dasar teori mekanisme endokrin pada udang

1. Controlledreproductionofpenaeidshrimp:
acontributiontoitsimprovement
JorgeAlfaroMontoya

2. Promotoren: prof.dr.E.A.Huisman
HoogleraarindeVisteeltenVisserij
Co-promotoren: Dr.J.Komen
Universitairdocent,leerstoelgroepVisteeltenVisserij
Samenstellingpromotiecommissie:
Prof.dr..H.J.Th.Goos(UniversiteitUtrecht)
Prof.dr.D.vanderHeide(WageningenUniversiteit)
Dr.J.H.W.M.Rombout(WageningenUniversiteit)
Dr.C.Cahu(UniteMixtedeNutritiondesPoissons
IFREMER-INRA,Frankrijk

3. f j u o U ^ ' ^ ^
Controlledreproductionofpenaeidshrimp:acontributionto
itsimprovement
JorgeAlfaroMontoya
Proefschrift
terverkrijgingvandegraadvandoctor
opgezagvanderectormagnificus
vanWageningenUniversiteit
prof.dr.ir.L.Speelman
inhetopenbaarteverdedigen
op18April2001
desnamiddagstevieruurindeAula
(G t ^ o b 5

4. Tableofcontents
Chapter1 Generalintroduction 1
Chapter2 Reproduction of the shrimp, Penaeus occidentalis
(Decapoda:Penaeidae)intheGulfofNicoya,CostaRica 23
Chapter3 Interaction of bacteria and male reproductive blackening
diseaseofcaptivePenaeussetiferus 37
Chapter4 Reproductive quality evaluation of male Penaeus
stylirostrisfromagrow-outpond 47
Chapter5 Developmentanddeteriorationofspermatophoresinpond-
rearedPenaeusvannamei 57
Chapter6 Effect of 17 alpha - methyltestosterone and 17 alpha -
hydroxyprogesterone on the quality of white shrimp
Penaeusvannameispermatophores 71
Chapter7 Ovarian maturation and spawning in the white shrimp,
Penaeusvannamei,byserotonininjection 83
Chapter8 Cooling, cryoprotectant and hypersaline sensitivity of
penaeidshrimpembryosandnaupliuslarvae 101
Chapter9 Generaldiscussion 117
Summary 131
Samenvatting 135
Resumen 141
Acknowledgements 147
Curriculumvitae 149

5. Stellingen
Belongingtothethesis
"Controlledreproductionofpenaeidshrimp:acontributiontoitsimprovement"
JorgeAlfaroMontoya
Wageningen,18April2001
1. Injections,with17-alpha-methyltestosteroneimprovequalityofshrimp
spermatophores.
Thisthesis.
2. Serotonininjectionsinduceovarianmaturationinshrimp,beitatalowerratethan
unilateraleyestalkablation.
Thisthesis.
3. Latestagesofshrimpembryoshaveabettertolerancetocoolingthanearlystages
ofbovineembryos.
ThisthesisandWilmutetal.(1975),Theeffectoncowembryosofcoolingto20,0
and-196 °C.J.Reprod.Fertil.45, 409-411.
4. Thepituitaryglandinfishandthebrain/thoracicgangliainshrimphavesimilar
functionsintheendocrinecontrolofreproduction.
5. Geneticselectionofshrimpforstresstolerancewillimprovethequalityofgametes
andtherefore,larvaeproduction.
6. Scientificadvancesinshrimpmariculturearecontributingtotherecoveryofover-
exploitedwildpopulations.
7. Humanityisincreasinglyaccumulatingscientificknowledge.So,thereisscopefor
understandingGod'screation.
8. Toreachthemoon,onemusthavebreakfast first.

6. Formydaughter,Valeria

7. Chapter1
Generalintroduction

8. Generalintroduction
Scopeofthethesis
Marine shrimp aquaculturehasbecome amajor industry sincethe pioneering
works in larvae culture andpond culturebyM. Fujinaga (Shigueno, 1975),eyestalk
ablationinapenaeidshrimp(Caillouet,1972),andclosingthelifecycleincaptivityof
someshrimpspecies(Aquacop,1979).
Globalproduction offarmed shrimphasgrownat8%annuallysince 1989.In
1998,theproductionreached1,113,887mt,representingaround32%ofworldshrimpand
prawn catches, based on FAO fishery statistics (www.fao.org) and FAO (2000).The
industry has expanded mostly in Southeast Asia and Latin America, where different
penaeidspeciesarecultured.Themostimportant speciesinLatinAmericaisPenaeus
(Litopenaens) vannamei, followed byP.(Litopenaens)stylirostrisandP. {Litopenaeus)
schmitii.InAsia/3
,monodon,P.chinensis,P.(Marsupenaeus)japonicus,P.penicillatus,
P.merguiensis,andP.indicusarecultured(WeiderandRosenberry,1992).
InAsia,shrimpculture startedback in 1962inJapan.By 1991SoutheastAsia
contributed 80.6%oftheglobal shrimpproduction;theleadingproducerswereChina,
Indonesia andIndia. In 1997,thetotalproduction of captured andcultured shrimps/
prawnsfromAsiawasestimatedat2.95millionmt.Culturedshrimprepresentedaround
18%ofthistotal yield,basedonFAOfishery statistics.Current leadingproducersare
Thailand, India and Indonesia (Weidner and Rosenberry, 1992; Anonymous, 1999).
Thailandhasbeentheworldleadingproducersince1992,withanestimatedproductionof
243,800mtin1998(FAO,2000).
InLatinAmerica,shrimpfarming startedaround 1962inEcuador.Duringthe
1980 decade, other Latin American countries initiated shrimp farming activities,
reaching orsurpassing infew years theirown shrimp catches (Colombia, Honduras;
WeidnerandRosenberry,1992).In1991thecontributiontotheglobalproductionwas
19.4%,andtheleadingproducerswereEcuador,Colombia,andMexico(Weidnerand
Rosenberry, 1992). In 1997 fisheries and aquaculture in Latin America generated
around0.63millionmt,withshrimpfarmingrepresenting34%ofthisproduction(FAO
fishery statistics; Anonymous, 1999). By 1998 the contribution of Latin American
countriestotheglobalshrimpproductionhadincreasedto29%,withEcuadorasthe
major producer and the second largest inthe world with 144,000 mt (Anonymous,
1999;FAO,2000).
Inrecentyears,commercialshrimpoperationshaveexperienceddramaticlosses
duetodiseaseoutbreaks,causedbyviruses,bacteria,fungi, andparasites(Brockand
LeaMaster, 1992; Lightner, 1992; Wang et al., 1998; Itami et al., 1998; Morales-
Covarrubias andChavez-Sanchez, 1999).Viral diseases especially havehad a major
impact on shrimp production worldwide. In 1988-89 Taiwan experienced serious

9. Chapter1
economic losses duetoMonodon-type baculoviruses anditsproduction decreasedto
extremelylowyields(4,812mtofP.monodonin1998;FAO,2000).Afewyearslater
Baculovirus penaei appeared in America and the infectious hypodermal and
hematopoietic necrosis virus (IHHNV) dispersed in Southeast Asia and America
(Lightner,1992).In1992anewvirusappearedintheTauraregionfromEcuador,the
Taura syndrome virus (TSV), which caused severe economic losses throughout
America (Hasson et al., 1999). In the same year the white spot syndrome virus
(WSSV), which has a wide host range and infects various decapods at varying
infectivityrates(Wangetal.,1998),affected SoutheastAsia.Asaresultproductionin
SoutheastAsiadecreasedto71%oftheworldshrimpproduction(Anonymous,1999).
WSSVhasbeenrecently(1999)identified inEcuadorandCentralAmerica, andlow
productions are expected. However, American countries may learn from the Asian
experience(Jory,2000).
Shrimpfarmingisbasedonwildandculturedpostlarvae,dependingonregional
characteristics for postlarvae availability. Reproduction of marine shrimp is now
performedincontrolledenvironments,wherewildorculturedanimalsmatureandrelease
theirgametes.Thistechnological approachallowsapredictableproductionofnauplius
larvae. In the past only wild animals were utilized, but nowadays pond-grown and
selectedanimalsarebecomingmoreimportantasspawnersbecause,assurvivors from
viraldiseases,theymightpresentgeneticresistancetothespecificvirus.
Thetraditionaldependencyonwildstocksfornaupliiproductionwillthuschange
towards a more controlled production of genetically improved animals, selected for
important aquaculture traits like growth rate and disease resistance (Hedgecock and
Malecha,1991).However,thegeneticimprovementofshrimprequirestheapplicationof
breedingprograms,whichrelayonexcellentgametequality,controlledfertilization,and,
if possible, cryogenic storage. For this a proper understanding of basic shrimp
reproductive biology, and the development of in vitro fertilization techniques and
cryogenicprotocolsforgametes,embryos,andlarvaeareneeded.
Femalematurationisachievedbyunilateraleyestalkablation.Thistechniqueis
appliedtoeachfemale atintermolt,selectingoneoftheeyestalks.Fewdaysafter the
procedure,ovarieswillstarttogrowandfirstspawningswilloccurwithinaweek,with
anincreasingrateinthesubsequentdays.Eachfemale generatesvariousspawns,but
eventuallythespawningactivitydecreases,untilaround2to3monthsafter ablation,
whentheyarereplacedbynewfemales.Eyestalkablationaffects allaspectsofshrimp
physiology (Quackenbush, 1986) and overtime ithas adeleterious effect on spawn
quality and quantity, as well as on survival (Emmerson, 1980; Primavera, 1985;
TsukimuraandKamemoto, 1991;Benzie,1998).Thisdependencyoneyestalkablation

10. Generalintroduction
isamajor bottleneck for the advanceofcontrolledreproduction, andmajor research
activitiesarededicatedtodevelophormonaltreatmentsforcontrolledmaturation.
Traditionally,penaeid shrimpmaleshavereceived lessattentionthan females,
assumingthattheywillalwaysgivehighqualitysperm.Whilethismaybesoformales
caughtfromthewild,itisnotthecaseforpond-rearedanimalsnorformalesfromthe
wild after a few months incaptivity. Male shrimp also showreproductive problems
(Brown et al., 1979; Chamberlain and Gervais, 1984), they are often affected by
infections ofthereproductivetissue,includingampoules,vasdeferens, andtestis;the
condition renders males infertile. Chamberlain and Lawrence (1981) stated that the
reproductive capabilitiesofthemalesmaybealimitingfactor incaptivity, indicating
thatabetterunderstandingofspermatophoreproductionandanalysisofspermquality
isimportantinshrimpmariculture(Leung-TrujilloandLawrence,1987).
In vitrofertilization is essential for selective breeding programs as it allows
controlled mating designs,but it has not been developed yet. Some data have been
published on this subject, giving contradictory results on fertilization rates and
indicating that our understanding of the fertilization mechanism in open and closed
thelycumshrimpisstillfragmentary. Clarketal.(1973)reported 10%fertilization for
Paztecus,Alfaro etal.(1993) gotnofertilization forP.occidentalis,Misamore and
Browdy(1997)obtained 2.48%forP.setiferusand3.88%forP. vannamei.A50%
hatching rate wasreported for P. monodon(Lin and Ting 1984 cited in Primavera,
1985).
Nauplii are shipped in plastic bags with chilled (20 °C) and oxygenated
seawaterat15,000to25,000nauplii1"'(Kungvankijetal.,1986).Internationaltrading
aswell asbreedingprograms would bepositively influenced bycryopreservation of
penaeid shrimp seedstock (Anchordoguy et al., 1988; Benzie, 1998), but these
techniqueshavenotyetbeendeveloped(Benzie,1998;SubramoniamandArun,1999).
Theaimofthepresent thesiswastoaddress someoftheproblems in shrimp
reproductionasdiscussedbefore.Variouspenaeidspecieswereselectedforconducting
theresearch(Table 1).Thisselectionofspecieswasbasedonseveralcriteriasuchas
commercial interest, ready availability, and ability to spawn in captivity. First, the
reproduction ofPenaeusoccidentalisinGulfofNicoya, Costa Rica, and an invitro
fertilizationprotocolusinggoodqualitygametesfromwildbroodstock,wereevaluated.
Thequalityofspermatophores, addressingbothpathological and cultureeffects, was
studied in 3 penaeid species. To improve spermatophore quality the effect of an
androgen and a progestagen was evaluated. Serotonin treatment was evaluated in
femalesasanalternativetoeyestalkablationforovarianmaturationandspawning.

11. Chapter1
Table1.Characteristicsofthepenaeidspeciesselectedforthestudiesofthisthesis.
Penaeidspecies Characteristics
Penaeusoccidentalis
Penaeusvannamei
Penaeusstylirostris
Penaeussetiferus
Trachypenaeusbyrdi
Thelycum
Open
Open
Open
Open
Closed
Habitat
GulfofNicoya
GulfofPanama/Farms
Farms
GulfofMexico
GulfofNicoya
Reasonforselection
Model/Fisheries
Aquaculture
Aquaculture
Model
Model
Finally, embryotolerance to cooling, cryoprotectants, andhypersaline exposure was
assessedasafirststeptowardsdefiningcryogenicprotocols.
In the next section of this chapter, the sexual characteristics of shrimp are
presented. The section on "reproductive endocrinology" describes the present
understanding of the hormonal pathways involved in reproduction of decapod
crustaceans.The section on "controlled reproduction of shrimp"defines the specific
aspects and environmental variables required for controlled reproduction of shrimp.
Finally,thelastsectiongivesanoverviewandanintegrationofthestudies performed
forthisthesis.
Reproductivebiologyofpenaeidshrimp
Primaryandsecondarysexualcharacteristics.
Penaeid shrimp are broadcast decapod-spawners (Dendrobranchiata). The
general structure ofthe female reproductive system consists ofapair ofmulti-lobed
ovaries(Fig.1),locatedinthedorsalregionoverthestomach,thehepatopancreas,and
theintestine.Theoviductsaresimpleandopentotheexterioratthecoxaeofthethird
pairofwalkinglegs(King,1948). Oocytematurationisdividedintwophases,primary
vitellogenesis and secondary vitellogenesis (Adiyodi and Subramoniam, 1983;
Charniaux-Cotton, 1985).Primaryoocytesstoreglycoproteinsproducedbythemselves,
then follicle cells proliferate around each oocyte and secondary vitellogenesis is
activated, accumulating vitellineproduced inhepatopancreas,hemocytesand ovaries
(YanoandChinzei,1987;QuackenbushandKeely,1988;Quackenbush,1989).
Marine shrimp females of the family Penaeidae show two distinctive
modifications ofthesternalplateslocatedbetweenthird,fourthandfifth walkinglegs
(Perez-Farfante, 1975;Browdy, 1992).Thisregion isknown asthe thelycum, andit
may present ornamentations for spermatophore attachment on the day of spawning
(open thelycum shrimp like P. vannamei, P. stylirostris, P. setiferus, and P.
occidentalis),oraseminalreceptacleforspermstorage(closedthelycumshrimplikeP.

12. Generalintroduction
Ovary
B Anteriorlobe
Laterallobe
Heart
Abdominalartery
Abdominallobe
Figure1.Femalereproductivesystemofpenaeidshrimp.A:Anatomicallocation
of the reproductive system. B: Isolated reproductive system (modified from
Shigueno,1975).

13. Chapter1
aztecus,P.monodon,andP.japonicus).
Malesarerecognizedbythepresenceofamodificationofexopoditesofthefirst
pair of swimming legs (pleopods). The modification consists of a membranous
developmentnamedpetasma,whichseemstoparticipateinspermatophoretransferand
attachment.Themalereproductivesystempresentsapairofdorsalmulti-lobedtestis
(Fig. 2).The spermiducts or vas deferens are very complex, showing the following
sections(startingfromthetestis):blindpouchnexttothetestis,ascendingmedialvas
deferens, descending medial vas deferens, distal duct, and terminal ampoule, which
opensatthecoxaeofthefifthpairofwalkinglegs(pereiopods,Talbotetal., 1989).
Twodistinctivechannelsareobservedinsidethemedialvasdeferens.
Spermatogenesis in the penaeid shrimp, Sicyonia ingentis, was extensively
studiedattheultrastructurallevelbyShigekawaandClark(1986).Inthetestis,sperm
cellsareimmature,andtheycompletematurationinthevasdeferens (openthelycum
species) orinthe seminal receptacles (closedthelycum species). Sperm cellsdonot
have flagella, insteadtheyhaveanon-motile "spike"(Clark etal., 1984;Shigekawa
andClark, 1986).Aparticularlyinterestingsubjectisspikeelongation,provedtobea
gradualprocessinP.stylirostristhattakesplaceinthedescendingmedialvasdeferens
(Fig.3).T.byrdimalespresentspikelessspermevenintissuesectionsfromthedistal
vasdeferens andampoules(Fig.3;Alfaro, 1994),butlightmicroscopicalobservations
ofspermfromspermatophoresrevealfullyelongatedspikes(Alfaro,unpublisheddata).
Therefore,asitwaspointedoutbyShigekawaandClark(1986)forS.ingentis,T.byrdi
sperm experiences rapid elongation that inevitably must take place within the
spermatophores.Recent lightmicroscopical observations inT.byrdishowthat sperm
morphology inspermatophoresremoved from males andinspermatophores removed
from female seminal receptacles issimilar. Inboth cases,sperm arepacked inmany
160um-capsules(spermatophores),thespikeisdeflected 80°from thebodyaxis,the
sperm body length is 7 - 9 urn, and the spike length 4 urn. These particular
characteristicshavealsobeen observed inXiphopenaeusriveti,which hasvery large
sperm: abodylength of 13urn with aspikelength of 6.5 xm(Alfaro, unpublished
data). InS. ingentis,sperm are capacitatedbythe female's thelycum, where further
development within the sperm cells takes place (Clark et al., 1984; Shigekawa and
Clark,1986).
Spermatophoreprimaryandsecondarylayers aresynthesizedintheascending
anddescendingvasdeferens,respectively(Roetal.,1990).Themainbodyandsperm
sac of the spermatophore originate in one of the two channels of the vas deferens
(Bauer and Cash, 1991). The terminal ampoule is histologically complex, showing
separated chambers where additional spermatophore sub-units (dorsal plate, wings,
glutinousmass,adhesive)areformedinopenthelycumspecies(Talbotetal.,1989).

14. Generalintroduction
Testes
Vasdeferens
Hepatopancreas
B
Blindpouch ^
Terminal
ampoule
Lobedtestes
Descendingmedial
vasdeferens
^. Ascendingmedial
vasdeferens
Distalduct
Figure2.Malereproductivesystemofpenaeidshrimp.A:Anatomicallocationofthe
reproductivesystem(modifiedfromShigueno,1975).B:Isolatedreproductivesystem
(modifiedfromKing,1948).

15. Chapter1
427nm
" 4gm
Figure3.A)Trachypenaeusbyrdispermatidfromthedistalvasdeferens.Thenucleus
presentsfibrillarchromatin(CF)andmembranelamellarbodies(CLM).Betweenthe
anterior granule (GA), from the acrosome, and the granular core (CG), from the
subacrosome,anewsubacrosomalregiondevelops,thesaucerplate(P).B)Penaeus
stylirostrisspermatidsfromthedescendingmedialvasdeferens. Spikeswith different
degreesofelongation(FromAlfaro,1994).

16. Generalintroduction
Penaeidswithclosedthelycumhavelosttheadhesiveandglutinousmaterials,andthe
dorsalplatefunctions asastoppertoplugtheopeningofthethelycum (Chowetal.,
1990;BauerandCash,1991).
Reproductiveendocrinology
Thereare6endocrinecentersbelievedtobedirectlyinvolvedinreproductionof
decapod crustaceans. The first one isthe ganglionic medulla terminalis -Xorgan -
sinusglandcomplex from theoptic ganglia.Othercenters arethebrain,thethoracic
ganglia,theandrogenicgland(males),themandibularorgan,andtheovaries(Fig.4).
Noendocrineactivityhasbeenidentifiedinthetestis.
Crustaceanswithpedunculatedeyeshaveagroupofneurosecretorycellsinthe
eyestalks,theXorgan.Thesecellssendthemajority oftheir axonstoaneurohemal
organ, the sinus gland (Quackenbush, 1986). The medulla terminalis - X organ
synthesizes polypeptides, that are packed in neurosecretory vesicles and transported
intra-axonallytothesinusgland,wheretheyarestoredandreleasedassmallpeptides
(Andrew, 1983). The whole complex is involved in the regulation of physiological
processes like calcium and sugar metabolism, heart rate, reproduction, molting,
osmoregulation, thermal seasonal acclimation, retinal pigments migration, and color
change(Quackenbush,1986).
The mechanism proposed for the control of gonadal maturation is an
antagonisticmodel(Fig.4),whichinvolvesthesynthesisofagonadinhibitinghormone
(GIH) from the X organ. This peptide was recently isolated and sequenced from a
lobster and a crayfish (Huberman, 2000). Thehormone inhibits or competes with a
hypothetical gonad stimulating hormone (GSH), produced in the brain and thoracic
ganglia.TheGSHactivatessecondaryvitellogenesisinfemales,andspermatogenesis,
hypertrophy ofvas deferens and androgenic gland hypersecretion inmales (Adiyodi
andSubramoniam, 1983).GSHisanabstractentity,butitsreleaseisstimulatedby5-
hydroxytryptamine and the red pigment-concentrating hormone; dopamine and
methionine enkephalin inhibit GSH release, and stimulate GIH release (Fingerman,
1997).
The androgenic gland isthe endocrine organ,which determines primary and
secondary male sexual characteristics (Charniaux-Cotton, 1960; Fingerman, 1987;
Charniaux-Cotton and Payen, 1988). Two androgenic gland hormones, AGH 1and
AGH2,were isolated from anisopodbyHasegawa et al.(1987).Other androgenic
glandextractslikefarnesylacetone andsteroidshavebeenidentified indecapods,and
theymayplaycomplementaryrolestoAGH(Sagi,1988).
10

17. Chapter1
OOCYTE MATURATION SPERMATOPHORE SYNTHESIS
Figure 4. Proposed pathways in endocrine control of reproduction in decapod
crustaceans. Continuous lines indicate a stimulatory effect; dotted lines indicate an
inhibitory effect; question marks indicate unconfirmed pathways. 5-HT: serotonin,
RPCH: red pigment-concentrating hormone, DA: dopamine, ME: methionine
enkephalin, GSH:gonad stimulatinghormone,GIH:gonadinhibitinghormone,MO-
IH:mandibular organ inhibiting hormone,MF:methyl farnesoate, AGH: androgenic
glandhormone.Themodelisbasedontheantagonisticeffect ofGSHandGIHoverthe
gonads.GSH induces oocyte maturation, presumablythrough steroid hormones.The
mandibularorganalsostimulatesoocytematuration.

18. Generalintroduction
It has been proposed that ovarian development and oocyte maturation in
crustaceans may be mediated by steroid hormones, as in fish and amphibia, where
estrogensstimulatevitellogenesisandprogestagensinduceoocytematuration(Fairset
al., 1990). In the lobster, Homarus americanus,these hormones are believed to be
producedinthefolliclecells(Talbot,1981).
The mandibular organ located near the esophagus, produces steroids and
terpenoids,thatstimulateovariandevelopment(TsukimuraandKamemoto,1991).The
terpenoid,methylfarnesoate (MF),wasidentifiedbyLauferetal.(1987).Itssynthesis
is increased by unilateral eyestalk ablation, and the compound is considered a
reproductive hormone as it induces ovarian maturation in the spider crab,Libinia
emarginata(Que-Taeetal.,1999).AmandibularorganinhibitinghormonefromtheX
organhasbeenpartiallyidentified(Huberman,2000,forreview).
Thefirstinvertebrate neuropeptides biochemically characterized werethe red
pigment-concentratinghormoneandthedistalretinalpigment lightadaptinghormone
ofcrustaceans(Josefsson, 1983).Othercrustaceanhormonesrecentlycharacterizedare
the molting hormone (20-OH-ecdisone; Chang, 1985), the molt-inhibiting hormone
(neuropeptide), and the hyperglycemic hormone (neuropeptide) (Benzie, 1998;
Huberman,2000,forreview).
Controlledreproductionofshrimp
InLatinAmericawildorpond-grownP.vannameiandP.stylirostrisarematured
incaptivity.Resistantshrimpareselectedfromtheculturedpopulationbasedongrowth
performance, keptinearthenpondsatalowdensity(1m"2
),andfed acombinationof
artificial feedsandfreshmarinefoodproducts.However,atpresentavoidanceof fishery
productsisrecommendedasapreventivemeasureagainstWSSVinAmerica(Joryand
Dixon,1999).Animalsfrompondsaregrownto45gforfemalesand40gformales,and
thentransferredtoamaturationfacility.
Males are stocked with females at a 1:1 ratio in large tanks (> 3.7 m in
diameter),underlowlightintensity,highwaterexchange(>100%daily),andavoiding
anykindofstress.Tomaturefemalesincontrolledenvironments,itisnecessarytofeed
marineanimalslikefish,oysters,worms,clams,andsquids.Theseproductswillgive
females an adequate level of essential nutrients: amino acids, cholesterol, and
polyunsaturated fatty acids(PUFAs).Oocytematuration and survival ofinitial larval
stagesareassociatedwiththeaccumulationwithinoocytesofsterols(synthesized from
dietary cholesterol), phospholipids, triacylglycerols, and diacylglycerols (synthesized
fromdietaryPUFAs;Ravidetal.,1999).Embryoqualityisaffectedbystorednutrients
sothatacceptablehatchingratesandnaupliiqualityareobtainedwhenbroodstock is
fed products from the marine food chain. Worms are particularly rich in PUFAs,
12

19. Chapter1
including20:5w3and22:6w3,whichareconsideredascrucialnutrientsfor crustacean
maturation(Harrison,1990).
Seawaterusedformaturationmustimitatethenaturalhabitat,whichisnormally
oceanicforpenaeidreproduction.However, somespeciesmayreproduce inestuarine
waterssuchasP.occidentalis(Alfaro etal., 1993).Generally, salinityismaintained
between28and36ppt,temperature intherangeof27to29°C,pHfrom 8.0to8.2,
dissolvedoxygenover5ppm,NH4-Nbelow0.1mg1"',andNO2-Nbelow0.05mgl"1
(Bray and Lawrence, 1992). To maintain a high quality environment, water is
exchangedatratesofover100%daily,inflow-throughsystems.
Reproduction ofmarineshrimpcanbeaccomplishedusingabroadvarietyof
lightsourcesandintensities.Ingeneralterms,itisadequatetousenaturalorartificial
coolwhitelightatlowintensity(<5ixErnV1
),withgradualincreaseanddecreaseof
light level,providing 14to 16hours of lightperday (Chamberlain, 1988;Bray and
Lawrence, 1992).Matingunderlaboratoryconditionsrequiresadequatespace(tanks>
3.7mindiameter)aswellasreducedturbulenceandnoisetofacilitate courtshipand
successofspermatophoretransfer,andhandlingshouldbeminimized(Browdy,1998).
When females have completed oocyte maturation, they will be receptive to
males to coordinate the mating behavior and spermatophore transfer, particularly at
dusk.Inseminationmayoccurundernaturalmatingorusingartificial insemination.A
few hours after spermatophore transfer, spawning takes place and sperm-egg
interaction initiates at the moment of water contact. Eggs show a cortical reaction,
characterized by the release of cortical rods containing jelly precursors and sperm
activators; at the same time, sperm undergoes acrosomal reaction, leading to a
secondary binding to the ovum's oolema (Clark et al., 1984). Eventually, germinal
vesiclebreakdownoccursandpro-nucleiarefused. Ahatchingenvelopeisformedby
thereleaseoftwodifferent typesofcorticalvesicles:densevesiclesandringvesicles
(PillaiandClark, 1988).Embryonaldevelopmentinpenaeidsleadstotheformationof
ahollow blastula, which evolves intothe larval stage named nauplius.The nauplius
emergesapproximately14hours(28°C)afterspawning.
Commercial facilities for nauplii production are integrated with maturation
facilities. Impregnatedandripefemales areindividuallytransferred tospawningtanks
(100-2001),whereeggsdeveloptheirembryonal stagesonthebottomofthetank.
Highqualitywater(1uafiltrationandU.V.disinfection) mustbeused(McVeyand
Fox,1983)andeggdensityshouldnotexceed3000eggsl"1
togetacceptablehatching
rates(Primavera,1985).
Eggswithhatchingenvelopeornaupliiarerinsedwithcleanseawaterfor5min,
concentratedinabucket,disinfectedwithformalin(eggs:100ppm/1 min;nauplii:400
13

20. Generalintroduction
ppm/30sec)andiodine(0.1ppm/ 1min),andwashedwithcleanseawaterfor 5min
(Browdy,1992).
Outlineofthisthesis
Theaimofthisthesiswastoprovidenewinformationforabetterunderstanding
ofpenaeid reproductivebiology, and to investigate some oftheproblems associated
with controlled reproduction of shrimp. The steps followed in this thesis are
schematizedinFig.5.
FirstweexploredthenaturaleventsinvolvedinP.occidentalisreproductionin
a tropical estuary, the Gulf of Nicoya, Costa Rica, where this species is the
predominantpenaeid.Thisstudyalsoinvestigated aprotocol for invitro fertilization,
usinghighqualitygametes,asanalternativetonatural fertilization (Chapter2).The
next step was to investigate spermatophore quality in penaeids. In captivity, male
shrimpdevelopreproductiveproblems.Thepathologyofadiseaseofthereproductive
system, which was not understood earlier, was studied in a susceptible species, P.
setiferus(Chapter3).Spermatophoreproductioninpond-grownmaleswasstudiedin
P. stylirostris (Chapter 4). Additionally, quality and natural deterioration of
spermatophores was further investigated in pond-grown P. vannamei(Chapter 5).
Next,theresearchwasfocusedonfindingahormonaltreatmentfortheimprovementof
malesexualcondition(Chapter6).Basedonthecurrentknowledgeaboutcrustacean
endocrinology, 17alpha-methyltestosterone and 17alpha-hydroxyprogesterone were
evaluated. Ovarianmaturation and spawning arecurrently induced through unilateral
eyestalk ablation. The effect of serotonin treatment on female maturation was
investigated inwildP.vannamei,asalessinvasivealternative (Chapter 7).A final
stepinthisresearchprocesswastogetaknowledgebaseforfutureembryoandnauplii
cryopreservation. Indoingso,Chapter 8explores embryoandnauplii sensitivityto
cooling,cryoprotectantsandhypersalineexposure,usingawildspecieseasytospawn
incaptivity, T.byrdi.InChapter 9the overall results are analyzed, and an integral
interpretationoftheseresultsisdiscussed.
14

21. Chapter1
Reproductivebiologyofwildpenaeidshrimp
-2Naturalreproduction P.occidentalis
Invitrofertilization
Factorsaffectingmalereproductivequalityincaptivity
-3MaleReproductiveBlackeningDisease
-4Spermatophorequalityinpond-grownmales
-5Spermatophorequalityinpond-grownmales
P.setiferus
P.stylirostris
P.vannamei
Improvinggametequalitybyhormonetreatment
-6Effect of17alpha-methyltestosteroneand17alpha-hydroxyprogesteronein
males P.vannamei
-7Effect ofserotonininfemales P.vannamei
Preliminaryinvestigationinembryoandnauplii cryopreservation
-8Cooling,cryoprotectant,andhypersalinesensitivity
Trachypenaeusbyrdi
P.stylirostris
Figure 5. Schematic representation of the research steps followed in this thesis.
Numbersindicatespecificchaptersaddressingthesubject.
15

22. Generalintroduction
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16

23. Chapter1
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17

24. Generalintroduction
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19

26. Generalintroduction
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20

27. Chapter1
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21

28. Chapter2
ReproductionoftheShrimpPenaeusoccidentalis(Decapoda:Penaeidae)in
Gulfof Nicoya,CostaRica
J.Alfaro,J.A.Palacios,T.A.Aldave,R.A.Angulo
EscueladeCienciasBiologicas,UniversidadNacional,
Heredia,CostaRica
Publishedin:Rev.Biol.Trop.(1993),41(3):563-572
(TheGeneralintroductionpresentsschematicfiguresofreproductivesystems;
therefore,originalphotographswereremovedfromthisarticle)

29. Naturalreproductionofshrimp
Abstract
ReproductionoftheshrimpPenaeusoccidentaliswasstudiedbetweenJune26andJuly
27, 1992nearCurazaobeach,ChiraIsland,GulfofNicoya,CostaRica.Thisisthefirst
documentationofreproductionofapenaeidshrimpinGulfofNicoya.Spawningbehavior
was divided in five stages based on these observations; a sperm-egg interaction was
evaluated using the in vitrospawning technique. Spawned eggs presented a cortical
reactionduringthefirst30minafterrelease,andthespermexperiencedprimarybinding
totheeggvitellineenvelope, butanacrosomereactionwasnotactivated.Thereappearto
be differences infertilization mechanisms between open and closed thelycum penaeid
shrimp.WildP.occidentalismalespresentedheavyspermatophores comparedtoother
openthelycumshrimps.Spermcount(49.52millionpercompoundspermatophore),and
spermabnormalities(22.0%),weresimilartothoseofwildP.setiferusfromtheGulfof
Mexico.
Key-words:Shrimp,Penaeus,reproduction,spawning,maturation
Introduction
TheGulfofNicoyaisatropicalestuaryandhasbeendividedintwosectionsof
different characteristics,theuppergulfandthelowergulf(DeVriesetal., 1983).The
upper gulf is shallow (< 20 m) with muddy sediments and is bounded mostly by
mangroveswamps;thetransitionbetweenbothareasislocalizednearSanLucasisland
andPuntarenaspeninsula. Somephysical-chemical aspects(Peterson, 1969;Valdeset
al., 1987),crustacean biology (DeVries et al., 1983;Epifanio and Dittel, 1984) and
shrimp fisheries (Carranza, 1985;Vitola, 1985;Palacios etal., 1992)have alsobeen
evaluatedintheGulfofNicoya.
Decapod crustaceans belonging to Penaeidae family are organized in four
subfamilies: Aristaeinae, Solenocerinae, Sicyoninae, and Penaeinae. The first two
includedeepwaterspeciesandthelasttwosubfamilies includecoastalshrimp(Imai,
1980).
Boschi (1979) describes the reproductive cycle of the genus Penaeus that
consistsoftwophases.Inthefirstphase,pre-adultsmigratefromestuarinewaterstoa
reproduction zone in deeper and higher salinity waters. Once the spawning occurs,
postlarvaemigratetoestuarinewaters.Thispatternisfollowed byvariousspecies:P.
duorarum,P. brasiliensis,P. aztecus,P. schmitti,P. stylirostris, P. vannamei, P.
setiferusandP.occidentalis(GarciaandLeReste,1981).
The quality of wild spermatophores during natural mating has been partially
documented.Thistypeofanalysishasbeenreportedformalesundercultureconditions
(controlled reproduction) and wild P. setiferus broodstock (Leung-Trujillo and
24

30. Chapter2
Lawrence, 1985; Leung-Trujillo and Lawrence, 1987; Chamberlain, 1988; Alfaro,
1990).
Observations about spawning behavior, cortical reaction and sperm-egg
interactionhavebeenpartiallyevaluatedinpenaeidshrimp(Pillaietal.,1988;Clarketal.,
1984).Mostofourknowledgeabout fertilization mechanismsinmarine shrimpcomes
fromSicyoniaingentis(Sicyoninae)andP.aztecus(Clarketal.,1980; Clarketal.,1981;
LynnandClark,1987;PillaiandClark,1987;PillaiandClark,1988;Griffin etal.,1988).
Following contact with seawater, penaeid eggs experience a cortical reaction,
characterizedbyamassivereleaseofcortical cryptsorrods.Thencorticalvesiclesare
released,whichgiveorigintothehatchingenvelope.Duringsperm-egginteractionsperm
bindtoeggs,andmoleculesreleasedbytheeggsactivatethesperm;spermundergoan
acrosomal reaction, characterized by spike depolymerization, and the release of the
acrosomalvesiclecontent.Theseeventsareaprerequisiteforfertilization.
The spawning behavior has been described for closed thelycum shrimp: P.
japonicus (Hudinaga, 1942),P.monodon(Motoh, 1981)andS.ingentis(Pillai etal.,
1988). However, in open thelycum shrimp (P. vannamei, P. stylirostris, P.
occidentalis),thespawningbehaviorhasnotbeenstudiedindetail.
Thisworkpresentsresultsaboutmaturationandmatinginthewhiteshrimp,P.
occidentalis,from Curazao beach, Chira Island, and observations on their spawning
behavior,corticalreaction,andsperm-egginteraction.
Materialandmethods
Captures
OnJune26,1992,azonewithmaturationandmatingactivityforthespeciesP.
occidentaliswasfound.ThereproductionzoneislocalizednearCurazaobeach,onthe
northeastsideofChiraIsland(Fig.1),atadepthof2-6m.
Six fishing trips were assigned to the reproduction zone. Once a week, a
monofilament fishingnet(meshsize=7.60cm)of212mlongand2.43mwide,was
castduringaperiodof20min.Thisprocedurewasrepeatedthreetimes.Captureswere
performed during afternoon hours and the degree of maturation and mating was
registered until the end of the closed fishing season (July 31). In addition to field
observations,sampleswerepreservedoniceforlaboratoryanalysis.
Maturationandmating
Female maturation was evaluated using an arbitrary scale based on our
observationsaboutthedegreeofdevelopmentandthecolorationofovaries:
1. Undefined:ovaryoutlineisnotdistinguishable.
25

31. Naturalreproductionofshrimp
2. Maturing:anterior,lateral,andabdominalovarianlobulesareclearlyvisible
andtheircolorationisopaque.
3. Almost mature: lobules are highly developed with a yellow to light red
coloration.
4. Mature:lobulesareturgidwithadarkredcoloration.
Natural mating was evaluated based on the presence of complete
spermatophores,spermatophoreresidues,orspermmass,attachedtofemalethelycum.
Controlledspawningandspermatophoreanalysis
During the fishing trips mature females with and without attached
spermatophores, and males with spermatophores were selected. These animals were
transported in isolated containers (7 animals/ container) with chilled (23.5 °C) and
oxygenated seawater to the Experimental Maturation Laboratory at Criadero de
CamaronesdeChomesS.A,whereexperimentsonspawningandsemenanalysiswere
performed.
Inthelaboratory,thepresenceofspermatophores orresiduesonthethelycum
was again evaluated and animals were gradually acclimated to the culture water
(temperature=27°C,salinity=35ppt).Impregnatedfemaleswereisolatedinplastic
tanks (40L)with seawatertreated byfiltration (silica sand and 1^m cartridge) and
sedimentation.EDTAwasaddedasachelatoratarateof 10mgl"1
(Chamberlainand
Lawrence, 1983)andaerationwasprovided.Bytaking3,0mlsamples(4replicates),
the number of spawned eggs was estimated. Also most of the impregnated females
completely removed their sperm mass and spermatophore residues, during the 3 h
transportationtime.Eightimpregnatedfemales andsevenmaturebutnot impregnated
femaleswereselectedforspawning.Theyweretransferredtospawningcontainersand
keptunderconstantobservationuntiltheirspawning.
Theeventsassociatedwiththesperm-egg interactionwere studiedthroughin
vitrospawningsusingthefollowingtwotechniques:a)plasticcones,andb)beakers.At
themomentofspawningfemaleswerecapturedandheldovera300mlplasticconeor
100mlbeaker.Thesecontainerswerepreviouslyinoculatedwithasperm suspension,
obtained by homogenization of two sources: a) sperm mass removed from its
spermatophoreormedialvasdeferens,andb)completespermatophores.Spermdensity
in spawning units was estimated by hemacytometer counting (Alfaro, 1990; Alfaro,
1992).Toensureadequatespermandeggsmixing,plasticconeswereprovidedwithan
aerationline,andsuspensionsinbeakersweremixedwithagoiter.Additionally,ovain
beakersweregentlyswirled,followingtheprotocolforinvitrospawningofS. ingentis
(Griffin etal.,1988).
26

32. Chapter2
Costa Rica
km
10 0 10
I l,„, i
10°00'N
Pacffico
9°30'N —
85°00'W
Figure1. Locationofthestudyarea(station 1)inthenortheastcoastofChiraisland,
GulfofNicoya,CostaRica.
27

33. Naturalreproductionofshrimp
Spermanalysisinvolvedtotalspermcountpercompound spermatophore(two
sub-units), compound spermatophore weight, and percentage of sperm with missing
spikes or malformed bodies, following the techniques previously described (Alfaro,
1990;Alfaro,1992).
Results
TheJuly7capturecouldnotbeanalyzedquantitatively(Figs.2and3).These
figuresshowthefrequency offemalesandmales(Fig.2),ovarianmaturation,andrate
ofmatureandimpregnatedfemales(Fig.3)inthereproductionzone.
Captureanalysisindicatesthatduringfourweeksofconsecutivesampling,there
wereadultmalesandfemalesinthearea.Femaleswerefoundinstages1,2,3and4of
ovarian maturation. Itwas found that over40.7 %ofmature females captured each
weekwereimpregnated. OnJuly22,onefemale and3maleswerecaptured,andon
July27maturingfemalesappearedagain.
Under laboratory conditions, P. occidentalis females showed the following
spawningpattern:
A) Resting.Femalesshowminimumornoactivity,restingonthebottomofthe
tank.Thisphaselasted5-6hours.
B) Ascension anddescension.Femalesswimtothesurface andrapidlyreturn
tothebottom.Thisbehaviorisrepeated,lasting1h.
C) Swimming.Femalesaresuspended inthewatercolumn,swimmingslowly
around the tank; pleopods are vigorously agitated and the abdomen is
curved.Thisphasemaybeshortorberepeatedsometimes.
D) Eggreleasing.Femalesreleasetheireggswhileshowingbehavior (C).Eggs
descendandaredispersedbythewatercurrentgeneratedbypleopods.The
ovarycontentisevacuatedpartiallyorcompletely;acompletespawntakesa
fewminutes.
E) Spawned.Femalesrestonthebottom,showingminimumactivity.Ovaries
have lost their dimension, outline, and color. In partial spawns, females
presentabdominalovarianlobes.
Spawning experiments showed that all impregnated and 5 non-impregnated
femalesspawnedatdawnthedayaftertheircapture.Only2impregnatedfemaleshad
abnormalspawns,characterizedbythereleaseofoocytesthatformmassesthatstickto
thebottomofthetank.Females(meanweight=63.4±8.7g)released26600to186600
eggsperspawn.
Aparticularaspectofthisspecieswasthatthemajorityofimpregnatedfemales
completelyremovedtheirspermmassduringtransport,possiblyasastressresult.For
28

34. z
0.
s
Of
X
m
100-1
90-
80-
70-
60-
50-
40-
30-
20-
10-
0-
66.94
^ B (7.81)
• f c = | (3.75)
^ 1 156
'89
^ H I (11.91)
• FEMALES
• MALES
72.24
(7.33)
46.79 H
(1.26) •
52.84
(3.10)
Chapter2
69.17
(13.43),
47.21
(4.31)
43.16
(5.80)
26/VI 02A/II 15MI 22/VII
CAPTUREDATE(1992)
27A/II
Figure 2.Number of shrimp and average total weight (g) of Penaeusoccidentalis
collectedduringthestudyinChiraIsland(northwestcoast),GulfofNicoya.Valuesin
parenthesisrepresentstandarddeviationofweight.
26/VI 02/VII 15/VII 22/VII
CAPTUREDATE(1992)
27/VII
Figure 3. Maturation stages (1, 2, 3 and 4) and rate of impregnated Penaeus
occidentaliscapturedinthenorth-eastcoastofChiraIsland,GulfofNicoya.
29

35. Naturalreproductionofshrimp
example,atthemomentofcaptureonefemalewasremovingthespermmasswithher
claws. Probably, females that arrived tothe laboratory with sperm masses, removed
them during the night because spawns were infertile. Fertilization in penaeids is
externalanditoccursatthemomentofspawning.
The technique of in vitro spawning allowed the evaluation of the cortical
reactionofeggsandspermcondition.Table 1summarizesthecorticalreactionevents
observedinfreshsamples.Thereleaseofcorticalcryptsisamassiveprocessthatforms
ajellycoatof660ixm indiameter,aroundtheeggs(220|im).Eggdiameterisreduced
Table1.CorticalreactioneventsforPenaeusoccidentaliseggsduringinvitro
spawnings3
Time(min) Eventb
0 Spawning.Eggdiameter=280ixm.
8 Eggssurroundedbycorticalrods.
15 Roddissipationandhatchingenvelopeformation.
30 Hatchingenvelopecompletelydeveloped.
a
Temperature=27°C,salinity=35ppt.
b
Thesequenceofeventsoccurredinsimilarwaywithandwithoutsperm.
duringthecorticalreaction andthisendswiththe formation ofahatchingenvelope;
theseeventsdonotneedthepresenceofsperm.Invitrospawningwaspracticedbytwo
techniques.Table2showstheconditionsandobservationsobtainedinthespawnings;
noacrosomalreactionofspermwasobservedundereithertechnique.Table3presents
thequalityparametersofspermatophoresfrom wildmalesduringanaturalperiodof
maturationandmating.
Discussion
This work documents a massive reproductive activity of the species P.
occidentalisnearCurazaobeach,Chiraisland.Theevidenceindicatesthatintheregion
P.occidentalisdevelopovariesandmatewiththeimminentreleaseofeggsinthesame
area.ThisprocesswasobservedbetweenJune26andJuly 15,1992(closedseasonfor
shrimpfishery);samplesfromJuly22indicateanabsenceofadultsinthearea,andthe
followingweek(July27),theactivityappearsagain.
Carranza(1985)establishedthattheupperGulfofNicoyaisnotareproduction
regionforwhiteshrimps.However,thisstudydemonstratesthatthereproductivecycle
ofP.occidentalistakesplaceinestuarineandshallowwaters.Theabsenceofgravid
femalesinCarranza'sstudymaybetheresultofselectedsamplingstations.
30

36. Chapter2
Table2.ObservationsonPenaeusoccidentalisspermininteractionwitheggs,during
invitrospawnings.
Spawning
technique
Cone
Beaker
No.of
spawn
1
2
3
4
Spermdensity
(cel./ml)a
150000b
305000b
185000c
185000c
(
0
N.C.
N.C.
N.C.
N.C.
spermmorphology
15
N.C.
N.C.
N.C.
N.C.
30min
N.C.
N.C.
N.C.
N.C.
a
Spermsuspensionswereprepared1or2hoursbeforespawning.
b
Spermremovedfromspermatophoresandmedialvasdeferens.
c
Spermfromspermatophores.
N.C.=nochangeinmorphology
Table3.SpermatophorequalityinwildPenaeusoccidentalis.
Animal
1
2
3
4
Average
Totalweight
49.4
40.6
46.8
46.0
45.7
Spermatophore
weight(g)
0.27
0.24
0.35
0.33
0.30
Spermcount
(millions)
44.80
45.80
60.75
46.75
49.52
Abnormality
(%)
29.8
22.0
16.9
19.3
22.0
It is interesting to indicate that during the study, captures were almost
exclusivelyofP.occidentalis.Thisseemstoindicatethatduringreproductionactivity,
penaeidspeciesdispersefromeachother,asithasbeenreportedforP.setiferusandP.
aztecus(ChamberlainandLawrence,1983).
InthesoutheastcoastofChiraisland(LagarteroandLagartitobeaches),twoP.
occidentalisfemalesinstage4ofmaturationwithspermatophoreresidues,andoneP.
stylirostrisfemaleinstage4,butnotimpregnated,werefound.Thisparticular finding
couldbeanindicationthat activitiesliketheonemonitorednearCurazaobeach,are
occurring inother areas ofGulfofNicoya, whereP.occidentalisandP.stylirostris
31

37. Naturalreproductionofshrimp
reproduce.Onlythrough constantobservationitwillbedefinedwhereandwhenthese
reproductive events occur. Knowing the location of spawning grounds has a great
importancefortheestablishmentofpermanentprotectionareas,thatcontributetothe
balancebetweenexploitationandconservation.
Mating ofP. occidentalisin the study area occurred during early afternoon
hours. Impregnated females were found in samples taken at 12:00. This finding
indicatesthatthereproductiveactivitywasveryintensebecauseopenthelycumshrimp
generallymate atdusk (Chamberlain andLawrence, 1983).Openthelycum shrimps
mateafewhoursbeforespawning,andthepresenceofaspermmass,atthemomentof
spawning,isaprerequisitefor fertilization. Onthecontrary,closedthelycum females
like P. aztecus (Chamberlain and Lawrence, 1983), P. notialis, P. subtilis, P.
brasiliensis(Martinezetal.,1984),matebeforeovarydevelopmentandstorespermin
their seminal receptacle (Browdy, 1992). Sperm mass removal by females, in the
presentstudy,isprobablyaconsequenceofstressassociatedwithcaptureandtransport.
However,otherwildopenthelycumpenaeidslikeP.setiferus(Brayetal.,1983)andP.
schmitti(Martinezetal.,1984)donotexpresssuchbehavior,releasingfertileeggsafter
captureandtransport.
The spawning behavior is similar to that observed inP. vannameigrown in
ponds(unpublisheddata)andS.ingentis(Pillaietal.,1988).However,thedescription
byphasespresentedinthisdocumentcontributestoabetterdefinitionoftheprocess.
Femaleremoval atthemomentofspawningforinvitroassaysresulted inthe
partialreleaseofeggs.Onthismatter,wildP.occidentalisdemonstratedtobehighly
sensitive tothe technique; onthe contrary, wild S. ingentis(Pillai et al., 1988), P.
vannameiandP.stylirostrismaturedinthelaboratory(unpublished data),generateda
massivereleaseofeggsbythistechnique.
Corticalreactioneventsaresimilartothosedescribed fortheclosedthelycum
species,S.ingentis(PillaiandClark, 1987)andP.aztecus(Clarketal.,1980).Toour
knowledge,foropenthelycumshrimps, thecortical eventshavebeendescribedonly
for P.setiferus(Clarketal., 1980).Ourfindings onP.occidentalisindicatethat at
contact with seawater eggsexperience amassive release of cortical rods, lifting the
vitelline envelope. Sperm cells are observed in primary binding to the vitelline
envelope;with the release of rods,eggsreduce their size from 280 um to 220 um.
Finally,rodsdissipate andthehatchingenvelopedevelops.Spermarenotrequiredfor
theactivationoftheseevents.
Duringsperm-egginteraction(Clarketal.,1981)orinvitroactivationwithegg
water(Griffin etal.,1988),S.ingentisspermexperiencean acrosomalreaction. InP.
aztecus the reaction is different and it has been observed during the sperm-egg
interaction(Clark etal., 1980).InP.stylirostris,theacrosomal reaction seemstobe
32

38. Chapter2
characterized by spike depolymerization, followed by acrosomal vesicle exocytosis
(Clark,W.andGriffin,F.,personalcommunication).
Inthisresearch,duringthesperm-egginteractionsofP.occidentalis,nosperm
were observed developing an acrosomal reaction. Onlytheprimarybinding between
spermandthevitellineenvelopeofeggsoccurredmassively.InS.ingentis mostsperm
experience an acrosome reaction when cells are mixed invitro.To our knowledge,
therearenoreferencesaboutacrosomalreactioninopenthelycumpenaeids;therefore,
theanalysisofourobservationsislimited.Basedonstudieswith S.ingentis,itwould
beexpectedthatamassiveacrosomereactionduringtheinteractionbetweenwildeggs
(goodquality)andwildspermof P.occidentaliswouldoccur,butitdidnothappen
thatway.Itseemsthatfertilization inopenthelycumshrimpshasparticularitieswhich
havenotyetbeenunderstood.
Wild males of P. occidentalis showed spermatophore quality parameters
similartothoseobtainedforwildP.setiferus(spermcount=45million,abnormality=
23%; Alfaro, 1990).P. occidentalisspermatophore weight (0.30 g) is considerably
heavierthanthe averagevalueregistered forP.setiferus(0.14 g).The sizeofthese
unitsmaybethecausefortheunsuccessful manualejaculation,whichcauseddamage
tothereproductivesystemineverytestedmale(n=24).Thisproblemdoesnotoccur
withP.vannamei,P.stylirostris,andP.setiferus,whichcanbesuccessivelyejaculated.
ThisparticularityofP.occidentalisisalimitationformalereutilizationinfuturestock
enhancementprogramsusingculturedpost-larvae.
Acknowledgements
Thisresearchispartoftheproject: White Shrimp,supportedbyCONICIT -
Ley de Pesca - Escuela de Ciencias Biologicas, UNA, and the project: Controlled
ReproductionofShrimp,supportedbytheprogramsUNA-JICAandUNA-LUW.We
thank Vicerrectoria de Investigation, UNA, Chira island community, Criadero de
Camarones de Chomes S.A., and to the students, Alex Molina, Sidey Arias and
GerardoZunigafortheircooperation.
References
Alfaro, J., 1990. A contribution to the understanding and control of the male
reproductive system melanization disease of brood stock Penaeussetiferus.
MasterThesis,TexasAandMUniversity,CollegeStation,Texas,USA.
Alfaro,J., 1992.Reproductive qualityevaluation ofmalePenaeusstylirostrisfrom a
grow-outpond.J.WorldAquacult.Soc.24(1),6- 11.
33

39. Naturalreproductionofshrimp
Bray,W.A.,Chamberlain,G.W.,Lawrence,A.L.,1983.Observationsonnaturaland
artificial insemination of Penaeus setiferus. Proc. 1st
Int. Conf. on Warm
Aquaculture,Crustacea.Hawaii,pp.392-405.
Boschi, E., 1974. Biologia de los crustaceos cultivables de America Latina. Simp.
FAO/CARPASobreAcuacul.enAmer.Lat,Uruguay,23pp.
Browdy, C, 1992. A review of the reproductive biology of Penaeus species:
Perspectives oncontrolled shrimp maturation systems for high quality nauplii
production. In: Wyban, J. (Ed.), Proc. of the Special Session on Shrimp
Farming,WorldAquacultureSociety,BatonRouge,LA,USA,pp.22- 51.
Carranza, F. P., 1985.Distribucion y abundancia del recurso de camaron bianco y
alternativas deaprovechamiento mediante unmetododepesca artesanal enel
Golfo de Nicoya. Tesis de maestria, Universidad Nacional Autonoma de
Mexico,D.F.,Mexico,60pp.
Chamberlain, G. W., 1988. Stepwise investigation of environmental and nutritional
requirements for reproduction ofpenaeid shrimp.PhD Thesis,Department of
WildlifeandFisheriesSciences,TexasA&MUniversity,USA.
Chamberlain, G. W., Lawrence, A. L., 1983.Reproductive activity and biochemical
composition ofPenaeussetiferusandPenaeusaztecusinthe Gulf ofMexico.
TAMU-SG-84-203,CollegeStation,Texas,35pp.
Clark,W. H.,Kleve,M. G., Yudin, A. I., 1981.An acrosome reaction in natantian
sperm.J.Exp.Zool.218,279-291.
Clark,W.Ff.,Lynn,J.W.,Yudin,A.I.,Persyn, H., 1980.Morphology ofthecortical
reactionintheeggsofPenaeusaztecus.Biol.Bull.158,175-186.
Clark,W.Ft.,Yudin,A.I.,Griffin, F.J., Shigekawa,K., 1984.Thecontrolofgamete
activationandfertilization inthemarinepenaeidae,Sicyoniaingentis. In:Engels,
W.(Ed.),Advancesininvertebratereproduction 3.Elsevier SciencePublishers.
NewYork,pp.459-472.
DeVries, M. C, Epifanio, C. E., Dittel, A. I., 1983.Reproductive periodicity of the
tropicalcrabCallinectesarcuatusOrdwayinCentralAmerica.Est.Coast.Shelf
Sci.17,709-716.
Epifanio,C.E.,Dittel,A.I., 1984.Seasonalabundanceof brachyurancrablarvaeina
tropicalestuary:Gulfof Nicoya,CostaRica,CentralAmerica.Estuaries7(4B),
501-505.
Garcia,S.,LeReste,L., 1981.Life cycles,dynamics,exploitation andmanagementof
coastalpenaeid shrimp stocks.FAOFisheriesTechnical ReportNo.203,116
pp.
Griffin, F. J., Shigekawa, K., Clark, W. H., 1988. Formation and structure of the
acrosomalfilamentinthespermofSicyoniaingentis.J.Exp.Zool.246,94-102.
34

40. Chapter2
Hudinaga,M.,1942.Reproduction,developmentandrearingof PenaeusjaponicusBate.
Jpn.J.Zool.10,305-393.
Imai,T.(Ed.),1980.Aquacultureinshallowseas:progressinshallowseaculture.A.
A.Balkema,Rotterdam,Holanda.
Leung-Trujillo, J., Lawrence, A. L., 1985. The effect of eyestalk ablation on
spermatophoreandspermqualityinPenaeusvannamei.J.World Marie.Soc.16,
258-266.
Leung-Trujillo,J.,Lawrence,A.L., 1987.Observationsonthedeclineinspermqualityof
Penaeussetiferusunderlaboratoryconditions.Aquaculture65,363-370.
Lynn,J.W.,Clark,W.H.,1987.Physiologicalandbiochemicalinvestigationsoftheegg
jellyreleaseinPenaeusaztecus.Biol.Bull.173,451-460.
Martinez, L. S., Torres, M., Remolina, R., Maldonado, J., 1984. Reproduction
artificial decincoespeciesdiferentes decamaronmarinoparalaobtencionde
semilla como alternativa en el repoblamiento de areas naturales y desarrollo
acuicola.CentrodeInvestigacionesPesqueras,INDERENA,Colombia,83pp.
Motoh, H., 1981.Studies on the fisheries biology of the giant tiger prawn,Penaeus
monodoninthePhilippines.TechnicalpaperNo.7,SEAFDEC,Filipinas.
Palacios,J.A.,Rodriguez,J.A.,Angulo,R.A.,1992.EstructurapoblacionaldePenaeus
stylirostris(Decapoda:Penaeidae),enelGolfodeNicoya,CostaRica.Rev.Biol.
Trop.(inpress).
Peterson,C.L.,1960.LaoceanografiafisicadelGolfodeNicoya,CostaRica,unestuario
tropical.Inter.Amer.Trop.Tun.Comm.Bull.4 (4),139-216.
Pillai,M. C. ,Clark, W. H., 1987.Oocyte activation inthe marine shrimp,Sicyonia
ingentis.J.Exp.Zool.244,325-329.
Pillai, M. C, Clark, W. H., 1988.Hatching envelope formation in shrimp {Sicyonia
ingentis)ova:originandsequentialexocytosisofcorticalvesicles.TissueCell20
(6),941-952.
Pillai, M. C, Griffin, F.J, Clark, W. H., 1988.Induced spawning of the decapod
crustaceanSicyoniaingentis.Biol.Bull.174,181-185.
Talbot,P.,Howard, D.,Leung-Trujillo, J.,Lee,T.W.,Li,W-Y.,Ro,H.,Lawrence,A.
L., 1989.Characterization ofmalereproductivetractdegenerative syndromein
captivepenaeidshrimp(Penaeussetiferus).Aquaculture78,365-377.
Valdes,J.,Brenes,C.L.,Solis,E.,Mendelewicz,M.,1987.Propiedadesfisicoquimicas
delasaguasdelGolfodeNicoya, CostaRica.Ing.Cienc.Quim.11(1),21-25.
Vitola,M.M., 1985.Camaronespeneidos(Decapoda:Natantia) delGolfo deNicoya,
Costa Rica: Un analisis de su distribution y densidad. Tesis de Maestria,
UniversidaddeCostaRica,SanJose.
35

41. Chapter3
Interactionof BacteriaandMaleReproductiveSystemBlackeningDisease
ofCaptivePenaeussetiferus
J.Alfaro',A.L.Lawrence2
andD.Lewis3
1
EscueladeCienciasBiologicas,UniversidadNacional,Heredia,CostaRica.
2
DepartmentofWildlifeandFisheriesSciences,TexasAgriculturalExperimentalStation,
TexasA&MUniversity,P.O.DrawerQ,PortAransas,TX78373.
3
DepartmentofVeterinaryMicrobiologyandParasitology,CollegeofVeterinary
Medicine,TexasA&MUniversity,CollegeStation,TX77843.
Publishedin:Aquaculture(1993),117:1-8

42. MaleReproductiveBlackeningDisease
Abstract
Thegenitalapparatusofmalepenaeidshrimp,Penaeussetiferus,blackenswithresulting
detrimentaleffectsonmatingforproductionoflarvaewhenanimalsarekeptincontrolled
maturation/reproduction situations. A progressive, melanized condition of the male
reproductive tract was shown to be associated with bacterial infection. At least three
different species (Vibrioalginolyticus, Pseudomonasputrefaciens,and an unclassified
strain),wereisolatedfromdamagedtissuesandsuccessfully developedthesamesignsin
challengeexperiments.Itissuggestedthattheconditioncouldbeaprogressivesyndrome
withbacterialinvasionperhapsonlyintheadvancedstages,orthatmorethanoneetiology
maybeinvolvedindeteriorationandblackeningofP.setiferusreproductivesystem.
Introduction
Pathologicalconditionsassociatedwiththemalereproductivesystemofpenaeid
shrimp have been reported. These conditions frequently affect Gulf of Mexico white
shrimp,Penaeussetiferus, whenthis speciesisheld incaptivity. Reproductive tissue
darkeninghasbeenalsodocumentedfromP.vannamei,P.stylirostris,andthefreshwater
prawn,Macrobrachiumrosenbergii(Chamberlainetal.,1983;HarrisandSandifer,1986).
It appears that P. setiferusis particularly susceptible to these conditions, which are
detrimentaltolarvaeproductionsincetheyrendermalesinfertile.
Signsofconditionsaffecting malereproductivesystemweredescribedpreviously
byChamberlainetal.(1983),Leung-TrujilloandLawrence(1987),Chamberlain(1988),
andTalbotetal.(1989).Untilnowthecauseoftheseconditionshasnotbeenestablished.
The study upon which this report was based, was designed to evaluate the possible
interaction between bacterial infection and a male reproductive system blackening
conditionofPenaeussetiferus.
Materialsandmethods
Broodstockandexperimentalconditions
SexuallymaturePenaeussetiferusmaleswithameanweightof40±5.2g(mean
±SEM)wereobtainedbytrawlingoffPortAransas(27°5'N,97°3,W),Texas.Animals
were transported in chilled (22 °C) and oxygenated seawater to the Texas A&M
University Laboratory at Port Aransas, where they were gradually acclimated to the
culturewater(temperature=30°C,salinity=31ppt).
Shrimpwerestockedat3.3animalsm"2
in2.4mdiameterlightblue fiberglass
tanks.Waterflowwascontinuousandadjusted to 151min" usingasemi-closedwater
recirculationsystemwithoutsterilization.Broodstockwerefedat3%bodyweightday'of
apelletedpreparedbroodstockdiet(60%protein;10%lipid).
38

43. Chapter3
Hydrologicalparameterswereoptimumandstableduringtheexperimentalperiod.
Watertemperaturewas29±1 °C,salinity30±1 ppt,dissolvedoxygen6.4±0.3mgl"1
,
andammonia-nitrogen0.107±0.053mgl"1
.
Bacteriaisolationandcharacterization
Three different types of reproductive tissue were classified based on tissue
appearance: A) tissue from wild males within 12 days of capture, showing
spermatophores,ampoules,andvasdeferenswithnormalcoloration,B)tissuefromwild
malesmorethan32dayspostcapture,showingdeterioratedspermatophores,butnoblack
pigmentonampoulesorvasdeferens,C)tissuefromwildmalesmorethan 12dayspost
capture,showingblackpigmentonspermatophores,ampoules,andvasdeferens.Various
regions ofthereproductive systemwereprocessed torecoverbacteria asfollows. The
malegenitalapparatuswasasepticallydissectedinsterilepetridishes,thenthesystemwas
divided intoampoule,distal,medial vas deferens segments 2Aand 2B (Talbot et al.,
1989).Somespermatophoreswereremovedfromtheirampoules.Reproductive system
sectionswerethenmaceratedin2mlsterilesaline(0.85%)andhomogenizedinasterile
blender.
Homogenizedmaterialswerestreakedontrypticasesoyagar(DifcoLaboratories,
Detroit,MI48232)containing2%sodiumchloride(TSA)andincubatedat25°Cfor2
days. Bacterial growth was observed 24 h after inoculation; negative plates were
incubatedforanadditionalfivedaystodetectslow-growingbacteria.
Bacterial characterization wasperformed usingthe API20E System (Analytab
Products,200ExpressStreet,Plainview,NY11803)asrecommendedbyLightner(1983).
Infectivityexperiments
Bacteriarecovered from blackreproductive sectionswerecultured intrypticase
soybroth(TSB,Difco Laboratories)containing2%sodiumchlorideat25°Cfor24h.
Threechallengesystemswereevaluated(n=6malespersystem):intramuscularinjection
(26-gaugetuberculinsyringes)of0.1ml5-fold-dilutedsaline(0.85%)-resuspendedisolate
1;surfaceexposure(preparedbycentrifugation ofbroth)ofisolate1appliedwithsterile
cotton swab to gonopores; and injection into the gonopore of 0.05 ml 5-fold-diluted
saline-resuspended isolate 1.Controlswereprovidedusingthesamechallengesystems
usingsterileTSB-salines(n=6malespersystem).Afourth challengesystemwasthen
evaluated:gonoporeinjectionof0.05ml50-fold-dilutedsaline-resuspendedisolate1(n=
10),isolate2(n=5),andisolate4(n=5);sterileTSB-salineswereusedascontrols(n=5
malesperisolate).Brothwaseliminatedbycentrifugationpriortobacterialresuspension
and dilution. Males selected for these experiments presented deteriorated reproductive
systems(morethan54dayspostcapture)withnovisibletantoblackcoloration.
39

44. MaleReproductiveBlackeningDisease
Results
Isolationofbacteriafrommalereproductivesystems
Bacterialisolationattemptsrevealedthatonlyblackreproductivetissuesections
(typeCtissue)containedbacteria (Table 1).Inoculatedplates from blackreproductive
sections gave positive and massive growth of pure colonies at 25 °C in 24 h from
incubation.NeithertypeAtissueortypeBtissueproduced bacterialgrowth.
Table1.BacteriarecoveredfromdifferentsectionsofreproductivesystemsofPenaeus
setiferus.
Reproductivetissue
condition3
Section Bacteria
growthb
TypeA
TypeB
TypeC
SP
AM/SP
SP
AM/SP
AM
MV2A
MV2B
SP
AM/SP
DV
MV2A
MV2B
3
1
6
2
1
1
1
2
1
1
1
1
(-)
(-)
(-)
(-)
(-)
(-)
(-)
(+)
(+)
(+)
(+)
(+)
a
Type A= tissue showing normal coloration. Type B= tissue showing deteriorated
spermatophores, but no black pigment on ampoules or vas deferens. Type C= tissue
showingblackpigment.
b
Recovering wasperformed in TSA 2%NaCl incubated at 25 °C for 6 days.AM=
ampoule,DV=distalvasdeferens,MV2A=medialvasdeferens2A,MV2B=medialvas
deferens2B,SP=spermatophore.
Organisms were identified based on morphological and biochemical
characteristicsofisolates(Table2)andthekeysof20EAnalyticalProfileIndex(Analytab
Products), Bullock (1971), Lightner (1983), and Frerichs and Hendrie (1985).
Identification suggests that isolate 1 and 3 are Vibrioalginolyticus,isolate 4 is
Pseudomonas putrefaciens,and isolate 2 could not be identified with the available
information.
40

45. Chapter3
Table2.Characteristicsofbacteriaisolatedfromblackreproductive
Reaction3
Morphology
Gramstainb
MacConkeygrowth
Cytochromeoxidase
Motility
Glucosefermentation
Glucoseoxidation
O-nitrophenyl-fi-d-
galactoside
Argininehydrolysis
Lysinehydrolysis
Ornithinehydrolysis
Citratehydrolysis
H2Sproduction
Ureahydrolysis
Tryptophanehydrolysis
Indol
Voges-Proskauer
Gelatinhydrolysis
Mannitolfermentation
Inositolfermentation
Sorbitolfermentation
Rhamnosefermentation
Sucrosefermentation
Melibiosefermentation
Amygdalinfermentation
Arabinosefementation
Nitratereduction
Nitrogengas
Saltrequirement
Growthat35°C
Penaeus
Isolate1
rod
-
+
+
+
+
+
-
-
+
-
+
-
-
-
+
-
+
+
-
-
-
+
-
+
-
-
-
+
+
setiferus.
Isolate2
Rod
-
-
+
+
+
+
+
-
+
-
-
-
-
-
+
-
-
+
-
+
-
+
-
+
-
-
-
-
~
Isolate3
rod
-
+
+
+
+
+
-
-
+
-
+
-
-
+
+
-
+
+
-
-
-
+
-
+
-
-
-
+
+
systemsof
Isolate4
rod
-
+
+
+
-
-
-
-
-
+
+
+
+
-
-
-
+
-
-
-
-
-
-
-
-
+
-
-
+
UsingAnalytabProducts,Inc.system.
b
UsingBBLGramStainKit,andFisherGramSlidesforcontrol.Staphylococcusaureus:
Gram-positivecontrol;Escherichiacoli:Gram-negativecontrol.
41

46. MaleReproductiveBlackeningDisease
Bacteriachallenging
Attempts to produce blackening of reproductive systems were initially
accomplished by challenging with Vibrioalginolyticus(isolate 1). Four different
infectivitymechanismswereevaluated.
Injectionbeneaththirdabdominalsegmentandsurfaceexposure(byswab)didnot
blacken reproductive tissue. Gonopore injection of 5-fold diluted culture caused83%
mortalityinlessthan24hwithnoblackeningofreproductive tissue.Controls didnot
inducethecondition.
Challengingbyinjecting 50-fold-diluted24-hcultureintoterminalampoulesdid
initiateswellingandblackeningofreproductivesystems.Inductionofmalereproductive
blackeningwasachievednotonlywith Vibrioalginolyticus,butalsowithPseudomonas
putrefaciens(isolate4),andisolate2(Table3).Inducedblackeningbeginswithswelling
ofampoules,thenspermatophoresbecomeblack,andinlessthan6daysfrominjection
blackeninghasextendedtomedialvasdeferens.Injection of0.05mlsterileTSB-saline
intoterminalampoules(controls)didnotinitiatethecondition.
Table3.InfectivityresponsetoinitiateblackeningofreproductivesystemsofPenaeus
setiferus.
Condition No.challenged
Males
10
5
5
5
5
5
No. shrimpdevelopinj
blackening0
10
0
4
0
3
0
V.alginolyticus
Controlb
Isolate2a
Controlb
P.putrefaciens*
Controlb
a
0.05ml50-folddiluted24-hTSBcultureinjectedthroughleftgonopore.
0.05mlTSB-salinecontrolinjectedthroughrightgonopore.
c
Swellingandblackeningdevelopedduringthefollowing6daysafterinjection.
Discussion
Under our experimental conditions, P. setiferus rapidly became infertile as
described in other studies (Bray et al., 1985; Leung-Trujillo and Lawrence, 1987;
Chamberlain,1988;Talbotetal.,1989).
42

47. Chapter3
IsolationofbacteriafromtypeCtissue(blackspermatophores,ampoules,andvas
deferens)suggestsassociationofbacterialinfectionswithblackenedreproductivesystems.
Types A and B tissue were aseptic, indicating that normal reproductive tissue and
reproductive systems in deterioration, as described by Talbot et al. (1989), do not
represent bacterial infection. The visual differences in reproductive system quality
betweenthethreetypesoftissuescouldindicateaprogressivesyndrome,withbacterial
infectionbecominginvolvedinadvancedstages,orthatmorethanoneetiologymaybe
involvedindeteriorationandblackeningofP.setiferusreproductivesystem.
Theinfectivityexperimentsindicatethatblackreproductive systemsignscanbe
inducedbychallengingwithVibrioalginolyticus,Pseudomonasputrefaciens,andisolate
2.Eachspeciesinducedtheconditionatdifferent levelsofpositivecases(Table 3);V.
alginolyticuswasthebestisolatedinducer(100%ofcases).Vibriospp.areanormalpart
ofthemicroflora ofpondandraceway-rearedshrimp(Vanderzantetal.,1971;Lightner,
1985) and are opportunistic pathogens (Lightner, 1977). Both organisms {Vibrioand
Pseudomonas)have been frequently isolated from other cultured prawn and penaeid
shrimpdiseases(Lewis,1973;Delves-BroughtonandPoupard,1976;LightnerandLewis,
1975;Lightner, 1983). Shell disease of crabs,lobsters, and penaeid shrimp,hasbeen
associated with those chitinoclastic genera (Cook and Lofton, 1973; Malloy, 1978;
Ciprianietal.,1980).
Theevidencepresentedhereandourcurrentunderstandingindicatethatbacterial
infectioninteractswithblackeningofspermatophores,ampoules,andvasdeferens.Some
opportunisticbacteria,ofatleastthreedifferent genera,penetratetheampoulethrough
gonopores.Theinvasionisacceleratedbyartificialejaculation(Chamberlainetal.,1983;
Sandiferetal.,1984),buttheconditionalsoappearsinnon-ejaculatedanimalssuggesting
that invasion mayoccur in shrimp with impaired defense mechanisms resulting from
stressfulcultureconditions.
Micro-organisms reach the surface of spermatophore acellular matrix and vas
deferens,whereblackdepositionsappear(HarrisandSandifer, 1986).Theyfindagood
supportingchitinoussubstratefortheirpropagation(Benton, 1935),andshrimp'sdefense
systemisactivated.Melanin,whichisadefenseandtoxicmolecule(Grahametal.,1978;
Bang,1983),isproducedbytheshrimp'sprophenoloxidasesystem.Thismechanismplays
amajor roleinforeign substancerecognitionbythehost (AshidaandSoderhall, 1984;
Soderhall and Smith, 1986), and it has been recently confirmed in P.japonicus, P.
monodon,Macrobrachiumrosenbergii,andPalaemonadspersus(Tsingetal.,1989).
Dougherty and Dougherty (1989) found black pigment droplets, identified as
melanin,notonlyconfinedtothesurfacetissueofspermatophores,butalsointhesperm
massmatrixofP.vannamei.Theseauthorsdidnotfindbacteriausinglightandelectron
43

48. MaleReproductiveBlackeningDisease
microscopy. It is of course possible that similar gross pathology could have different
etiologyinpenaeidshrimp.
Acknowledgements
Wethank the Texas A&MUniversity Mariculture Program staff and graduate
students:BillBray,JoannaLeung-Trujillo,FrankCastille,TzachiSamocha,KarenHall,
HeHaiqi,MarcoAraujo,JoseValverde,LuisMena,AllenDavis,TammyThompsen,and
James Biedenbach. To Matt Mireles whohelped usin some experiments and animal
maintenance;toVickiChandler,project secretary,forhercontinuouscollaboration,and
Mr.JimTucker,Captainof"TuffEnouff.
References
Ashida, M., Soderhall, K., 1984.The prophenoloxidase activating system in crayfish.
Com.Biochem.Physiol.77B(1),21-26.
Bang,F.B.,1983.Crustaceandiseaseresponses.In:Provenzano,A.(Ed.),Thebiologyof
Crustacea,v.6.Pathobiology.AcademicPress,Inc.,NewYork,NY,USA,pp.113
-149.
Benton,A.G.,1935.Chitinovorousbacteria-apreliminarysurvey.J.Bacteriol.29,449-
465.
Bray,W.A.,Leung-Trujillo,J.R.,Lawrence,A.L., 1985.Preliminaryinvestigationon
theeffects oftemperature,bacterial inoculation,andEDTAonspermqualityin
captivePenaeussetiferus.J.World Marie.Soc.16,250-257.
Bullock,G.L., 1971.Diseasesoffishes.Book2B.In:Sniesko,S.,Axelrod,H.(Eds.),
Identification offishpathogenic bacteria. T.F.H. Publications,New York,NY,
USA,43pp.
Chamberlain, G. W., 1988. Stepwise investigation of environmental and nutritional
requirements for reproduction ofpenaeid shrimp.Ph.D. Thesis, Department of
WildlifeandFisheriesSciences,TexasA&M University,USA.
Chamberlain,G.W.,Johnson,J.K.,Lewis,D.H.,1983.Swellingandmelanizationofthe
malereproductivesystemofcaptiveadultpenaeidshrimp.J.WorldMarie.Soc.
14,135-136.
Cipriani,G.R.,Wheeler,R.S.,Sizemore,R.K., 1980.Characterization ofbrownspot
diseaseofGulf Coastshrimp.J.Invertebr.Pathol.36,255-263.
Cook,D.W.,Lofton, S.R.,1973.Chitinoclasticbacteriaassociatedwithshelldiseasein
Penaeusshrimpandthebluecrab.J.Wildl.Dis.9,154-159.
Delves-Broughton,J.,Poupard,C.W.,1976.Diseaseproblemsofprawnsinrecirculation
systemsintheU.K.Aquaculture7,201-217.
44

49. Chapter3
Dougherty, W. J., Dougherty, M.M., 1989.Electron microscopical andhistochemical
observations onmelanized sperm andspermatophoresofpond-cultured shrimp,
Penaeusvannamei.J.Invertebr.Pathol.54,331-343.
Frerichs, G. N., Hendrie, M. S., 1985. Bacteria associated with diseases of fish. In:
Collins,C.H.,Grange,J.M. (Eds.), Isolation and identification of micro-
organismsofmedicalandveterinaryimportance.AcademicPress,Orlando,FL,
USA,387pp.
Graham,D.G.,Tiffany, S.M.,Vogel,F.S.,1978.Thetoxicityofmelaninprecursors.J.
Invest.Dermatol.70,113.
Harris,S.,Sandifer, P., 1986.Spermproduction andtheeffects ofelectrically induced
spermatophoreexpulsionintheprawnMacrobrachiumrosenbergii(DeMan).J.
Crust.Biol.6(4),633-647.
Leung-Trujillo,J.R.,Lawrence,A.L.,1987.Observationsonthedeclineinspermquality
ofPenaeussetiferusunderlaboratoryconditions.Aquaculture65,363-370.
Lewis,D.H.,1973.ResponseofbrownshrimptoinfectionwithVibriosp.Proc.4thAnn.
Workshop,World Marie.Soc,pp.333-338.
Lightner,D.V.,1977.Crustaceandiseases.In:Sinderman,C.J.(Ed.),DiseaseDiagnosis
and Control in North American Marine Aquaculture. Elsevier Scientific
PublishingCo.,NewYork,NY,USA,pp.8-77.
Lightner,D.V.,1983.Diseasesofculturedpenaeidshrimp.In:McVey,J.P.(Ed.),CRC
handbook ofmariculture,vol. 1,Crustacean aquaculture.CRCPress,FL,USA,
pp.289-320.
Lightner,D.V.,1985.Areviewofthediseasesofculturedpenaeidshrimpsandprawns
withemphasisonrecentdiscoveriesanddevelopments.In:Taki,Y.,Primavera,H.
J., Llobrera, J. A. (Eds.), Proc. 1st Int. Conf. on the Culture of Penaeid
Prawns/Shrimp,AquacultureDepartment,SEAFDEC,Iloilo,Philippines,pp.79-
103.
Lightner,D.V.,Lewis,D.H.,1975.Asepticemicbacterialdiseasesyndromeofpenaeid
shrimp.Mar.Fish.Rev.37,25-28.
Malloy,S.,1978.Bacteriainducedshelldiseaseoflobsters (Homarus americanus). J.
Wildl.Dis.14,2-9.
Sandifer,P.A.,Lawrence,A.L.,Harris,S.G.,Chamberlain,G.W.,Stokes,A.D.,Bray,
W.A.,1984.Electricalstimulationofspermatophoreexpulsioninmarineshrimp,
Penaeusspp.Aquaculture41,181-187.
Soderhall, K., Smith, V. J., 1986. The prophenoloxidase activating system: the
biochemistryofitsactivationandroleinarthropodcellularimmunity,withspecial
reference to crustaceans. In: Brehelin, M. (Ed.), Immunity in invertebrates.
Springer-Verlag,NewYork,NY,USA,pp.208-223.
45

50. MaleReproductiveBlackeningDisease
Talbot,P.,Howard,D.,Leung-Trujillo,J.,Lee,T.W.,Li,W-Y.,Ro,H.,Lawrence,A.L.,
1989. Characterization of male reproductive tract degenerative syndrome in
captivepenaeidshrimp(Penaeussetiferus).Aquaculture78,365-377.
Tsing,A.,Arcier,J.,Brehelin,M.,1989.Hemocytesofpenaeidandpalaemonidshrimps:
morphology,cytochemistry,andhemograms.J.Invertebr.Pathol.53,64-77.
Vanderzant, C, Nickelson, R., Judkins, P. W., 1971.Microbial flora of pond-reared
brownshrimp(Penaeusaztecus).Appl.Microbiol.21(5),916-921.
46

51. Chapter4
ReproductiveQualityEvaluationof MalePenaeusstytirostrisfromaGrow-Out
Pond
J.Alfaro
EscueladeCienciasBiologicas,UniversidadNacional,Heredia,CostaRica
Publishedin:J.WorldAquacult.Soc.(1993),24(1):6-11

52. SpermatophoresofPenaeusstylirostris
Abstract
SeveralmalePenaeusstylirostriswereselected from a3-hacommercialearthenpond
and were individually evaluated for reproductive performance. Indicators measured
werecompound spermatophoreweight,spermcount,andspermabnormalities.Itwas
found thatspermatophore qualitywassignificantly betterfor 30-40gshrimpthan for
20-30gshrimp(PO.05).Thehigherfrequency ofabnormalitiesmeasuredinyounger
malesandtheinverserelationshipbetweenabnormalitiesandspermcountindicatethat
the vas deferens could be the tissue responsible for producing highly abnormal
immature semen. Wepropose that male maturation has at least three independently
controlled levels of organization: testes maturation, vas deferens maturation, and
spermatophoresynthesis.Theindividualevaluationshowedthateachmalefolloweda
particularresponseinreproductivequality.Changesinspermatophoreweightwerenot
an indicator of sperm density within spermatophores. Male reproductive tract
degenerativesyndrome(MRTDS)andmalereproductivesystemmelanization(MRSM)
didnotdevelopinanyshrimpduringourexperiments.
Introduction
Theprogressofshrimpmariculturedependsonabetterunderstandingofspecific
biologicalaspects,particularlyinreproductivebiology,toensurethatadequatenumbersof
laboratory-reared postlarvae can be produced at high levels of efficiency. Although
significant improvementsincontrolledreproductionhavebeenmadeinthepast5to10
years, much of the interest has focused on female maturation (Primavera, 1985;
Chamberlain,1988).Knowledgeaboutmalematurationandsemenqualityisfragmentary
(Leung-TrujilloandLawrence,1987a).
Theevaluationofreproductivequalityinmalepenaeidshrimpwasfirstreported
by Leung-Trujillo and Lawrence (1985),who measured spermatophore weight, sperm
count,andpercentagesofliveandabnormalspermforevaluatingtheeffect ofeyestalk
ablation in P. vannamei.Using the same parameters, Leung-Trujillo and Lawrence
(1987a)studiedadeclineinspermqualityinP.setiferusheldincaptivityandBrayetal.
(1985)usedthesameapproachtoevaluatetheeffect ofwatertemperature,EDTA,and
VibriobacterinonspermqualityincaptiveP.setiferus.Chamberlain(1988)assessedthe
effect ofdietaryrancidityandvitaminEongonadalmaturationofP.setiferus.Leung-
Trujillo andLawrence(1987b)evaluatedspermatophoreredevelopmenttimesinwildP.
stylirostris,P. vannamei,and P. setiferus;and Leung-Trujillo and Lawrence (1988)
studied the effect of ascorbic acid on reproductive quality in P. vannameiusing the
previousmentionedqualityindicators.Recently,Alfaro(1990)measuredspermatophore
weight,spermcount,andpercentageofabnormalitiesforevaluatingnutritionalvariations
48

53. Chapter4
on the onset of the male reproductive tract degenerative syndrome (MRTDS) of P.
setiferus(Talbotetal,1989).
Thus,theknowledgethathasaccumulatedonthissubjectisbasedontheanalysis
ofvarioustreatmentprotocols,withlittleinformationavailableconcerningthewayamale
producesspermatophores.Thepurpose ofthisstudyistoevaluatereproductive quality
duringsuccessivespermatophoreregenerationsforindividualmaleP.stylirostrisselected
fromcommercialgrow-outponds.
Materialsandmethods
Animals
Male P. stylirostris with spermatophores (n = 12) were selected from a
commercial 3-ha earthenpond duringthe densityadjusting phase at2.5 monthspost-
stocking.Shrimpwererearedatastockingdensityof5shrimpm"2
withaminimumwater
exchange of 10% daily using inorganic fertilization and supplemental feeding in a
polyculture strategy, but primarilyusingP. vannamei.Selected males were packed in
plasticbagsofoxygenatedandchilledseawater(25 °C).Bagsweretransported inice
cheststotheAquacultureLaboratoryatUniversidadNacional,Heredia,wheretheshrimp
wereacclimatedtotheculturewater.
ExperimentalConditions
Maleswerestocked(12shrimpm2
) ina 1.0 m2
recirculating cultureunitwith
externalbiologicalfilter andautomaticcontroloftemperature(EarthShyokaiCo.,Ltd,
Tokyo,Japan).Water exchange was limited toreplacement evaporative loss.A fresh-
frozen diet initially consisting of shrimp heads (P. occidentalis), marine bloodworms
(Nereissp.), fish (Opistonemasp.), and clams was provided at a ratio of 1:1:1:1,
respectively, at 10% of wet body weight per day. After the third spermatophore
regeneration,thedietwascompletelyreplacedbyfresh-frozen squid.Thetotaldailyration
was distributed over 4 feedings at 09:00, 12:00, 14:00, and 17:00 h. Salinity was
maintainedat32ppt,temperatureat29°C,pHat7.7,andtotalNH4-Nlevelatlessthan
0.01mgl"1
.
ExperimentalDesign
Selected males were initially analyzed immediately after stocking in the
recirculating unit. Spermatophores were removed by manual ejaculation following a
modified procedureofthatdescribedbyKing(1948)inwhichthespermatophorewas
partiallyejectedwithgentlepressureandthencompletelyexpelledemployingdisinfected
finetweezers.
49

54. SpermatophoresofPenaeusstylirostris
Total body weight was measured to the nearest 0.1 g and compound
spermatophore weight to the nearest 0.01 g. In addition, sperm count per compound
spermatophore andpercentage ofabnormal sperm were determined. Sperm countwas
performedbyhomogenizingthecompoundspermatophoreinaglasstissuegrinderwith
3.0mlofartificialseawater(Cavanaugh,1956).Thesuspensionwasmixedseveraltimes
to insure homogeneity, and 3 samples were counted using the total 25 fields of a
hemacytometer(0.0001ml).
The percentage of abnormal sperm was obtained by recording the number of
normalsperm,i.e.withasphericalbodyandanelongatespike(Talbotetal., 1989),and
numberofabnormalsperm,i.e.withmalformedheadsandbentormissingspikesforat
least100spermcells.Percentabnormalspermwerecalculatedasfollows:
%abnormalsperm
=100[abnormalcells/(abnormalcells+normalcells)]
At stocking, five males were individually marked bycutting the uropods in a
similarwaytothatdescribedbyMakinouchi andPrimavera(1987).Femaleswerenot
stockedinordertoallowtheevaluation ofmalereproductive performance withoutthe
enhancingeffectsintroducedbyfemalepresence.Markedmalesweremanuallyejaculated
every13days,until5regenerationswereobtained.
Initialdata onshrimpsizewerestatisticallyanalyzedfor significant differences
amongmeansusingthet-testforindependentsampleswithunequalvariance(Ott,1984).
SpermcountdataweretransformedusingsquaredrootofY+3/8(Brayetal.,1985)and
anarcsinetransformationwasusedintheanalysisofabnormalspermpercentages(Leung-
Trujillo andLawrence, 1985)tomakethevariance independent ofthemean. Dataon
spermcountsandabnormalitieswereanalyzedforlinearregressionandcorrelation(Ott,
1984).
Results
Penaeus stylirostris grown in an earthen pond were found to produce
spermatophoresinindividualslargerthan23.6g(totallength=100mm).Spermatophores
weresignificantlysmallerinweight(PO.05)for20-30g(100-111mm)shrimpthanfor
30-40 g (112-116 mm) shrimp. In addition, spermatophore quality was significantly
improved(P<0.05)inthe30-40gshrimpcomparedtothe20-30gshrimp(Table 1).A
linearregressionbetweenspermabnormalitiesandspermcountwascalculated,including
theinitialdataandthelaboratoryregeneration data.Itindicatesaninverserelationship,
whichisrepresentedbythefollowingequation:
Y=63.4-3.4X,
whereYisthepercentabnormalspermandXisthespermcountinmillions.Acorrelation
coefficient of-0.52wascalculated.Confidenceintervalswere63.4±12.3(intercept)and
50

55. Chapter4
-3.4±2.5(slope;P<0.05).Thelinearrelationshipbetweenthesetwoparameterswas
statisticallysignificant(P<0.05)inatestforslopes.
Table1.Comparisonofcompoundspermatophoreweight,spermcount,and
abnormalitiesbetweensizeclassesforPenaeusstylirostrisselectedfromacommercial
grow-outpond.Dataarerepresentedasmean±standarderror(N=4).Meanswith
differentlettersarestatisticallydifferent(P<0.05).
Parameter Weightclass
20-30g 30-40g
Spermatophoreweight(g) 0.03a
± 0.01 0.06b
± 0.03
Spermcount(million) 1.62a
± 1.02 4.83b
± 2.10
Abnormalsperm(%) 75a
± 2 50b
+12
The individual evaluation of spermatophore quality is presented for 5
regenerations.Table2showsdataonspermcount;Table3summarizesthespermatophore
weight data, andthepercentages ofabnormal sperm are shown inTable4. Individual
shrimpevaluationindicatesthateachmalegeneratedspermatophoreswithin13daysafter
each manual ejaculation. Theonlyexception tothis statement wasmale Datits fifth
regenerationtime(Table2).
Table2.Spermcounts(million)forfivespermatophoreregenerationsofindividually
markedPenaeusstylirostris.
Male:
Bodyv
A:37
B:35
C:32
D:35
E:29
reight (g) 1
3.18
6.84a
2.85
6.42
1.29
2
2.76
2.55
3.09a
5.85
1.89
Regeneration
3
4.89a
0.27
1.98
8.82
3.63a
number
4
4.53
0.06
2.07
14.22a
1.89
5
4.26
0.81
2.85
0.00
_b
Highestvaluemeasuredforeachmale.
b
Deadbeforeterminationofregenerationtime.
Fromthesedata(Tables2,3,and4)weseethateachmalefollows aparticular
responseinreproductivequality.Byanalyzingthechangesinspermcount,spermatophore
weight,andabnormalities,nocommonpatternwasfound;whereasmaleCshowsaclear
51

56. SpermatophoresofPenaeusstylirostris
incrementinspermatophoreweightinsuccessiveregenerations,maleApresentsadecline
ofthesameparameter.Theothermalesexhibitirregularfluctuations.
Oneinteresting finding wasthat changes in spermatophore weight for agiven
malearenotanindicator ofspermdensitywithin spermatophores.OnlymaleEhada
higherspermcountasthespermatophoreweightincreased.
Domesticated males, as for our pond-grown laboratory postlarvae, show an
encouragingreproductivestatus.MalesA,C,D,andEproducedeverynewcompound
spermatophorewithaspermcountsimilartoorbetterthanpreviousregenerations.MaleD
improvedfrom6.42millionsatfirstregenerationto14.22millionsatforthregeneration.
Table3.Spermatophoreweights(g)forfiveregenerationsofindividuallymarked
Penaeusstylirostris.
Male:
Bodyv
A:37
B:35
C:32
D:35
E:29
weight (g) 1
0.06"
0.08a
0.03
0.09
0.03
2
0.04
0.05
0.04
0.08
0.04
Regenerationi
3
0.04
0.07
0.04
0.10a
0.06a
lumber
4
0.03
0.08a
0.05
0.08
0.04
5
0.03
0.04
0.07
0.00
_b
a
Highestvaluemeasuredforeachmale.
b
Deadbeforeterminationofregenerationtime.
Table4.Abnormalsperm(%)forfivespermatophoreregenerationsofindividually
markedPenaeusstylirostris.spn=Spermatophoreswerenotregenerated.
Male:
Bodyweight(g)
A:37
B:35
C:32
D:35
E:29
1
56.6
62.2
45.1
33.6
64.3
2
88.2
73.7
56.1
39.9
33.5
Regeneration
3
60.0
90.0
61.8
36.1
35.2
number
4
31.3
81.8
62.4
19.2a
31.3a
5
25.7a
43.1*
31.2a
spn
_b
"Lowestvaluemeasuredforeachmale.
b
Deadbeforeterminationofregenerationtime.
52

57. Chapter4
Successiveregenerations alsohadapositive effect oversperm abnormalitiesin
everyexperimentalmale.Regardlessoffluctuations inspermatophoreweightandsperm
count, each male improved its proportion of normal sperm by the forth or fifth
regeneration.
Discussion
The observed increase in sperm count and the reduction in abnormalities in
relationtosizeappeartobefeatures ofthematurationprocessinmalepenaeidshrimp.
Young males of P. monodon(Motoh, 1981) and P. vannamei(Leung-Trujillo and
Lawrence,1985)havealsobeenobservedwithhighlevelsofabnormalities.
The maturation process in P. stylirostris,as documented by the regression
equation, involves aprogressive intensification of spermiogenesis: mitotic andmeiotic
division (King, 1948). Testes produce higher numbers of spermatids as the organism
grows;nevertheless,thehighfrequencyofabnormalitiesfoundinyoungeranimalsseems
to indicate that the vas deferens, which is the place for final sperm maturation by
completionofthesubacrosome:spikeformation (ShigekawaandClark, 1986),doesnot
matureatthesametimethatthetestisbegintoproducesperm.Inthisstudy,theminimum
shrimpsizeshowingimmaturespermatophoreswas23.6g.
With the purpose of understanding male maturation, some other experimental
observationsarepertinent.WildP.stylirostris(meanweight=49.4g)usedasbroodstock
for5weekspresentedlowlevelsofabnormalities(15.0%),yetspermcountswerevery
low(7.5millions;unpublished data).However,wildP.setiferus(meanweight=40g)
brought intocaptivitypresented deteriorated spermatophoreswithnospermat35days
post-capture.Thevasdeferensatday47showedhighspermcounts(263millions/bothvas
deferens)andincreasedabnormalities(61.0%)comparedtothespermatophorecondition
atcollection(spermcount=45millions;abnormalities=23%;Alfaro1990).
Byanalyzingthepreviousinformation, weproposethatmalematurationhasat
leastthree independent levels.Thefirst istestesmaturation, whichproduces immature
sperm. The second isvas deferens maturation, which completes sperm maturation by
spikeformation. Thethirdlevelisspermatophoresynthesiswithinampoules,wherethe
final product is packed. It is suggested that factors like stage of development,
environment, andnutritionmayaffect thefinal qualityofspermatophores byunknown
mechanisms,whichactatoneormoreoftheselevels.
This hypothesis would explain the higher abnormalities observed in younger
males; it also would explain how mature males produce spermatophores with low
abnormalitiesandlowspermcount,andhowspermatophoresynthesiscanbecompletely
stoppedinmalespresentinghighnumberofspermwithinvasdeferens.Theimprovement
innumberofnormalspermindependentlyofspermatophoreweightandspermcountin
53

58. SpermatophoresofPenaeusstylirostris
successiveregenerationsappearstosupportthishypothesis.Ejaculationandregeneration,
underourexperimentalconditions,hadapositiveeffectovervasdeferensperformance.
Thefindingabout spermatophore weight and its quality is very important for
commercialmaturationunitsbecauseitmeansthatevaluatingspermatophoresforsizeand
appearance doesnot yield information about spermcount. Thiswas corroborated ina
commercial maturation unit, where we found that wild P. stylirostrisbrood stock
spermatophoreswithanexcellentexternalappearanceandweight(meanvalue=0.16g)
hadspermcountsofonly7.5million.
Thecultureconditionsunderwhichweevaluatedouryounggroup(meanweight=
33.5g)werenottheoptimumforpenaeidshrimpmaturation;nevertheless,thewaythey
responded sexually,evenwithouttheenhancinginfluence offemales,appearstobean
indicatoroftheexcellentreproductivequalitiesofdomesticatedandselectedmales.
Malereproductivetractdegenerativesyndrome(MRTDS)andmalereproductive
systemmelanization(MRSM),astheyoccurincaptiveP.setiferus(Alfaro,1990),didnot
developinourexperimental group.Thiscouldbetheresultofspecies-specific defense
responses,thedegreeofphysiologicaladaptationtocaptivity,ortoejaculatoryprocedures.
Melanization has been reported as a problem for P. stylirostris, where
Chamberlainetal.(1983)foundahigherincidenceofmelanizationinmanuallyejaculated
shrimp.HarrisandSandifer (1986)alsoreportedmelanizationofreproductivetissuesin
electricallyejaculatedMacrobrachiumrosenbergii.Thelackofmelanizationinthisstudy
indicatesthattheprotocol followed for artificial ejaculation mayhaveaneffect onthe
incidenceofmelanization. Byejaculating with gentlepressuretoavoid damagetothe
gonopore and by using disinfected tweezers for final extraction, reproductive tract
melanizationinpenaeidshrimpbroodstockcanbeeliminated.
Acknowledgments
Theauthorisgrateful forthesupportoftheAquacultureProgram:UNA-JICA,
EscueladeCienciasBiologicas,andVicerrectoriadeInvestigacion.IwishtothankMr.F.
Vives,productionmanagerofCriaderodeCamaronesdeChomesS.A.,forhispermanent
collaborationwiththisresearch.
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DevelopmentandDeteriorationofSpermatophoresinPond-Reared
Penaeusvannamei
J.AlfaroandX.Lozano
EscueladeCienciasBiologicas,UniversidadNacional,
Heredia,CostaRica
Publishedin:J.WorldAquacult.Soc.(1993),24(4):522- 529