petersit. TIlesc were not recorded for M. scabrtculum larvae, Prominent chromatophores occur on the antedor carapaceof the three species mentioned. These are present as a red pair in M. ItpldQclyluJ, brownish inM. scubricuillm and II dense mass of red and bluc dendrites in M. ptttnlL Additlon:ll prominent chromatophores arc assoclated wilh thecyes ofM. peunll and M. Itp/daclyills, lind the pereiopod endopods ofM. peltrsllandM. scabrtculum.

FomiV No data isavatlable for M. australe and M. rude.

Carapace and tostrum. A single tooth Is present on rhedorsel margin of the rostrumof M. lepldactyiu: and M. peterstl, uswell as II single epig3strlc tooth In both species. In M. peterstt therostral tooth, epigastric tooth lind supra-orbltal teeth have serrations on the inner distal margin,

Telson. The outer pairofspines on the posterior margin Is longer than the other 4 p:lirs (Fig. 16/).

Pleopods. Bulges on the ventral abdomen in the positionof thepleopods was recorded In someM. peterst; and M. lcpldactylus larvae of this form.

Chromatophoes. Although M. lcpklactylus and M. scabrlculum larvae have :111 overall transparentappearance. increased chromatophore development W:lS recorded in thisform for the lattertwo species nnd for M. pClersil. The chromatophores associated with theeyes are prominent in M. lepldactylus and M. petersil but less prominent in M. scabrlculum larvae. TIle chromatophores on the an tcrior region of the carapace arc avery prominent mass of red and blue dendrites in M. pctersil: These chromntophores are paired InM. scabrlculum and M. lepklactyliu, with the colour rJnging from red to brownish In M. scabrlculum, lIlId only red In M. /l:pltlact)'llIs.

Chromatophores -m abdominal segment 3 are prominent ill M. I'tlersl/. where the dorso­ lateral pairhas distinct centres and few dendrites nnd in all three chromatophores the dendrites have nfragmented nppcarance, These chromatophores are present in the other two species, but developed to a lesser degree. Abdominal seSIIlent4 bean a single. red, vcntrnl chromatophore in all three species, densely branched In M. ICl'/dact)'ltls. and C.drly 138

prominent in this species IIncJ M. Stabr/nllum. This chromatophore is less prominent In M. ptlcrsll. TIle perelopods ofM. pttmlJ lind M. seabncutum have fnirly prominent red chromatophores on the endopods,

Fonn VI No datn isavailable for M. australe.

Antenna. The endopod ofthe nntennals as long liS or slightly longer than the exopod and the scgmentatlon has Increased to S or 6 segments (Fig.17B).

Rostrum andCarapace. No data Is available for M. scabrtculum:

A single dorsal rostnl tooth and single epigastric tooth were recorded for M. rude and M. petent: (Hg. J7C). Setae nrc sltuatcd anterlor to the base of the rostral tooth, numbering 2-3 forAI. I'tNC,slland 3-4 forM. rude. InM. pttmll two very smatl setae were recorded anterior to the epigastric tooth. M. le-pldacl)'llIs larvae have2 cJorsal rostral teeth and 2 epigastric teeth, although one of these epig3stric teeth may in fact be a proximal rostral tooth. Setae were situnted anterior to the fir-it rostral toothlind numbered 1-2.

Perelopods. Rudimentary development of a chelate condition was recorded only for M. petersil. The development of this condition appeared to be delayed in M. tefl/derc/yills, M. rude andM. scabttculum:

Pleopods. No data isaV3i1:lble forM. scabrtculum:

Abdominal bulges liS wellas unlramous buds lind 3 pairs ofl'lool'OOs were noted for M. leptdactylu: larvae, M. peterstt larvae hnd biramous buds on 41,alrs of pleopods initially fFIg. 17D), butlater these developed intoexopcd and cndopod, wllhout setatlon. M.ntclc larvae de­ vclopcd uninamous buds in this form although biramous pleopods with setese cxopods were also recorded.

Telson. M. Itpldac/ylll.f, M. rude, M. srabr/cuillm nntl M. ptttnlll.1rv3e have S &>r1 of posterior marsJnal .plnes with the outer p:lir donS-lted, almost twice the lenglh of the other spines. Short splncs on the posterior laleral mnrsin of the relson occur in the above­ mentloned species and number 2 painfH,. 17A). 139

"'ramarop/lOrt! Development or ehromarophores ·juring the61h and 7th Iarvll ronns differed In degree rillher than in kind,and chromatophore development for both fomls Js presented here. No data is nvailable for M. scabriculum form VII brvao.

The overall appeilrlnCe of the lurvac Is transparent, to light pink, with tho exception or M. ptltrsl/ I:uvae, which are a grey brown colour. Chromatophores are present on the eyes and/or eyepeduncles ofM. /tplc/aCI)'/III, /.t. rude, At. prlmll and /.t. scubricuium (form VI). These are prominent in At. /tpic/aelylllland M. pelersll. where a red branched chromnophore appellrs In Itt. Itp/daef)'/lls and u brownish chromatophore In /.t. ptfenlL

The anteriorcarapace bears prominent paired red chrom:ltophores In M. /tpldael)'/m and very promlnent chrornatophores InM. ptrtrs//. which extend dorsally onto the carapace and venlrally 10 the eye peduncle, wllh on overall brownish appearance. These chroma­ tophores all: prominent but less developed InM. scobrtculum (form VI) and consist of. red. blue and brown mixture of plgmenls.

Single chromatophores occur on the perelopods ofM. Itplc/aefY/us, Itt. melt, Itt. pt/mll and Itt. scabticulum. TI1CSC are present on some or "IIof the endopods ofperelopods 1-5 ofM. petentt, M. lepldactylus and M. scabrlclI/"m,nnd arc fairly prominent In ."'. peterst; but more prominent Inlei. srabrlc"III"'. Prominent chromutophores occuron the long segment ofpcrelopod S ofM. lepidactylus and M. ruelt larvae, being red Inthe former and red and blue In the latter species.

Prominent red chromatophores occur In M. lepldactylu« on abdomlnal segment 3. In M. p~l~rslllarv3e a mass of red and blue dendrites covers most ofthissegment and three distinct centres may be present. AI. scabticulum larvaedo nol have as prominent chroma­ tophores on this segment. "5 on the ventral surface of abdominal segment 4. The chromate­ phore on abdomln:J1 segment 4 Is prominent In all four species mentioned.

TIle chromatcphores lit the basis of the antenna and antennule Ire fairly promlnent In M. /('plc/aclylu!

Form VII Nodata Isllvall.1ble forM. scabrlcu/"", and M. austml« 140

UuaI'Qct and rostrum: The rostrum bears a single dorsal rostral tooth in M. p~ttn/lllnd M. rude wilh (rom 3-5 setae anterlor 10 IIlI: tooth (Jolgs. J7FamI GJ. The epiS:lSlric 100lh ofM. pctusi/ has 2-3 very smnll setae anterior 10 iI. Macrobfrlcllllltll teptdacrylus Imllc have 3-4 dorsal roslral teeth wilh (rom 1-2 setae assoclated wilh each tooth (Fig. 1711). The episaslrlc tooth ofM. It"pidactyills larvac hu 3 small selac below it.

Antenna. The endopod is longer than exopod with more: IIum 6 segment••

Peretopods. No chclll development WIlS recorded for M. ItpldQct)'lus. Macrobracl,'um nltl, and M. pttml/ I:lrv~e had rudimentary todeveloped hUgils equal In length) chelae on perelopods I lind 2(Fig. / 71J.

Pleopods: Appendices Interne are present onM. rude llnd M. pctmll endopods, with the exopods and most ofthe cndopods bc:lng setose (Fig. J71J. However variations were noted for the latter two species and csrecilllly(or M. le/Jldact)'lus larvae. Incomplete development of the cndopods liS well as asymmetric: appe:lrnnce of structures were recorded for M. Itp/. dactylus larvae :"\11 only a few showed full development of pleopods with appendices lntema and sctose exopods lind cndopods,

Telson. Posterior ntugin:lI spines number from 4-5 palrs, the outerpair being twice: the length or more, of the other spines. Short lateral margin:ll spines occur, one pair distally and I pair medially. Inaddition to these spines the elongated spines on the posterior margin have short spines situated at their bases (Hg. 17£).

4.6 POST·LARVAL REARING

A total of 11959 post-larvae of 4 Macrobrrzclll"m species were produced during the course of the project. The numbers for each species were as follows:

• M. row,bugll : 2424 (Juring I'"ast / 01 tire larvalrtarl,,, progN17,me and J32Jduring phas« 2. • ",. pctersl/ : 806.5 (Juri", pl,ast 2 • M. rud«: ISduri", phas« J m,d 114 duri"8l'ltast 2 • Itt. l(plc/dctY/lls: /8 tlur/II, p',ast 2. J4J

M. rosenbc'lll Out ofa total 0(2424 post-larvae produced in phase I, 1000 were reared us described in Chapter 3 section 3.1.1. Menn temperatures in the re:tring tanks ranged from 26,7-28,l oC.

Survival after6 monthsof rearing was 18,8% of the initialtotlll, by which time the prawns ranged from 16-123 mm in length, taken from eye orbitto telson tip. Mean lengths (or the prawns in the3 fibre glnss tanks (l,6 x 1,6 01) mnged (rom 107,1-111,8 nun While prawns in the original tanks had a mean length of81,3 mm.

The stocking density at this stage was between 18 and 33prl.wns/m 2• Prawns reached sexual maturity between 8 and 9 months after hatching, with males showing sexual characters before then the appearance of berried females.

During phase 2,1323 M. roscnbergil were produced, The Initial batchof these were lost shortly after stocking in freshwater.

2 The remaining post-larvae were stocked at 2014 01 and reared for 3 months, as described in Chapter 3section 3.11, until most were lost by accident. At a mean temperature of 27,60 e for this period, post-larvae grew to a mean length of3,8em from eye orbit to telson tip.

M. rude 111e fifteen post-larvae produced in phase I wen: reared ina single tank at a stocking density of 102/m2for roughly 80 days, after which they were transferred to the /II. rude communal tank. Afler 80 days of rearing the nine survivors ranged from 3-6.5 Col wilh a mean of4,5 em, an approximate rate of0,6nlln/day over Ihls period. Temperatures during the period ranged from 27,5-31°e, with a mean of 29,4°C.

M. rude post-larvae produced during the second phase of the rearing programme numbered 114. These were stocked at 706/m2 inasingle tank. High morlulilies occurred in the lank after 30 dllYs. No further recants lireavallable on these post-larvae.

M. pctersll M. I'clersll posr-l:trvae were only produced during the second phase of the rearing pro­ grnmrnc. A 101111 of 8065ftI. pc/Cnll posl·larvae were produced during Ihis phase. These J4Z

were reared asdescribed in Chapter 3 section 3.11. Data Is presented (or a batch of 2257 post-larvae which were initially., stocked in3 x 47 2 and I x 115 2tanks, lit densities be- tween 2540 and 3880/m-. Mean temperatures in these tanks ranged between 27,4 lind 28,60 C. Prawns were held under these conditions (or 3-4 weeks after which they were transferred to asingle tank. Survival until transfer was 77,3% of the initial total. Stocking density in thesingle 250 2 tnnk was 3180. At 6 months of age the density had declined to I 5S0 juvenlles/m2,and survival was 37,8% of the initial total. Juveniles ranged in !izefrom

15-35 nun with II mean of 20,2 mm, an approximate rateof0,11 nun/day. Temperatures in the single tank ranged from 26-30,SoC with n mean of 28,20C. At this stnge juvenllc males could berecognised as M. petersl! by the chela development on legs 1 and 2.

M. lepidnctylus M. lep/dactyl/ls pcu-larvae were produced during phase 2 of the rearing programme, The

18 post-larvae produced were stocked In II single 472 aquarium tank at II stocking density of I201m2 and reared as described in Chapter 3 section 3.1 J.M. ltll/dactylus post-larvae showed hardly any growth and none survived after 41 days. Temperature ranged between 20 and 290C with amean of 26,1 °e.

4.8 TREATMENT OF DISEASES

Disease conditions, similar to certain conditions described intheliterature referred to, were encountered during the course of the project. However these did not take on epidemic proportions, lind the losses of prawns which occurred were hugely 11 result of aggressive interactions, high stocking densities among post-larvae, and possibly inadequate nutrition.

Higil mortalities among larvae, with short survival times, were characteristic of the early stagesof the project. Subsequent experience revealed that these mortalitics were the result of unsuitably low S3linilics for rearing the larvae. Larvaeexamined under a microscope showed no obvious signs ofdisease, andonce the salinity of the culture watcr h3d been raised nbove 8 S0/00 survival improved.

Subscquently,losscS3g:1ln occurred amongl3rvac in a muhlcontalner ~arins unit. As previously mentioned (Chpntcr 3, section 3.12) these Il\rvac were trc3ted for 2 hn with formalin at 40 1\I~/2 prlor to transfer to another rcarins unit. Although survival Improved after transfer, low Icvcl mortalities continued. The condition of thc rearing unlls nt this lime mny have influenced survlv:l1 as these unitshad poor circulation of water throurh the 143

biofllter, and had been in use for approxlmately 4 months at the IImc. As J)r~viously mentioned (Chapter 3, section 3.4.2) the mulrlcontalner unils were fitted with lengths of 20 mm polyethylene piping, under the gravel, to improve water circulation at a later stage.

Mortnlilics 1I111ong post-larvae in the second phase of the project can be directIy attributed to the high slocking densities in the holding tanks. Post-larvae lind juveniles bee..me opaque and moved with dirliculty before dying, often in the process of moulting. Post-larvae were not treated chcmically and the mortulilics decreased to a lower level with the decrease in numbers.

The devetopmcnt ofa "rust" colouratlon among adults was noted during the period when breeding stock were housed in observatlon tanks equipped wilh mechanical nitration unlls. Certain Iemeles wilh this colouratlon produced orange coloured eggs which gradually dropped from the pleopods, while other "rust" coloured females produced normally coloured green eggs which proved to be fertile. The cause of the condition isuncertain, but it did not appear 10 be fatal, The condition appeared again in the latter phase of the project, mainly among M. petersllnnd M. scabrtculum adults.

Black gills appeared among individual M. rosenbergtl breeding slock and at II later stage among Itt. lepidactylus adults. M. rosenbergll with black gills were also opaque in appearance. The gill caville! were examined by Dr. J. Van As of the Zoology Department of the Uni­ versity, whofound that the gills were inlestcd with parasites.Chemical treatment wilh a 0,25 m~/£. Formalin and 0,1 ntgJ'J. malachite green combination, and later a 0,3 mg/£ treat­ ment with an Israeli compound, contalnina furazolidone and chloramphenicol, had no effect on theM. rosenbergii adults which subsequently died. Macrobracl,illln lepidactylus adults, which had only blackened gills bUI notan opaque appearance, recovered after a I week treatment of formalin lind malachite green at the same concentration as above. Rcsi­ dues of black coloration were lost with moulting.

Drown patches appeared on the exoskeletons of M. 1('!,ldact)'lus adults in certain holding tanks. Examination of these prnwns by Dr. vlln As revealed nulll filled cavities lying below eroded p3tchcs ofthe exoskeleton. 15013tlon of these prawnsinwater treated with an Ismeli preparation conlaining chloramphenicol and furazolldon e.at 0,3 ml%le. was ..UcIIlJlled. TIle hrown palches were lost wit h moulting and it Is possihle thaI thechcmical treanncnt was unneccssaty, The incidence of this condllion decreased once thehiofilters in the hold· inlt tanh were buffered by the addition of crushed sea shells. 144

3.9 SUMMARY

• Anlllyses of culture water showed that the biofiltratlon systems employed were functioning s:alisfuctorily,generally providingsultDble water (,ualily condltlons forbreeding and n:aring theMacrob'acl'/Ilm species Investigated. although. deterioration cUd occur In larval rearing unllslifter prolonged usc.

• Specimens of the indigenousMacrob,aclllwn species lent to Professor LB. lfollhulstLelden, Ne,herland~,for ldentlflcatlonwere Identified by hlll1l1l M. /tpltlac/yllls, /tI. rude, Itt. scabr/clI/llm lind Itt. alls/",/t. /tI. perenlilidulls could beseparated from those of Itt. scabrlculum once the tatter species had been posl/vely ldentlflcd.

• Results of the solar heutingexperiment lndleate that the apparatus used could heat approximately 50002 ofwater to temperatures suitable for culturing prawns, provided the reservoir pool was adequately insulated.

• Prawns collected from the wild survived and reproduced in Ihe Inborntory, al­ though bothsurvival and fecundity tended to decrease with lime in e:tplivlly forcertain of the species. This could be ascribed largely to aggressive inter­ actions and possibly inadequate nutrition.

• Generally poor larval survival was encountered during the first phase of the project, wilh the exception ofM. rosmbcrgllllnd M. rude larvae.

• TIle results of salinity preference experiments showed th3t the larvae of the indigenous species require saline conditions for survival and development of the early fonns, with minimum requirements of between 8 -lOS 0/0 0 •

• Surviv31llnd morphological development improved for the larvae of :III nvc species, once the S3l1nily of th~ culture medium used was Increased to levels above: 8 S 0/00 wilh the most satisfactory levels being above: 10 S0/00•

• I~earlng techniques employed were: adequate for the produeuon of l,o'C·!:tme of

M. rosmbC''KII, M. /(fllt/aerY/lls, M. rude and M. f1rtm /L 145

• Morphologicallkvelopnu:nt of the indigenous species followed patterns described (or other Macrobrachlum species.

• The period required for completion of l.nnl development W.:IS shortest for AI. petmt! and longest forM. leptdactytus lind AI. rud« larvae, where the period taken was approximately twice that for M. ptlmll.

•. Althollnh losses occurred liSIIresult of disease conditions, these did not reach epidemic proportions during Ihe project. Chemical treatment and Improved manaGement practices were employed in the treanuent of disease conditions wilh some success, 146

CIIAPTER FIVE

DISCUSSION

The production of freshwater prawn post-larvae Is usually lin intensive culture process ln­ volving a large degree of environmental control and relatively high densities of prawn larvlle In culture systems (Johnson, 1980). The need for such culture systems for post-larval grow­ ou t, under temperate climatic condilionshas been mentioned InChapter 2, section 2.1.1.

Most MacrobroeIJ/rm, species nrc Inhabilants of tropical regions (Goodwin and Hanson, 1975) lind therefore require hi;th water temperatures for survival nnd growth. There Isagreement that 28°C isthe optimum level for M. rosmbugl/ (Goodwin and Hanson, 1975) and most Macrobrachium speclcs require temperatures In the range l>f 25-30oC (Guest nnd Durocher, 1979). Thusthe maintenance of high water temperatures IS:1I1 Important conslderatlon for the culture ofMacrobracl,/um species.

Culture water was heated by means of immersion heatersduring the present study.with satisfactory temperature levels being malntalncd. The development of electronic conlrol units for finer manipulation of temperatures is considered to be Important for experimental work. as theaquarium immersion heaters tended to range around a given temperature and in many cases, these could not be set exactly at a selected level.

For the intended commercial production of post-larvae involving much larger volumes of culture water, reliance on electrical energy for heating will of necessity have to be reduced. Of the threemajor alternative energy sources listed inChapter 2,solar energy is the most likely alternative additionul source in the Transvaal, with ilsdry,sunny winters. McSweeney (1977) reported tlu heating of 5 000 gallon (22 5002) tanks of water for about nine months of the year using solar energy.

In the present study, 5 0002 of watercould be heated to temperatures suitable fur culturing Macrobrachlum species (2S-30oC~ thus dcmonstratlng the fCJs:Jbility of this source or ener~y as an addltlonal means of heating culture water,under Transvaal conditions, However, certain modifications would be required wlth respect to thecomponents of the system used In this study.

The components of the system used toobtain the results in Hgrm 98 arc not Maitlhle for use in a pool containing prawns for the roJlowlns reasons: 147

The doublelayer of plastlc on the surf3ce would make managemen! of the prawns difficult

as well liS decrease light penetrutlon. The presence of theheal exchanger in the pool would hinder managemen! of the prawns byobstructingactivities such asneUlng and c:Je3nlng.

The solar healed pool could however act as II reservoir of heated Wilier for other pools containing prawns. It Is thus recommended that the solarhealed pools should be silualed Indoors 10 reduce heat loss. Solar panels could then be placed outslde and would be connec­ ted to the pools In which hen t exchangers had been placed. This system would reduce Ihe need for Insulating the water surface wilh ndouble layerof bubble plnsllc and ellmlnate the need for he3t exchangers In the prawn-contalning pools.

Knowlton (1974) drew attenlion to Ihe effect of photoperiod on larvnl metamcrphosis 10 post-larvae,TIlls cffect was recorded III Ihls study. and the need for photoperiod control in larval rearing Is considered lmportant for year round 13rval production,

Photoperiod conlrolls required for the continuous production of berried Mtlcrobrac1llll/ll females (Wick ins and Beard, 1974; Dugan '" 01.• 1975; Read, I982.) Read (1982) found

that the onset of the reproductive cycle In M. pC/~rsl/ coincided wilh :l rise in water tempe­ rature end an increase in daylcngth,

Records of the indigenous species collected for this study suggcst tha}, with the exception ofM. rude, the breeding season isduring the summer months and that photoperiod control would therefore be required for year round production of berried females. As reportedin Chapter 4, section 4.2.1, berried females ofM. rude occurred in winter in Lake Cubhu, and the first batch ofM. rude collected during the slimmer months came into berry in the laboratory wilh theapproach of winter.However, during the second phase of the I:m:1I rearing programme, berried females of Ihis speciesoccurred during the slimmer months in the laboratory aswell. It is thus possible thatM. mde adulls breed throughout the year, with peaks al certain tlmcs of the year, and do not require n'bed photoperiod to stimulate reproduction.

1.I1~ht Intensilies in the vicinity of the larval rearing units r.lIIgc,1 from 2 000 - 2 800 lux, wlthln the recommended Iimil~ of 200 - 4000 lux set byl.iao ancll.iu (1982).lnllddllion the usc of whilc coloured con talners, allowlnj; light pcnetratlon, did not appear to be dc:­ trimental. Neverlheless, In the light of lIv:lllable inforrn:tllon (Sandlfcr et al..• 1977; C1lao and Llao, 1971; Read, 1982; Schoonbce,ptrs. comm.) the favmmblc C:(feci of Iw~rlli£J1t 148

on the orientatlon of larvae 10 food,llnd the need for opaque sides on rearing contalnell should be taken Into account in future modification of theculture systems employed In thls study,

Intensive to very intensive culture syslcms housed Indoors, lind ulilislng bloflltratlon systems for the malntenanee of water quality, have been proposed and Investiglltetl (or use in the culture of tropical prawns such liS M. rostl/be,,1/ in temperate climates (Forster lllld WickIns, 1972, S:andlfer and Smilh, 1978),

Closed water reclrculatlon systems employlns blontlcrs forwater purinclltion were de­ veloped for most of the culture systerm used In this sll~dy. Resulls obtained are considered satisfactory nnd lend further support to the sulCnbilily of these syslems for use In temperate climates, where hatcheries are situated lon8 distances from natural supplies of sea water.

Sick and Beaty (1974) found that the hardness of the water used to prepare saline culture media for larvae W:lS an important factor. From the work of these authors It appe:us Ihlll a hardness of SO 111F1~ Is dctrlmcntal to the survival of the early larval forms ofAI. roscl/bcrgli. Borehole water used in the present sludy, of hardness 12 mgJ~, proved satlsfactory forthe preparation of 5.,line culture media. However the boreholeand tap-water supplies were occasionally mixed, as the supply systems were connected. "'acrobraclllulII roscllbC'rgll larvae nevertheless reached post-larval form with reasonable survival rates In the present study, at a lime when the supplies were mixed. Allhollgh the hardness of the tap-water (38 rnrJ~) is below the 50 mrJ2 referred 10,it is surprising that noobviousdetrimental effects occurred. As the brackish waterused in this study in:II1Y case had a hardness of above I00 mgJ~ (Tablc 2). it appears that the hardness of the freshwater supply used may - in fact be less critical than W.IS previously anticipated.The effect of freshwater hllrdness In this respect requires further investigation. Nevertheless, water wllh a low conductivity anti hardness ispreferred as replacement walc~ inorder to counter rhe effects of blofiltration processeson conducllvlty, resulting from increasing Ionic concentratlons.

The lise of nrtificlal sea salts for the rca ring ofMacrobrlJe!llll1n I.mae Wl1S found to be s.,lIs­ f:tctory In Ihi!! project, liS well as In previous Investigaliolls (Sandlfcr anti Smith, 1975, 1978). Therefore the fcas.,bllily of :l Macrobrrzc/lium h:ttchery at the present life, roughly 700 km from the nearesl ~lIJlplies of naturnl seawater Is confirmed. 149

Greater control or5.1linily levels In rt~rlnl units is required, liS nuctunllons occurreddurin, the rearing programme for lhe following reAsons:

• AsQ mllil olovt>r/ll1ln,"lilts when replacing WilIer losl.

• hi/1m overflows occurred liS Q result 01 blockl", olollliet screens. • Whtlllan'Qe were trallsltrrtd10rtarlllguntts In IrtshwQltr.

The latter was or minor Importance usu.llly, because or the small volumes of woterInvolved. Possible solutions would be to equip rearing unlts wilhsupply pipes Iltted with boll·valves so thnt water lost through evaporation Is replaced mechlllllc:llly rather than manually.In addition, larvae could first be accllmtused to the correct snlinlty before transfer to rearln. units. The problem of blocking of outlet screens would have to be resolved by further modl­ flcatlons to Ihe design of the retiring contnlners, as repeated cleaning of the screens Is a time

consuming rllclor. TIIC outlets wert In r:lct modlfled towards the end of the project by In­ c1udlng screened bypasses on the sides of theoutlet plpes,lIbovc the basal outlet screen.

Although the literature reviewed contained few references 10 theI'll requirements forthe culture of freshwater prawns, pillevels of 7,5-8,5 have been recommended for larv..1rearing (Ling and Costello 1976;Suwannatous lind Sukapunt 1980). Fnilure to con trol pit wllh II resulting low plllcvcl (pll 5,5) has been lmpllcated In reduced reproductive ability of M. rosenbergil (Wkklns lind Beard, 1974). Since bloflltration systems may cause lowered plt lcvclsas II result of nitrification processes (Wickins and Beard, 1978; Spotte, 1979;

Stickney, 1979) this factor requires ..ttentlon from II management point of view.

The decline in pllas a result of nitriflcatlon processes was underestimated in freshwater tanks, as reported (Chapter 4, section 4.4.3), The importance or burfering biofiltrntlon units becomes even more critical when the water supply has a low buffer capacity, such as the borehole supply used. The use ofcrushed sea shells in thisproject was S:llIsfactory for fresh­ water tanks, bUI was not employed In Ihe hr:Jckish water rearing units. Although there WIIS ndecline In pll in the latter units with tlme, Ihls was more8r:Jdu3111nd the pH values reo maincd wlthfn accept..ble limits of 7,5·,8,5 recommended In Ihe literature by lhltl and Costello (1961) lind Suwannntous lind Sukapunt (1980). Ncverthc:lc~., Ions ten" usc of such bloflltrntlon units fer InrY31 fC:arlns could result In low pl] VaIUCS,llllll the usc of hurfcrs such as sodium carbonate or sodium bicarbonate.recommended bySpotte (1979),could be Invesrlgated. ISO

ImbalancesIn conditioned bloflltratlon systems Are known to cause increases in ammonia and nitrite levels, which may even become permanent (51)0110, 1979). Ammonla " the most toxic of the nllrogen compounds whleh lire present inculture systems (Spotte, 1979) and relatively low levels of Ihis compound,ln the region of 0,1 mgJe NII4-N ore considered unsatlsfactory In prawn culture systems (Wicklns and Beard, 1978).

Although measured ammonia levels were generally satisfactory, (epproxlmutely 0,1 msll as total NIf4-N), failure 10 monitor ammonia tevelson II regular bllsis during the I,resenl project created Blips In the data avallable, for example addlllonal d:ltr. which could hllve been obtained during the conditonlng process of blolilters. TIle data available wasmainly from nlrelldy condltloned blorllters, and served primarilyos an Indication that levels were not rising.

Data for ammonia levels during the condilloning of the first 3 single container rellringunltl, which were conditioned by the direct re:lrlng of larvae in these unlts is however available (Chpnter 4, section 4.4.3). In spite of mortalities of larvne liS a result of low ~lInily levels, the ammonia levels In these units were acceptable overan eighteen dny period. Thissuggests that the ren,ring techniquesemployed during conditioning were satlsfnctory.

Although filter capacities were not cstlmatcd in the design of blofllters used, the 8enerolly satisfactory ammonia levels suggest that the size of the filters was adequate for the purposes intended. Detailed ann lysis of the culture water from the three multl-container fellring units, just prior tocompletion of the rearing programme, revealed that a build up of ammonia occurred at the time (Table 2). Undesirable levels r:Jnglng from 0,2-0,4 mpJe were recorded. These nrc considered to be the result of the reduction in the water exchange rate. to minimise the loss or Anemia n:mplil to the filter. as well as the prolonged period of continuous use (4-6 months).

While ammonia isthe most toxic or thenitrogenous compounds encountered In euhure systems, the presence of nitrate and nitrite in culture systems clln give risc 10 loxlcity lIfrects nt certain I""cls (Annstmns ~I al; 1976; Wickins nnd Dennl, J978; Spotte, 1979). Nitrate Is not acutely toxic lind relntlvcly hlSh levels of 20-50 mgJe h:ave been considered themaxl­ mum acceptable limits for nqunria and prawn culture system\ (Spottc, 1979; \Viddns lind Beard, 1978). Nitrite toxicity is afrected hy I'll lmd salinity (5polle, 1979: ""nstroIlS tl aL, 1976) nndSpotte (1979) conslden II unlikely (hlll It would pose a threat to.,nIrl1:l1s held In brackish or seawater nqunrin. Upperaeceptable limitsfornitrite r.tnRe frolJl f),1 III'" (Spottc, 1979) -I mvJi (Wickins And lIeArd, 1978). lSI

Nitrate plus nitrile levels reported in detailed analysesof water from the multlcontalner rearing unils(TQb/~ 2) can be consldered to represent theN03-N levels as a separate value for N02-N was less than 0,1 mg/2. Thus the NOJ-N levels reported for these units were slightly in excess ofthe 20 109 N03-N/2 Iimil suggested by Spolle (1979) but well within the SO mcl2limit chosen by Wickins anti Beard (1978). Although Lemnasp. was employed for removal ofnilrogen in freshwater tanks, no at tempt was made to remove nitrate from the salinewater inlarvnl rcnring units. Brackish water pl:tnts need to be investigated for this purpose, The use oflemma sp. in freshwater tanke appenred to be satisfactory, if thecopious quantities of Lemna harvested from freshwater tanks Is taken liSan Indlcatfon,

Failure to monitor levels of nitrate anti nitrite on n conlinuous basis throughout the study created gaps in thedata available on the functioning of thebloflltratlon systems, and more attention to these aspects of water qualily isrequired.

Aeration in culture tanks was kept high, and it was therefore considered unlikely that either oxygen depletion ortoxic effects of carbon dioxide would occur. Nevertheless, dlssobed oxygen concentrations should be monitored to insure that adequate levels are maintained in the filter beds.

From the literature referred to, iI is clear that the design of culture systems for the rearing of prawns varies to a large extent, depending on the type of culture practised and the level of sophistication involved. The culture systems developed during the course of this project were satisfactory for the level of operation that was necessary at the time. In the light of practical problems experienced, certainmodifications arc required in the design of some of the systemsdeveloped.

Undergravel filters built into aquarium tanks arc commonly used for aquarium fish-keeping. These worked well for the holding of adults and post-larvae, providing a more natural substrate than a nllt tank bottom, The usc of perforated pipes, lald in the gravel, for water circulation can be Improved upon. Spotte(1979) has recommended the usc of cormgned flbreglnss shl:Cls asa filter plate.on which the gravel Is placed. The filter plate is placed on spacers in the bottom of the tank and water isdrawn by airlift Irom the space created below the plate. It isfelt thllt this would provide a morc even now "f water through the filter bed than the pipes used In the present project. Inaddilion, thiswould, eliminate the need for a coarse layerofgravel to cover the pipes anti thus reduce thedepth of thc filter bed, :ISa slnglc l:lyer of uniformly slzed crnvel could then be used. 152

Artificial habitats and/or substrates need to be provided In tanks housing adults and juveniles to reduce 3ggrmive interactions between prawns as well as to make full utilisation of the water column (S3ndifer lind Smith,1976,1978;WlIIisetaL,1976;Mancebo.1978; McSweeney, 1977;\Vickins and Beard, 1978; Kneale and Wang,1979). Most eulturisn rearing prawns intensively make use ofsome form ofartificial substrate to Increase the surface area available to prawns (McSweeney, 1977). Artifici:1I habitats, In the fonn of sections of plastle piping were not utilised equ:l1ly by all species In the present study. Macro­ brachium rude :mdM.uustrale for example tended to rnnge widely across the tank during the dllY­ time, only occasionally occupyplng pipes. M. lepidactylus, M. permit and M. scabrlculum usunlly tooksheller In these pipes during the daytime, also burrowing under feeding plat­ forms placed on the filter bed. Horizontal shelves of netting material may be more suitable substrates for /It. rud« and M. australe adults, and are recommended for use in placeof vertical netting material in tanks in which post-larvae are held.

The basic facilities required for any Intensive culture system are the culture vessel and filter, with II means for circulating the water between the two (McSweeney, 1977). Although prawn larvae have been cultured in various shapes lind sizesof vessels, the shepe of the container is important from the point of view of management nnd the creation of clrculatlon patterns for the suspension of food partlclcs (Zielinski et al., 1974;Sick and Beaty 1974; Dugan tt oL, 1975). TIle filtrntion unit is commonly separate from theculture vessels.according to lltera­ ture referred to.and the design appears to vary. Hatcheries commonly usc mechanical pumps for the circulation ofwater between filler unit and culture vesscl(s) although airlift pumps are also employed for thls'purposc (McSweeney, 1977). Thc hcating of culture water, where necessary, requires consideration in the design of the culture system. The system employed bySandifer lind Smith (1978). in which a heat exchangerisplaced in the blofiltratlon tank. appears to be :I practical solution to thisquestion ofdesign.

TIle single container rearing units first developed in this study were provided with 2 min sized gravel particles which became clogged with time.as particulate mauer was not :It first removed from the return pipe. This was to allow for circulation ofArremla naupll! through the filters and back to the rearing container. Ifthegravel used WIlS of 11 coarser gmin, the Anemia clreulatlon could be improved and thefilters would be less likely to clog up. TIlesc units were intended for small scale experimental rearing.and are considered suitable for this purpose, provided the gravel used in the blolilter Ischanged toacoarser gmin. Sinr,lecon­ tainer I'e:lrlng unils functloncd ~1tisfactorily until the liIters beg.ln to clo' up. ISJ

No problems of this kind were experienced with the multlcontalner rcaring units where coarser gravel of 1-2 cm diameter wasused. However, the water circu 1:11 ion in these filter beds needs to be Improved. During the final stages of the larval rearing programme, now rates to the rearing containers from the filter unit were reduced to minimise the removal of Artemla from therearing containers, As theairlifts wereconnected to perforated pipes running the lengths of the filter bed, thishad the effect of reducing the now through the filter and thuslocallslng the area of filter In use as well as the nltrlllcation capacity, This was 'overcome In one of the multicontalner units to some extent. TIlls unit was used for the tesling of an electrlcally controlled Immersion heater designed by the J.D.U. of the University. Inthis unit two sets of airlifts were In operation, (Fig. 3D) one set clrcu­ laling water through the renrlng containersand one set circulating water through the heat exchange container, provided for the large single 2 Kw Immersion heater.

The principle ofseparate nirlifts has been Incorporated into modiflcatlons to these units since the termination of the project. In addition corrugated fibregla~s niter plates have been installed to rest on the reinforcing platesat the bottom of the flbreglass tanks. Heatlng ln the units is by means of undcrfloor heating cables strung through the relnforcing struts below the filter plates. Temperature control isachieved by the usc of:1 transistorised unit developed by the Instrument Development Unit of the University. Airlifts positloucd across the centre of the filter circulate water through the filter bedonly, ata constant flow rate. Separate airlifts supply filtered water to the rearing containers.

The conflgurationof the rearing containersused was unsuitable, asthe bottoms were nat. This made the removal of settled food particles time consuming, and could be overcome by. using cither conlcal containers or cylindrical containers with conical bottoms (Sick and Be:1ty, 1974; Dugantlal, 1975).

The circulation patterns in the rca ringcontainers used, were adequate for suspending food partlclcs but required a relatively strong stream of air from the slr rinB attached Co the out­ let pipe. It is likely that this would be reduced In a conical tank, providing :l less turbulent circulation pattern.

The design of the screened outlet pipe was responsible for occi\slollal overflows from re:uing containers, when thescreens became blocked with food particles. While the solution to Ihls problem liesI':lrtly In feeding techniques, thedesign should include 11 larger surface :ueaof screeningaswell as the: emergency ()VCrnows th:tt were Incorporated In the flnul staltl'_' l)( the programme. 154

The arrangement of the rcaring containersaround the filterunit provided an Increasedsur­ face area frol11 whleh heat In the system could be lost. Thishcatloss was exacerbated by reducing water turnover rates in rearing containers, to maintain Anemia densities. Poorin­ sulation of the bulldlng housing renrlng units would further aggravate the problem In winter. A possible solullon would be to insulateeach Individual rearing container. This would how­ ever be lmpractlcal lfa number of containers were Involved. lind the IncJuslon of the feulna containers In I reservolr-blofllter tank could beconsidered.

The removal of particulate matter from water entering the filter unit remains 0 problem, liS the renring tcchnlques employed in this study rely on the reclrculation ofArtemta nauplll In the rearing unit. D3g5 used to trap particulate matter (Sandifer and Smith, 1978) would also removeAneml« from the system. The solution to this problem lies In the reduction of the water turnover rate, as mentioned above.

Under suitableconditions of water quality, temperature, photoperiod, and In certain cases, salinity, adult Macrobraclllll11l prawns will mate and spawn readily incaptivity throughout the year (Dugan et al; 1975; Sandifer and Smith, 1974, 1978). Under artificially controlled environmentalconditions, where a natural diet Is not avellablc, these prawns require a well balanced prepared diet to ensure satisfactory levels of survival and fecundity (McSweeney, 1977; Sandiferand Smith, J978). Experience has shown thatadult M. rosenbergll can be stocked at between 30and 90 prawns/m2of floor space, in tanks equipped with artiflclal habitats and connected to closed water recirculation systems, in either freshwater or brackish water up to 3salinity of 8 S 0/00 (Sandiferand Smith, 1978). Adult Macrobrac!ll/lm females can beexpected to lay from 500-140000 eggs(Dugan el 01., 1975) depending on thc.lndivldual size and the species concerned (Dugan et al., 1975). Berried females may be held in either freshwater or brackish water during the entire lncubatlon period of the eggs (Ling and Costello, 1976). At temperaturesof 2SoC the Incubation period Is from 16 lind 20 days for most Macrobracl,llIm species (Dugan CI al., 1975). Dug3n nnd co-workers (1972, ~ 1975) have demonstrated that spawning rn3Y be Induced in certain Macrobracl,lunr species, by mnnlpulating the temperature.

The survival of wild prawns In the laboratory. over the course or the present study, was approximately 50% of the total collected. TIle losses expericced arcllllributed to aggres- sive Interactionsbetween prawn, and possibly Inadequate nutrition with ",: rosc"btr;:11 losses being the hlghcst as a result of higher stockingdensilies. lnadequate nutrition I'mhably plnyed a role In the decline In fecundity reported for certain ofthespecies Invesligatt'''. ISS

The nutritional requirements of breetllng prawns remains a problem until suitable commer­ cial diets are developed. The m.yor problem In this study was less one of nutrient composi­ tion than oneofpoor binding properties. The pellets either broke down rapidly or bluely at oil, when In water. Rapid breakdown releases nutrients (Farmanfannaian ~t al..• 1982). The strongly bound pellets were found tobe unpalatable to the prawns. Local animlll feed producersareat present working on theproblem of water stability for prawn feeds, aswell assuitably formulated diets. The University will be co-operatina wilh these producers In the development of prawn feeds at 11 later stage.

Management of breeding anti the productlon of berried females did not present nny problems. As for other /IIacrobraclllllln species held under laboratory conditions, the five Indigenous species reproduce readlly in the laboratory. Mllcrobracl,illln eustral«, and to a lesser extent M. rude adults, In the present study, exhibited a loss of fecundity with time in captivity. M. australe females moulted and spawned regularly, but tended todrop their eggs gradually, so that the size of hatches was usually small, and larvae from different females were usually comblncd. M. lepidactylus females appear to have potential toproduce large numbers of eggs, as demonstrated by the example ora 7 cm female from which 10,500 larvae were hatched. Larger females of the latter species collected from the Limpopo river tended to drop some of their eggs in the laboratory, during Incubation, lind therefore their fecundity could not be measured.

During the initial stages of the present study, poor resultswere obtained in the rearing of larvae obtained from the indigenous species. Investigations of the salinity preferences of the early larval formswere therefore conducted during the second phase of the rearing pro­ gramme. These investigations should rightly have preceded the second phase and are there­ fore discussed before the results obtained from rearing the larvae intheculture systems developed. The methods employed, modified from the techniques used by Choudhury (1970b, 1971 b) for M. QCQIIII"mlS and M. carclnus larvae, provided Infonnation on the salinity preferences ofthe early larval forms, which was applied with reasonable success to the rearingof the Indigenous larvae,

In the studi~softheeffects of!nUnlty and/or temperature on survival and development of pataemonld larvae under laboratory conditions. Nlf4··N, p02 and pe02 are usulllly not monitored, particularly If water Is changed daily. (Choudhury,1970b, 1971b; Sandifer, 1973; Knowlton,1974; Dugan er al., 1975;Guest and Durocher, 1979.) Choudhury C1970b) did not monllor I'll In the experimental rearing of M. carel",u, lIS water was ch3nr.c" dally. 156

Duganet 01.,(1975) transferred larvaedaily to clean contaln~n, wh~n deteminin, opUmal crowth and sun-Ivai conditions for certain Macrobraclllum sp~c1es,1O thllt daily water quality an:lIysis WI1S not required. In the prescnt study the monllorlng or the abovementlcned water qUlllity parameters, :IS well as temperature, salinity and pll was undertaken, and re­ sults showed that Sllllsfllctory water quality was maintained, for the most part, durin. tho course of the experiments. Results or the experiment uslnclater lan-al ronns ofM. ptttnil showeJ that problems would be encountered using the apparatus and techniques (or these Iorms, lIS NII4-N levels were consistently higher than (or the clirlier larval forms, Reduc­ tions In the number of larvae os well as the density nt which Arumla are fed, would be neee.. I:Iry If the same :Ippllrl1fUS nnd techniques were to be used for Il1ter larval (orms.

Experimental results showed that the IlIrvlle of all five Indigenous species require sallne con­ dllions for survivl11 and development of the early larval fonns. Larvae held at low 5:llInlllcs, or In freshwater, exhibited poor survival and limited morpllologlcal development at thepre­ vulllng water temperatures in the experiments.

oC, The survival time of M. p~tcrslliarvae In freshwater, at a mean temperature of 29,I WI1S within the range obtained by Read (1982) forM. pcttrsll, thaI is3,4 days median survival 0C time at 29,7 In O,OS S 0/0 0• The survival lime of the larvae of the two M. Icpldoctylul populations in freshwater Isof interest. Larvae of the Limpopo river population exhibited a distinctly longer survival time in freshwater than the coastal population from LakeCubhu. Numbers of larvae of the Lake Cubhu population had dropped to 50% between the fourth and fifth daysof the experiment, while survival of the I.lmpopo river population was stili close to 90% forthe same period. Noneofthelarvae from the Ll1ke Cubhu population sur­ vived beyond the 6thday while survival ofthe Limpopo river population larvae wns be­ tween 60 and 70% onthe 6th day of the experiment. Since adults ofthe Limpopo river population were collected several hundred kilometers from the c03st,and since the larvl1e require saline conditions for development, Itseems likely th:!t theability to survive for. longerperiod of time In freshw3ter would beImportant for survival, until the larvae reached brackishcondilions. It Is uncertain at this stnge whether larvae arc tran5Portcd durlnll nood periods to the COllSt, orwhether the adulls are catadromous, mIBnalln8 down the l.impop river to saline conditions for breed!n,. asIs thecase for M. IOscllbtrrlL

M. lcabrlc:ulum larvl1e exhibited a limII.., survivnl time to the l:arvac of the ,\1. it'1'/,II:ct"/1I1 populallon (rom Lake Cubhu,th3l is I 50% mortality between days 5.nd 6. IS7

The morphologkal development of the larvae of nil five species did not progress beyond the first larval fonn In freshwater, Although the first larval fonns ofItIacrobraclllum species sur­ vive on the contents of the yolk sac and do not ingest food from the outside, Artemta nlluplil were provided to the larvae in freshwater. This wasdone so that food would be available should any of the larvae progress beyond the first larval fonn In freshwater. This, however, had a possible negative effect, as the Arteml« were unable tosurvive in freshwater and their decomposition may have negatively Influenced the survival limes oflarvae held in freshwater and provided with Anemia nauplli,

Survival of the larvae of all five species was poorest at low s:llInitles between 4-5 S 0/00, evert where food was provided. Although larvae were able to moult to the second larv:tl form at these salinllies, survival of the second larval fonn wasnot favoured by the low salinity. Macrobrachium petmlt larvae were able toreach form III at4S 0/00 where Artemia were provided but were unable to survive. This is inagreement with the results obtained by Read (1982) which showed that low salinity and high temperature do not favoursurvivalof M. petersitlarvae, Macrobrac1,lum lepldactylus larvae from the lake Cubhu population moulted to the second larval form at 4 S 0100 butdied very soon after moulting.

Read (i982) found that salinities from 8-35 S 0/0 0 were favourable for the survivalof M. petersli larvae and that a minimum of8S 0/00 was required for complete larval develop­ ment. The results obtained for M. petersltlarvae at 8 S 0/00 arc inagreement with thls finding, but the poorer survival of larvae ofthis species at 12 S0100 appears to be contra' dictory. Morphological development was favoured at the 12S0/00 salinity level however, with larvae reaching fonn V by the eighth day. The sudden Increase in mortalities with the appearance of form V larvae may have been as n result of theunsuitable nature of the experimental system for later larval fonns. This was the case for form V and VI larvae of AI. petersllin experiment 2,whcre survival was found to be poor.

Salinities below 8S 0/00 did not favour, survival ofM. lep/dactylus larvae from the Lake Cubhu populalion and M. scabrlculum larvae. Salinitiesbelow 10 S0/00 did not favour survival ofM. leptdactylu« larvae from the limpopo river population nor M. rude and AI. australe larvae. It seems reasonable toassume that snlinUles In the region or 8 S 0/00 ­ 10 S 0100 areclose tolile lower limits for survival of larvae ofall nvc indigenous !lipcc:lcs at temperatures in the vicinity or 28°C. IS8

In all the experiments, morphological development was most rapid (or larvae fed Arlttnla and held at the highest s..,linily level ineach experiment. As bothsurvival and development of larvae was favoured by the highest salinity levels In the experiments, it may be concluded that the oprlmum sillinity levels for survival and development may In fact exceed the levell chosen for theexperiments. The range ofsalinities tested In the apparatus used was limited by the size of the experimental set-up and should Ideally include IiIlinlUes from freshwater to fuJI strength sea water. Thus further InvesligaUon of thesalinity preferences of the five Indlgenousspecles Isrequlred, In addltlon, theeffects of temperature In cornblnntion with S3l1nity should be Investigated, so that optimum temperature and 5:llinity combinations for survival and development can be established (or all the stages o( brval development.

The poor survival oflarvae of the Indigenous species, during the first phase of this Investi­ gation, Isattributed mainly to the lowsalinity levels chosen for rearing. With the exception ofM. rude larvae, 5.1l1nity levels chosen for rearing were below lOS0/0 0, and often below 8 S 0/00. TIle results (rom the s:llinlty preference experiments demonstrated that higher salinity levels were required, than thoseemployed. It was nevertheless possible to rear M. rosenbergl/ and M. rude larvae to post-larval form, during this first phase, using blo­ filtration systems. TIlls Indicated that the rellring techniquesemployed, which included a diet ofan ega custard mix and Anemia niluplil, would be a satisfilctory base for the further rearing oflarvae, once the results ofthesalinity preference studiesbecame available.

Adramatic change In the survival and morphological development ofM. peicrsillarvae reo suited from increasing the rearing ~linity to levels above 8 S0/0 0 , considered the minimum required salinity fordevelopment of this species by Read (1982). As In results obtained by Read for the latterspecies, a relatively wide range of sallnlties was suitable (or metamor­ phoslsto post·l3rv:::,. M. pcttrslliarvae were reared to post-larval form at mean salinities ranging from 8,3-14,ISo100, at tempera lures of approximnlcly 27°C and mean s:lIinlties of approximately lOS0/00. /tIacrobraclll"", ptl(f'$II'arvae completed development in 21 days in several batches which reached post-larva! form. Thlscompares favournbly with resultsobtainedby Reild (1982) for thisspecics, of 20-22 days at 25 t 1,20 C And sallnllY 14range: :t 2,2S0/00•

Minimum levels of8-10 S 0100 were: ncccwry for the survival :lnd development of the other four species, asthe re:sults of the salinity experimentsdemonslnted:However, the salinity levels chosen for reAring these IflCdel, on the basis o( the pn:(crtnces o( tttr (',uly larval fonns, did not produce dramatic changes In surviv:lllind developmenl,as wilh 159

M. petersil. Read (1982) demonstrated Ih:lt metamorphosis 10 posl·l:lrvne could takeplace over a wide salinity renge for Itt. peltrsl/larvae, and th:at the r:lllgc was probably wider than for most Macrobraclllllm species. It isqulte likely that the salinlly requirements of the other four indigenous species Investigated in the present study may be more restricted in range, as isthecase wllh certain other Macrobraclllllln species. Further investigations into the salinitypreferences of the later larval fomlS ofItt. lepldactyllis. Itt. rude. M. scabrlClllum andItt. austrd« arc required. This should include the investlgallon of the effect of different sallnlty temperature combinations as previously mentioned earlier in this chap ter,

Attention was drawn to the wide ranse oflarval feeds employed alvarious hatcheries for rearinglarvae on (Chapter 2, seclion 2.7.2). Thediet fed to larvae in the present studyen­ abled development topost-larval form, and was therefore considered adequate at thisstage. Dietary studles,including the investigation of mlcroworms (Panagrelllls sp.) as a substitute for Artemla In the diet, nrc Intended for a later stage, Microwonns were fed to the larvae on occasions and were taken by them. In addition, the nematodes appear to tolerate snllnilles up to lOS 0/00 for fairly long periods.

The eggcustard provides a useful matrix for other nutrientsand can be mixed with vitamins forexample, toprovidc n more balanced prepared diet.

The need for maintaining a continuous supply of food nt :I suitable density In rearing containers has been outlined in Chapter 3,section 3.9.4). Two maier problems were expe­ ricnced In practice, namely:

• Loss01Artcmia In fllrt!n,ln spltt ot« degrtt offeclrcu/allon • Accumulation 01 prepared food onth« ~ar/ng container bottoms.

As mentioned in Chapter 2, seclion 2.1.3, areduction In the turnover rate of water through the rearing conllliners would reduce the loss ofArtemla in the filter. Sandifer and Smith (1978) have found 2-5 tank turnovers perdlly to be satisfactory in Ihis respect. This Is considerably leu than the turnover rateofroughly 50 lank lurnovers/day employed in the present study.

Modification of the rearing containers to include I conical or tapered bottom, would nllow more frequent feeding of prepared food, scdimcnted particles of which could then be re- 160

moved In a short time. Due to the need for high densities of food particles (Chapter 2, section 2.7.2)for effective feedins, much of the prepared food provided is not eaten and becomesunpalatable to the larvae aftera lime In the water. Effective densities of food need to be investigated to resolve this problem. although other methods of feeding could also be Investigated, for example the use of light to concentrate larvae Into one area of the eon­ tainer, where they could be fed more precisely, at a lower density offood particles. This would however require more frequent feeding of the larvae ifreduced densities of food arc to be maintained between the times at whleh food is provided. Mechanical feed Ina dcvlccs could be developed towards this end to minimise the time consumlnglnbour which would be required.

Although stocking densities In the present study were not adjusted toany speciOc levels, they were generally within the levels of 100-150 larvae/2, reported In a review of freshwater prawn cultureby Ling and Costello (1976). Large batchesofM. lep/dactylus larvae were usually not divided up Into more space due to anticipated losses, lind stocking densitieswere . In the region of 500/2. High initinl stocking densities for rearing Macrobraclr/um larvae are not detrimental, as larvae tend to be gregarious in the early stages (Dugan et al., 1975) lind in fact high densities have been recommended to facilitate fceding (Chiao and Llao, 1977). However both Dugan etal., and Chiao lind L1ao (1977) recommended a reduction In numbers once the gregarious habit is lost. In the present study, only I reduction in numbers was made in the beginning If necessary, and thereafter no further reductions were made, assur­ vival of larvae was not high enough to warrant this. In fact low densities of larvae in the laterstagesof development made the feeding of these larvae difficult, as a relatively high density of food was provided to ensure effective feeding, with a resulting poilu tion risk. In such cases larvae from different females were combined, to increase the stocking density, and thus enable more efficient utilisationof the available food.

Dobkin (1971)considered that the selection ofa number of morphological stqes (or des­ criptive purposes was of necessity an arbitrary process. TIle compil:lllon ofa Brid of seven larval forms, forthe purposes of the present study, frorn II survey ofthe larval morpholon' of other Mocrobroclllum species, was an arbllrary one. TIds did not take into account facton such as moulling ormorphological chall,es occurring at moultin,. Nevertheless, the division of larval developmental processes into a grid ofseven morphological fonns provided :I useful framework forc:ompnrins larval development In the five species studied. TIlls h;IlJ the draw­ back that certain developmental processes, normally distributed over scver;al SI;l;1<:$ of develop· ment by other authon,arc regrouped Into two larval forms In this ,rid, namely (unns VIand 161

VII. The latter Iornu cover development from the inili:ulon of pleopod development In fonn VI. 10 the development o( setatlon on the exopod andendoped o( the pleopod, as w~1I as chela development on pereiopods I and 2 in Ionn VII. In M. Irpidact)'/lls DIUIM. ",dt larvee, the appe"rance and development ofpleopods ami perelopod chelae took much longer than inM. ptttnll. lind was Accompanied by a degree o( variability. particularly with M. ItpldaCI)'/uJ larvae. where no sign of chela devl."lopment was recorded on larvae showlng distinci characters o( form VII such AS complete denticulation on the dorsal roslral murgln. setae on bOlh Ihe exopods and some endopods of thepleol,O(ls, lind an extremely tllpered telson, TIlallhlsvnriabi:ity occun In the Inter Inrvl1l (onns Is In ltsreernenl wHh Ilnd­ Ings of other workers. (Choudhury. 19703; I'illai lind Mohllmed, 1973; Reeve, 1969; Knowlton, 1974), who reported constant or synchronous development of a population o( larvae during wly I:lrval development, wilh llsynchronolls development and v:ui:abilHy of fonn at the same moull during later larval development. In the case ofM. nICJ~ and M. Itpl. dactylus this Is considered to be 0 result ofunsuitable ~linily levels during larval ~arinl. However. Sandifer (1979) hns postulated that varialion In the plank Ionic larvlll phase of Palaemonldae lI1ay increase the probabililyof recruitment to exlslinK parental populations, as well as the likelihood of successful colonisation of freshwater environments. Ileredltary varinbilily should be borne in mind In the case ofM. IcpldactylllJ, which was collected roughly 700 kill from the coast in the Limpopo river. and the larvae of which require s.,lIne conditions for developmcn t.

Morphological development ofM. IJCICrslilarv3c took place r3pidly and although the dura­ tlons of fonns VI and VII were longer than Ihe other larval fonns, these were much shorter than in M. Itpldaclyl"s and M. rude larvae.In nddltion.developmcnl conformed to alllrge extent to the seven form grid that was used for the study, Ihus differing from the nine stascs identified (or Ihls ~pecics by Read (1982). Read (1982) relied onchanges In the number of setae associatedwith the first rostral teeth and changes Infonn of the pleopods for ldentl­ fylng the 1:lII:r sl:lses o( development. The seven fonn grid used In Ihls study comblnos stages V-VII of Read into a slnslc developmental form, (0"" VI. :11111 stoges VIII alltllX of Read into form VII.

MacrobracMllnJ scabticulun: lind M. alulrole laMe were not reared beyond form VI. Poor results (or these specie, may be: attribuled 10 unsuitable SJllnlly levels, ;although rhi\ requires fUrlher Invcstl~tlon. TIle reproductive condition o(M. allJ/mlc adul', seemed l() llt-rcrlorale wllh transfer to the Illbonuory. and this may be II (aclor to beconsldered in the P\,IOf dCYtlop­ ment of Ihe larvae lIS well. 162

The sludy of the morphological formsoflluv3e of lite indigenous species revealed differences between the species, which may be of use inidenlifying the larv3e In the laboratory. Characters which may be of use are the chromatophones and thedevelopment ofslmclures on the anteriorcarapace and rostrum, While chromatophones are rather variable characlers, used for larvae rellre~ under similar conditions, they mllY be of v:tlue. Characteristics of Ihe perelopods, antenna, pleopuds and relson may be of use, nlthough 1I detailed study of these was not made allhls slilge. These structures were monitored forappearance and develop. ment, rather than detall of structure, In the present study.

Post-larvae produced durlng the larval rmlns programme were malntalned at high stockln, densltles due to alack of space, but In antlclpatlon of expanded fl1cilllles for the rellring of juveniles, However, expansion of post-larval rearing facilities was not possible before the termination of the present prolcct.and as a result mortalitieswere high nmong the post­ larvae produced. Only among the first balch of M. roscnbt'rgll, reared to sexual maturity In Ihe laboratory, was II possible to maintain stocking denslties:ll ~:lfc levels. The Initilll stocking of 733/111 2 for these prawns lies below the level of 900/m2above which a break­ point mny beexpected to occur (Kneale lind Wang, 1979). Survlv:.1 of these prawns to sexual maturlty, roughly 6 months after stocking the post-larvae out, was I 8,8~. TIlls low survival rate can be auributcd to the fact that, nlthough periodic rcductlons ln dcnslty were made, a point was reached where no further reductions could be made, Thus at the tcrml­ nation of the project stockingdensities of the batch ofM. rost'llbc'811 had declined to be­ tween 18 and 33/012 In the rearing tanks, This Is above the figure of 12-18 considered a probable optimum in llawailan pond grow-out (Goodwin lind Jlanson, 1975), and the level of 141m 2 chosen by Mancebo (1978) for the last two months of:l six month growth stUdy Involving tank reared M, rosenbergil juveniles. A wide mngc ofsizes existed among these M. rosenbergi! toward theend of the present study r:tn£il'S from 66-123 mm,from eye orbit to tclson top. The means for three rearing tanks of these prnwns ranged from 107,7- II1,8 IIInI, roughly equivalent to 0,4 nun/dllY overa nine monlh fearing period. "ntis Is al· most twice the rate presented by Hanson lind Goodwin (1977) for Ihis species in a t3ble of comparative growlh r:3lcs for tank rearedAlacrobrocltlll'" species. A second hAtch of 2 /II, rosenberzt! slockell:lt 2014/m illill,llly, reached :I mean of3,8em after 3 months, equivalent to 0,42 11Im/day. Survival of these pr:awns W:lS not calcul:llcd :as most were hnt accidentally.

Siockinlt densltlcs for M. pclCnll post-larvae were e'luaUy hls:l\ or even higher In f III~ pre­ 2 sent study. Sun-lvIII after II 3-4 week pcrlod III high Initial densities of 2540 ..38:;0(01 163

was a satisfactory 77,3 % for 4 batches of post-larvae monitored forgrowth. Survivalofthese 4 batches after combination and at 6 months ofage was 37,8%with stocking densityreduced to I 550/m2. Although this is considerably higher than the 900/m2 breakpoint level des­ cribed by Kneale and Wang (1979) for M. rosenbergii, the fact thatM. petersii is a smaller species, ranging in size from 5-6 em, should be taken into account. Thus densities of I 550/m2 may not be as detrimental to M petersiijuveniles as to M. rosenbergii juveniles. Mean length of theM.petersii larvae under discussion was 20,2 mm after 6 months, an equivalent of 0,11 mm/day which is lower than for Me rosenbergii post-larvae reared for 3 months at an initial stocking density of 20 I4/m2. The rate of 0,11 mm/day is also lower than most of the growth rates presented by Hanson and Goodwin (1977) for tank reared juveniles ofthree Macrobrachium species. It is not clear at this stage whether high stocking densities were responsible for the slow growth or whether the development ofM. petersii juveniles is a slower process than for M. rosenbergii. However ifM. petersii juveniles exhibi­ ted the same growth rate as those ofM. rosenbergii, that is 0,21 mm/day (Hanson and Good­ win 1977), they would reach adult size in 23,8 - 28,6 days, which seems unlikelv..

The first fifteen M. rude post-larvae produced during the course of the rearing programme exhibited rapid growth but less s~tisfactory survival, stocked at I02/m2. The level of 102/m2 is much lower than the 400/m2 level at which Sandifer and Smith (1976) found poor survival and growth for M.· rosenbergli, in spite of shelters added. The growth rate of 0,6 mm/day forM. rude juveniles is well above growth rates reported by Hanson and Goodwin (1977) for those of three otherMacrobrachium species.

M. rosenbergii and M. rude post-larvae exhibited high growth suggesting that nutritional requirements were satisfied.

From a management point of view, the use of glass sheets as feeding platforms above the filter bed proved to be effective for the removal of unw~nted food. However, the use of aquarium tanks with built in gravel filters is considered unsuitable for rearing post-larvae and should be replaced by tanks with separate filter units. This would eliminate the danger ofuneaten food clogging the filter as well as enable excess food to be more easily re­ moved.

The protein level in the prepared diets used.ranged from 36%- 48%, the latter levei being most commonly employed. New (1976) concluded that protein levelsof 27-35% were suit­ able for rearing shrimps ofvarious sizes, while Balaz and Ross (1976) obtained results which 164

suggest thDtlevels in excess of 35% lII11y be necessary(orpond feuing M. rostnbtrgll juveniles. Willis tl0/., (1976) obtained higlll:st growth ratcs fOrlank reard M. rosellbfrgll populations, providing a 40% protein diet. The high growth rates obtained in the present study may be the result of the hlSh protein level of 48% employed.

The waterstability of the prepared diets used in the present study presented problems IlS mentioned earlier In this chapter. This was especially true for the 48% trout starter used for feedin" most of the juveniles.

The 'practlce of"djustlr'!: the quantity of food provided at each feeding according to the amount eaten, WIIS adopted for practical purposesof excess food removal. 8y 1I1>l'lyln8thls practice thequantity of excess food was reduced. but still created difficulties as pnrllcles were disturbed bymovement ofjuveniles and inevitablysome food accumu latcd on the filter beds. This procedure of g:luging quantity by observation of feeding h3S been adopted by Balaz and Ross (1976) and Mancebo (1978), after Initially feeding juveniles a fixed quantity based onthe percentage of prawn biomass.

While McSwecney (1977) has recommended feeding AI. rosmbcrgll juveniles several portlons of food each day, others have limited fecdins frequency to 1-2 feeds per d3Y. (Ollla1. and Ross. 1979; Kneal and Wang 1979; Piyatiratltivokul and Mcnasvcta, 1980.). Ling (1962) claimed that/II. rosenberzt! juveniles were active night feeders.and it seems reasonable to assume that a small quantity of food provided during theday and a larger quantity at night would besuitable.

TIIC temperature levels at which the post-larvae of AI. pc/mil and M. rude were reared, were temperatures suitable for M. roscllbrrgll and may in fnct not be optimal for the two lndigcnous species. Further investigations nrc needed in this respect.

Results forthe few M. lepldactylus post·I;1rv:le were inconclusive as they were slocked over an extended period of time. making clllcul:ltions diflicult.llowever.very little growth occurred over the two month rearing period.

The resultsobtained during the present Itudy have been useful for pl:lnning further modlfl­ cations of Ihe halchery facilities developed for Mocrobroc!lluIII species. More detailed In· vestigations ofcertain factors such assalinity, temperature, nutrition ami Itud-II1C denslly. 165

for the selected lndlgenous ~fQ(robTQ(1JIum speelesare required Ifthese arc to be mass'reared for comparlson wilh M. rosenbergl/. or the five species Investi~ated. two arc relAtively small and may not be ofInterest commercially. Macrobrachtum.leptdactylus, M. rud« and possibly M. QIIstrale warrant furlherInvesllgatlon In thisI't,anl.ln addilion ~'. tqrlldtlll. not Included In this study. may beofInterest for brackish water culture. 166

UTERATURE crrae

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