W it der

REPRODUCTIVE MECHANISMS IN MACROBR4CHIUMROSFXBERGII AND PFNAFUS JAPONICUS: ENDOCRINOLOGICAL

RESEARCH AND POTENTIAL APPLICATIONS IN AQUACULTURK

Marcy N, WilderJapan lntemational Research Center for Agricultural Sciences

Ministry of Agriculture, Forestry and Fisheriesl -2 Oh washi, Tsukuba, Ibaraki 305-8686, Japan

tel. 01141-298-38-6630; fax 011-81-298-38-6316

c-mail: marwi!@jircas.affrc.go.jp

ABSTRACT

The giant freshwater prawn Macrobrrrcriiurn roseobergii, cultured extensively throughout SouR Asia, and thekuruma prawn Penorusjaporurus, targeted ponmpally in Japan and Taiwan, are species of commercial itnpor-tance which have been widely studieil in terms of basic physiological function. ln Japan, P. japortiius isaddiuonally asignificant target of artificial seed producuon operations for restocking of coastat areas. ln order toensure a sustainable means o artificial seed production for significant crustacean species, i is important toeffectively control female molting and reproduction under artificial conditions. ln decapod Crustacea, thcphysiological processes of molting and reproduction are linked and are under honuonal conuol, The role ofecdysteroids which serve as molting hormones are well-established, but the physiological significance of juvenilehormone-related substances is Just beginning to become clear, Endocrtnoiogical research in I rosenbergii and Pjuponfciis Can be putenttany applied tO aquaCulture OperatiOnS m the future, inCluding anifiCial Seed prOduCtiOnprograms for P japnnicus.

INTRODUCTION

RKPRODUCTIVK ENDOCRINOLOGY IN

M. ROSENBERGII AND P. JAPON1CIjS

BackgrounrIIn decapod Crustacea including prawns,

shrimps, lobsters and crabs, the physiological

The establishment of sustainable prawnculture depends on many factors and requires theintegration of variOus fields of expertiSC. Alpresent, inducing reproduction in captivity andcontrolling disease remain obstacles to successfulculture, and solving these technological problemswill depend greatly on basic research relating tothe biology and physiology of the animals beingtargeted. The giant freshwater prawnMacrobrachirarrt rosertbergii, cultured extensivelythroughout South Asia Chavez Justo 1991!, andthe kuruma prawn Penaeris japonicus, targetedprincipally in Japan and Taiwan Liao and Chen1994!, are species of commercial importance whichhave been widely studied in terms of basicphysiological function. While widespread viralinfection has become a problem of increasingmagnitude in the culture of saltwater Perraertsspecies, disease outbreak has not been of sigruficantconcern in freshwater species such as M.

msenhergii. However, in all prawn species, il isimportant to be able to cffecti vcly control moltingand reproduction under artificial conditions in orderto produce larval seed for further aquaculturalgrowout. In Japan, Pj aponicusis additiorially asignificant target of artificial seed productionoperations for restocking of coastal areas,

This paper addresses the current state ofendocrinological research in M. roserrbergif andP j aponi ctas, focusing on the roles of ecdysteroidsand juvenoids in molting and reproduction, anddiscusses how basic research in this area can bc

potentially applied to aquaculture operations in thefuture. The status of artificial seed productionprograms for P japorsicus and current aquaculturein Japan in this context are also discussed.

tan UJNR Technical Report Ala. Zn

rl at factOrsExter

!t-organSinus =tan=

Cen.rai .-e.;ous systen.

IV I H

Y-organ

e:D

Ibl'Iar o a t

Vo0I-,Ovary

Vg

vg syn:rtetl csite

Figtsre l, General scheme for the endocrinological control ofmolting and reproduction in Crustacea, Abbreviations areindicated in the figure for molt-inhibiting hormone MIH!,viteliogenesis-inhibiting hormone VIH!, ecdysteroid ECD!, vitellogenes is-stimulating hormone Vt H!, methylfarnesoatetlVlF!, nnd vitettogenesis-stimu!anng ovarianhor-mone VSOH!.

processes of molting and reproduction areinextricably 1 inked and under hormonal control.Ecdysteroids such as 20-hydroxyecdysone serveas "molting hormones" in Crustacea and areexcreted from a tissue known as the Y-organ, Onthe other hand, peptide substances such as rnolt-inhibiting horrnonc MIH! and vitellogenesis-inhibiting hormone V[H! originating in the sinusgland complex of the eyestalks exert negativeinfluence on molting and ovarian development.There is evidence for thc existence of positivestimulatory factors, including a putativevitcllogcnesis-stimulating horrnonc VSH!,vitellogenis-stitnulating ovarian hormone VSOH!,and molt-stimulating hormone MSH! which maypossibly be secreted at the brain and thoracicganglion Takayanagi et al. 1986, Meusy and Payen1988!; however, such factors have not beensufficiently isolated and identified. A generalscheme for the control of molting and reproductionin Crustacea is shown in Figure 1. Structures ofrepresentative ecdysteroids in M. rosenbergii areshown in Figure 2.

In insects, juvenile hormone JH!, a larvaldevelopmental hormone, also appears in the adultfernale to stimulate yolk protein production anduptake. To date.. JH itself has not been identifted

in any crustacean species. Methyl farnesoate MF!, the un-epox idated precursor of JH, has beenfound in a limited number of' crustacean speciessuch as the American lobster Hornarusamericanus Tsukirnura and Rorst 1992! and thespider crab Libinia ernerginara Laufer el al.1987!. In prevt'ous studies of this author and co-workers, MF was detected in M. rasenbergii Wilder ct al. 1995! but was not found in P.

japonicus. MF has been shown to bc secretedfrom the mandibular organs Sagi et al, ]99ti.While MF is considered to be the crustacean

equivalent of JH, its role in crustacean reproductionremains unclear. MF may possibly function as aVSH to stimulate yolk protein production and uptakeas suggested in Figurc 1. Thc structure of MF isshown in Figure 3

Eedysteroids and juvenoids in M. roserebergeiand P jrrponicus

In both M. rosenbergii and P, japonicus,an ecdysteroid surge occurs in thc hemolymph inthe late pre-molt stage and the predominantccdysteroid species is observed to be 20-hydroxyecdysone with lesser amounts of highlypolar ecdysteroids high polarity products HPP!!.In M. rosenbergii, peak titers are about 40 ng/ml Okumura et al, I 992!, and in P j aponicus, theselevels reach nearly 200 ng/ml Okumura et al.1989!, 20-hydroxyecdysone is generallyconsidered to be the acti ve form of the hormone inmost crustacean species, and it regulates the moltingcycle.

In addition to involvetncnt in molting,ecdysteroids are found in newly laid eggs andmature ovaries of numerous insect and crustaceanspecies. In general, eggs ecdysteroids during theearly embryonic stages are ovarian in origin andserve as a stock for purposes of early developmentuntil embryonic prothoracic glands or Y-organsdifferentiate and produce ecdysteroids de novo Spindler et al. 1987!, In M. rosenbergii,ecdysteroids are present in mature ovaries �5 ngfg! and newly spawned eggs �6.9 ng/g! Wilder etal. 1990!. These ecdysteroids are accutnulated inovaries during the reproductive molt cycle mottcycle accompanied by maturation of the ovaries!to levels of 50 ng per ovary. In contrast, during thecommon molt cycle mott cycle in which ovaries

wilder lZ7

EcdysteroId structures

> ~ rluvIII <II IO

Reproductive

{2E6E!

Methyl farssesoate

so

Figure 3. Structure of crustacean juvenotd substance, me-thyi famesoate MFl.

4ocnID

remain immature!, ecdysteroid content is 1.5 ngper ov ary Wilder et al, 1991! Fig. 4!. This ovarianecdysteroid accumulation which occurs insynchronization with molting may signify a role forthese ecdysteroids in inducing germinal vesiclebreakdown GVBD! and subsequent ovulation.

This author has examined hf. rosertbergiiand P. japorricus for the presence in thehemolyrnph of juvenoid substances includingjuvenile hormone Ill and methyl farnesoate MF!.MF was present in females during both therepruductive and conunon molt cycles and in lnalesin M. roserrbergi r Wilder et al l995!, but was notdetectable in P jrrporticus Wilder and Aida 1995!.In M rosenbergii, MF fluctuated during the moltcycle without corlnection to ovarian development,being highest in the early pre-molt stages Wilderet al, 1995!. These results suggest that MF may

ra 3D

O crCL

aDC

CI0isIas soOI�

D CII CI DII D tsI Dr a 8Molt stage

Figure 4. Ovarian ecdysteroId accumulauoo m ovaries dw-ing tbe repraduCtive molt cycle and eedytterOid coIIlentduring the common molt cycle in ftf. roserrbrrgri on ltasi sof a 25-g individual. Ecdysteroids are abbreviated as ECD.

Figtt re 2. S UDC tureS Oi representative ecd ysteroid s in hf. roseahergii Incl tIdi stg the acb ve form Of the hOrm one. M-hydrus yecdy sone,preCurSOr eCdySOne, and rnetabOliteS 20,2b-dihydrOXyeedysnne and 20-dihydrOxveedysonotc aCid.

UJNR recbnieal Report .'so. zs

be involved in the molting process, In Arremia,MF has been found to elevate Na/K-ATPase

activity in larval homogenates, additionallysuggesting a role in osmoregulation Ahl and Brown1991!.

In a separate investigation, this author andco-workers tested the effects of MF injection onvitellogcnin production in eyestalk-ablated juvenileM. rosenhergii Wilder et al. 1994!. Althoughvitcllogenc»is in hf. rosenbergii will not bediscussed in detail here, there were no observedincrease» in vitellogenin production in response toMF treatment, These results indicate that MF

alone could not promote increased vitcllogeninproduction, but do not rulc out a role for MF incrustacean reproduction. Whether MF plays a rolein inducing patency of the ovarian follicles, therebyallowing the uptake of vitellogenin in developingoocytes, as JH does in insects Davey and Huebner1974, Davey et al. 1993!, needs to be furtheraddressed.

Current perspectives in endocrinologicairesearch

At present, it is we! 1-established that 20-hydroxyecdysone and other ecdy steroids functionas molting hormones in Crustacea, but it is stillunclear what role ecdysteroids play in conjunctionwith juvenoid substances in stimulating reproductiveprocesses, Much progress has been achievedrecently in the isolation and identification of eyestalkhormones, particularly of MIH, A Japanese grouphas succeeded in isolating a putative MIH in P.japoiricas Yang et al. 1996! and has demonstratedit to have molt-inhibiting activity by assessingecdysteroid synthetic activity under Y-organculture, The nature of VIH in both M. roseirbergiiand P. japonicus and MIH in hf. rosenbergiiremains unclarified, Morc information concerningthe structures of these hormones and how titers

fluctuate during the molt cycle should greatlyimprove knowledge of endocrinologicalmechanisms controlling molting and reproduction.It is also of importance to further elucidate thephysiological roles of MF, and to detemme whetherputative brain and ovarian factors are involved inhormonal processes. Finally, an understanding ofhow such mechanisms operate in context ofenvironmental factors is expected to contribute

significantly to the development of techniques forcontrolling maturation and reproduction in captivity.

The remainder of this paper will introducethe current status of Pj aponicus culture in Japan,and artificial seed production programs for thisspecies and related applied research being carriedout by the Japan Sea-Farming Association JASFA!, The author is engaged in a combinedfarming systems project in the Mekong Delta regionof Vietnam, focusing on M. rosenbergii seedproduction and aquaculture, but this will not bcdiscu ssed in detail.

PFNAEUS JAPONICUS CULTURE INJAPAN

The following information is based on a1995 Fiscal Year Report of thc Norinc hukin Bank,Fisheries Division sce Fujiwara 1995!. In 1995,total marine culture production in Japan wasapproximately 1.284 million tons with a marketvalue of Y575,6 billion, Major species of interestin Japan include ycllowtail, sca bream, flounder,and the kuruma prawn P. japorricus, Of this totalproduction, P. japonicrrs culture accounted forapproximately 2000 tons valued at Yl 3,514 billion.During 1991-1994, production of P, japorricusdecreased from nearly 2500 to 1500 tons due tosevere viral outbreaks in western Japan, butevidence of recovery is beginning to be seen ascauses of viral outbreak have come to be

elucidated, The number of operators in total hasremained fairly constant during this period, around150-160 enterprises nationwide. Table 1summarizes changes in enterprise number,production volume, and market volume from 1991to 1995,

P, japonicas culture began as early as1962 with the development of artifrciaJ propagationtechniques, and production levels peaked in 198gat about 3020 tons. Typically, culture operationsare begun in the spring, between March and May,and prawns are reared to market size of about 30-50 g by the end of the year or following spring.Culture is thus carried out on the basis of yearlycycles, Common feeds include minced sardine,squid or clam, and artificial pellets Culture isfocused predominantly in western Japan, Kyushu,and Okinawa. In 1994, of a total of 151 operators,52 were based in Kumarnoto Prefecture, 25 in

year

No, enterprisesProduction volume tons!hiarket volume billions of en

1511, 519

76

1562, 1877. 11-t

1612, 491

17, 176

1631, 71215.

a2, 000. 514

~ 'estimated

Table 1. Changes in enterprtse number, product ton volume and marketvolutne lnr Lurunia prawn culture in Japan during1991-1995

P ecture No. ent r ' To La I ea tn' Avera e a ea m'

Kutaamoto

KagoshitttaOkittasra

Ehime

Yama uchi

1, 549, 000997, 000715. 000642, 0005 000

52

25

20

15

l.3

30, 00040, 000

36, 00049, 000

9 00

Table 2. Number of enterprises engaging in kuruma prawn cuhure, and total and average areas under operation.

stocking or on an experimental basis. Theseprograms will be introduced in the next section.

Kagoshima, 20 in Okinawa, 15 in Ehitnc, and l 3 inYamaguchi. Table 2 sununarizes these figuresalong with total and average area per enterpriseunder culture. Three forms of culture arc typicallypracticed: artificial pond culture, net culture, andtank culture. Artificial pond culture and tank cultureare most predominant with only a minority ofoperators engaged in net culture.

In 1992, widespread viral outbreakoccurred as a result of the introduction of infectedseed imported frotn China. Causes of viral outbreakhave since been elucidated, and control measures,such as the disinfecting of culture ponds, havehelped bring the situation under control. Operatorshave had to rely increasingly more on domesticsources of seed, In Okinawa, parent prawnsobtained from Miyazaki, Oita, and NagasakiPrefectures are used to secure seed. While thereis some technical assistance and cooperationcarried out between governtnental agencies andprivate operators, the fortner is not permitted toproduce and sell seed to the private sector;therefore, culturists need to rely on other privatc-sources for obtaining seed. Goverrtment-sponsoredprojects relating to artificial seed production areimplemented explicitly for purposes of coastal re-

A RTIFI CI A I. S FFD P RO DUCTIONPROGRAMS FOR P. JAPO/VICUS INJAPANCurrent status

Artificial seed production prograins for P.japrrnicus are implemented by the Japan Sea-Farming Association JASFA!, an auxiliaryorganization of the Japan Fisheries Agency.Programs relating to kurutna prawn production aremamly earned out at JASFA's Momoshima Stationin Hiroshima Prefecture, and Shibushi Station inKagoshitna Prefecture. Actual operations areprincipally carried out at Shibushi while workrelating to the cultivation of female spawners isbeing done at Motnoji ma.

Figure 5 shows statistics for artificial seedproduction and release during the years 1977-1995.Production levels have generally ranged between400 and 600 inil lion seeds/yr, with figures for actualrelease fluctuating around 300 million seeds/yr JASFA statistics, personal communication, M.Kobayashi, Japan Sea-Farming Association, Kanda.Tokyo!. At present, JASFA relies entirely on thc

130 1,:JVR Technical Report Kin. 26

p

//

//

X

l j! 1

'i"~' 78 7'9 h ! Hl 8',~ h,", 8 1 jl;=, Ht' 87 Nit Hjl tl ! 9 ji'> H.'> Sil

Yc tr f r !jtt It>77 � ]HH~!

Figure S. Statistics for artificial seed production and release of P japonicus in Japan from 1977 to 1995 courtesy of the Japan Sea-Farming Association!.

use of natural spawners for obtaining seeds whichare generally raised to a size of 12-18 mrn beforerelease, although "large-size" seeds frotn 20-40 mmare occasionally produced. Seed productionoperations are conducted principally from April toSeptember, when water temperature is warm about 26'C! and parent spawners are readilyavailable, In general, prawns with developedovaries are purchased from commercial fishermen,and are brought to JASFA premises while chilledslightly or kept on sawdust, and are then put intostocking tanks. With tetnperature in the stockingtanks raised back to higher temperatures, spawningusually occurs in the evening or by the following

day. At Shibushi in 1995, purchasing operationswere carried out 20 times between 25 April and 7September, during which time 6283 parent prawnswere obtained Miyajima 1995!, and actual seedproduction operations were implemented on a totalof 15 occasions. Of total prawns purchased, 6120individuals survived transportation to ShibushiStation, and 2005 individuals actually spawned atan average spawning rate of 39,4%. This yieldedan initial total of 236 million seeds with a final

harvest figure of 92 million seeds. Table 3 showsactual figures for each spawning occasion, withsize of tank used, date, number of seeds obtained,

and density for initial stocking and harvest.

% elder 131

Prod.Bo.

2 4 56 79 910li121314

Tant4<rtt'!

5, 850, 0006, 390, 000

2, 660, 000

31, 420, 00010, 210, 0009,200,000l6, 340, 000

2.500 26-27 Aar.400 7 � 9 aey400 11 Bay400 19 � 2I Ityy

2. 500 25-26 Nay2. 500 8 June

40IJ 30 June400 30 June

2, 500 29 July400 11-12 hu8.400 11-12 hug.400 19-20 Au8.400 19-20 Atut.400 7 Seat.

7

34, 000, OQQ9, 070, 0003, 020, 0007, 30D, 000

I I. 480, OOQ66, 920, IIOO18,640, OOQIB, 4 10, OQO32, 090, QOO4. 230, OQO4. 430, OOO3. 210, 0003. 150, 000

1 I. 000. OOQ

0. 5I. 6

3. 43. 6I. 53 7I. I6. 7T. 27. I3. 2I. 7i.6I. 2I. 24. 4

I:.279. 2

19 aey12 June12 Bey6 June3D Bay12 July4 Aut7 Aug.31 Aug,

8 I14. 4

0. 7 1'T. 9

14. 514, 514. 613, T

27. 4

1,82. 62. 3O. 9

45. 954. 750. 051. 0

6. 170. 000 56. II

21 Seat,5

6. 99' 0

Table 3. Artificial seed production at JASFA Shibushi Station in 1995: initial production, density, body length and tinal harvestvalues.

not discuss these areas in detail, but one potentialmeans of marking is uropod-cutting, whereby theregenerated uropod differs in color and pattern fromuncut ones, making individuals of artificial origindistinguishable Miyajirna et al. 1996!.

Operations were similar in the previous two yearsof 1993-1994 Sato 1993, Sato and Yoseta 1994!.

Transport of artificial seeds is usually doneby truck, and seeds are supplied to variousprefectural users. Some trips take up to 17-1g h.Shipping density ranges from 5.6 - 55.7 millionindividuals/m' and is adjusted according to lengthof the trip and size of the seed. Temperature iskept between 19.0-22.5'C in order to suppressmetabolism. Mortality during shipping is virtuallynil and seeds are observed to be in good conditionupon arrival.

At present, it is not difficult to secureparent females during rnid-spring to early autumn;however, the availability of spawners obtained fromnatural sources makes it difficult to conduct

operations earlier than April. In addition, whileseed production is generally successful based onplacing these females in holding tanks prior tospawning and collecting the seed, it is difficult tocontrol spawning time or to synchronize thespawning of many individuals which would makeoperations more efficient, It is still thereforenecessary to improve technology in order to providea stable supply of seed for coastal restocking.JASFA is engaging in basic and applied researchin order to address these problems. It also remainsdifficult to assess the effectiveness of seed release

programs. At present, JASFA is investigatingmeans of marking seed destined for release in orderto determine the proportions of artificially-producedprawns in the natural habitat, This manuscript wiH

RESEARCH RELATING TO SEED

PRODUCTION IN P. JAPONICUS

BackgroundThe Mornoshirna Station of JASFA has

developed a biopsy method for determining the stateof ovarian deve!Opment in Pj aporticus Miyajimaand Matsumoto 1996!. It was previously necessaryto rely on assessing prawns for maturity byobserving the visible development of the ovaries,and classifying them into A, B, C, and D ranksbased on relative size and visual appearance ofthc ovaries. The A and B ranks in which ovaries

are enlarged were considered mature and the Cand D stages in which ovaries were still elongatedwere considered immature Miyajima andMatsurnoto 1996!. However, with these methods,it was difficult to observe fine differences in

ovarian maturity which would serve as an indexfor spawning potential. The biopsy methods permitdetailed observations of developing oocytes. Inthis method, a syringe is inserted into the ovariesvia the soft area between the first abdominal

segment and the carapace. Oocytes are thencollected and positioned onto a glass slide andobserved under light microscopy at a magnificationof 100-200X. Oocytes can be differentiated into

13$ tlJNR Technical Report tsa. zs

Irrducliort of rnatttrationIn order to secure seed at specific, desired

times, it is considered essential to elucidate theenvironmental factors that are involved in

controlling fernale maturation. In this experiment,the effects of light and temperature on ovarianmaturation were examined below, see Miyajima1995!. The study was conducted during the seasonin which female prawns are not normally obscrvcdto mature. Three groups � 2PC lights-on 14 h,25 C lights-on 14h, and 2' natural day length�

iatt st e batch size Hatchin rateHo. rastts go. s aaners S avnin ate No. da s3rd Yolk globuleEarlr aatttration

t t'o

l. 261, 000 32. 2%214, 000+ 101, 000 57. 1~36. 2%

+84 000 78. 9~ . 2%

7. 1%1422

122

3

e 4. Spawning rates, number of eggs per batch and hatching rates for spawning stimulation experhnent in kurutna prawnat >ASFA.

three stages which correlate with histologicalexamination. In the third yolk globule stage, corticalal veol i cannot be seen, but other features are sinu! arto those of the early maturation stage. In this stage,the cortical alveoli become apparent. Finally, inthe maturation stage, the cortical alveoli becomeelliptical,

These methods are a useful tool in selectingspawners for seed production operations and inimplementing experiments relating to induction ofmaturation. In JASFA during 1995 Miyajirna1995!, a series of experitnents carried out at theMomoshima Station relating to artificial inaturationin context of selection of prawns, transportconditions, and rearing conditions are highlighted,These studies, irnpleinented in order to dcvcloptechnology for the control of maturation andspawning, are briefly described below,

Development of technology for the control ofmaturation and spawningMtrsseLariom of spawrriIsg

In part I! of this study, the relationshipbetween maturation stage of the ovary andspawning rate and the effects of eyestalk ablationon inducing maturation were examined� below, seeMiyajima 1995!. One-yr-old females showing Arank ovaries were selected and biopsied in orderto classify prawtis into one of the three maturationstages third yolk globule, early maturation, andmature! as described above, Prawns were putinto individual aquariutns maintained at 25 C.Spawning rates, number of eggs per batch, andhatching rates were observed for each group. Inyolk globule prawns, 7,1% of the individualsspawned while in the latter two groups, this was100% and 75%, respectively. Egg batches rangedbetween 200,000 and 300,000 eggs per prawn forthose individuals which spawned, and hatchoutra.tes were 32.2%, 57,1%, and 78.9%, respectively,for the three maturation stages given above. Theseresuhs are shown in Table 4. Fifteen additional

individuals in the third yolk globule stage wereunilaterally eyestalk ablated right-side!, ln theseindividuals, there was a 40% spawning rate withabout 200,000 eggs per batch and 73.7% hatching.In individuals not ablated, spawning was 7.1% withno differences between the ablated group regardingbatch-size and hatching rate. These resultsdemonstrated that biopsy can be used to reliablyselect prawns which will spawn, and that unilateralablation is effective in increasing spawning ratesin individuals prior to reaching the maturationstages.

In part �! of this study, the use ofenvironmental factors to induce spawning in the thirdyolk globule stage females was examined. FifteenI-yr-old females with A rank ovaries in this stagewere selected and used experimentally for a periodof 4-7 days. Three groups with differing lightconditions, 24 h lights-on, 14 h lights-an, and 0 h lights-on were maintained bctwecn 18.6-24.2'C. As a

result, one individual in the l4 h lights-on group onlyspawned during the experimental peri<xi �.7%!. Inall groups, clear development of the cortical a}veoliwas not seen, but in contrast, there was degenerationof the oocytes in 10%, 33.3%, and 80.0% of theindividuals in the 24, 14, and 0 h lights-on groups,respectively. These results indicated that shorteningday length has adverse effecLs on ovarian matunuion,and suggested that light treatinent can be used tocontrol maturation processes,

Wilder i33

were elnployed. Prawns were reared for a periodof about 2 months and then exatnined for ovarianlnaturation and evidence Of mating deposition Ofsperm case!, Mating rates were 50.0%, 83.7%,and 93,8% in groups 1, 2, and 3, respectively.Maturation rates were 6.0%, 26.5%, and 0.0%.Thus, treatment 2 was most effective. Prawnswith lnature Ovaries were further examined bybiopsy for the presence of the cortical alveoli, butno individuals exhibited this. Actual spawning rateswere 0.0% and 42.9% in groups 1 and 2, Thus,only group 2 individuals spawned with hatchoutrates of 86,4%. This is the first time, however,that females were induced to mature and spawnoutside of their norlnal spawning seaSon withoutusing eyestalk ablation, by manipulatingenvironmental parameters,

PERSPECTIVES ON ARTIFICIAL SEEDPRODUCTION AND CONCLUSIONS

The above studies carried out by JASI'Ahave demonstrated that it is possible to controlmaturation and spawning in P. japorticus byunderstanding the effects of the environment onthese processes, At present, JASFA is cooperatingwith universities and other research organizationsto increase knowledge of mechanisms ofmaturation and to improve existing technology, lnfish, knowledge of the interaction of theenvironment and endocrinology of significantspecies has formed a basis for the development ofuseful technology. In many species, it is knownthat following ovarian maturation, the secretion ofsteroid hormones which serve as maturation-inducing substance MIS! is triggered byenvironlnental cues, Other basic knowledge hasallowed the developlnent of hormonal treatmentsto stimulate final maturation and spawning, suchas in the use of human chorionic gonadotropin. InCrustacea, while much progress has been achievedin elucidating hormonat mechanisms, much remainsto be elucidated on how environmental factorsinfluence the secretion of hormones which controlmolting and reproductive processes. Similar to fish,whether an M IS exists is still unc! ear. In the future,it will be important to link basic studies to explainobservations and results of fieldwork and practicalexperiments, Cooperation between persons

working in these respective areas should bc activelypursued.

ACKNOWLEDGMENTS

The author expresses thanks to ToruFurusawa, Managing Director. Masato Kobayashi.and Kouichi Saotornc of the Japan Sea Farming,Association JASFA! for generously providingJASFA in-house lnaterials and guidance.

LITERATURE CITED

Ahl, J.S.B. and J,J. Brown. 1991. The effect ofjuvenile hormone III. methyl farnesoatc, andrnethoprene on Nw'K-ATPasc activity inlarvae of the brine shriinp, A rtemi a. Comp.Biochem. Physiol. 100A: 155-158.

Chavez Justo, C. 1990. Ecology, reproduction,and culture of the giant freshwater prawn.Macrobrachium rosenbergii. pp. 163-191.ln; C. Chavez Justo ed!., Thc Aquacultureof Shrimp, Prawn and Crayfish in thc World.Midori Shobo Publ., Tokyo. in Japanese!

Davey, K.G. and E. Huebner, 1974. The responseof the follicle cells of Rhodnius prolixus tojuvenile hormone and antigonadotropin invitro. Can. J, Zoot. 52: 1407-1412,

Davey. K.G., V.L, Sevala, and D.R.B, Gordon.1993. The action of juvenile hormone andantigonadotropin on the follicle cells ofLocusta tnr'grataria. Invertebr. Reprod.Dev. 24 39-36.

Fujiwara, T. 1995, Shuyo yoshokugyo saochichosa hokuku. Annual fiscal report of theNorinchukin Agricultural! Bank, 1-13-2Yurakucho, Chiyoda-ku, Tokyo, l 00-0006,Japan, 106 p. In Japanese!

Laufer, H., D. Borst, F.C. Baker. C. Carrasco. M.Sinkus, C.C, Reuter, L.W Tsai, and D.A.Schooley, 1987. Identification of a juvenilehormone-like compound in a crustacean.Science 235: 202-205.

Liao, I,C. and Y-H. Chien. 1994. Culture ofkuruma prawn Penaeus japotticus Bate!in Asia. World Aquacult. 25 I !; 18-33.

Meusy, J-J. and G.G. Payen, 1988. FemalereprOduction in lnalacoStracan Crustacea.Zoot, Sci. 5:2l7-265.

t:JNR Teehaieol Report 4o. Za

Miyajima, Y. 1995. Artificial seed production inkuruma prawn at Mornoshima Station, pp.43-51. In: Nippon saibai gyogyo kyokaijigyo-nenpo. Annual report of the JapanSea-Farming Association JASFA!, 3-14-8Uchikanda, Chiyoda-ku, Tokyo 101-0047,Japan. In Japanese!

Miyajima, Y. and Matsumoto, A. 1996. Maturityclassification using biopsy in pond-rearedbroodstock of kururna prawn Penaeus

japvnicus and efficient egg removal. SaihaiGiken 25: 37-40. In Japanese!

Miyajima, T., K. Toyota, Y, Harnanaka, and H.Komaki. 1996. Efficiency of uropodcuttingfor marking young kuruma prawn Penaeus

japvnicus. Saibai Giken 25: 41-46.Mizuta, Y, 1995. Artificial seed production in

kururna prawn, pp. 42-43. In: Nippon saibaigyogyo kyokai jigyo-nenpo. Annual reportof the Japan Sea-Farming Association JASFA!, 3-14-8 Uchikanda, Chiyoda-ku,Tokyo 101-0047, Japan. In Japanese!

rishi, A. 1995. Promotion of kuruma prawnartificial seed pmduction at Shibushi Station,pp. 336-338. In: Nippon satbai gyogyokyokai jigyo-nenpo. Annual report of theJapan Sea-Farming Association JASFA!,3-14-8 Uchikanda, Chiyoda-ku, Tokyo 101-0047, Japan. In Japanese!

Okurnura, T., K, Nakamura, K, Aida, and I. Hanyu,1989. Hernolymph ecdystemid ]evels duringthe molt cycle in the kuruina prawn Penueus

japvnicus. Nippon Suisan Gakkaishi 55:2091-2098.

Okumura, T�C.H Han, Y. Suzuki, and K. Atda.1992. Changes in hernolymph vite]logeninand ecdysteroid levels during thereproductive and non-reprocluctivc moltcycles in the giant freshwater prawn,Macrvbrachium irisenbergii. Zool. Sci. 9:37-45.

Sag i, A.. E. Homo]a, and H. Laufer. 1991. Methylfarnesoate in the prawn Mucrvbrachiumrvsenbergii: synthesis by the tnandibularorgan in vitro and titers in thc hemolyrnph.Comp, 8iochern. Physio]. 99B: 879-882.

Sato, H. 1993. Promotion of kuruma prawnartificial seed production at Shibushi Station,pp. 361-362 In: Nippon saibai gyogyo

kyokai jigyo-nenpo. Annual report ttf theJapan Sea-Farming Association JASI'A!,3-14-8 Uchikanda, Chiyoda-ku, Tokyo ] 01-0047, Japan. In Japanese!

Sato, H. and K. Yoseta. 1994. Promotion of

kuruma prawn artificial seed production atShibushi Station, pp. 324-326. In: Nipponsaibai gyogyo kyokai jigyo-nenpo. Annualreport of the Japan Sea-Farming Association JASFA!. In Japanese!

Spindler, K,D., A. Van Worrnhoudt, D. Sellos, andM. Spindler-Barth. 1987. Ecdystcroid ]eve]sduring embryogenesis in the shrimp,Palaenton serrarus Crustacea Decapoda!:quantitative and qualitative changes. Gen.Comp. Endocrinol. 66: 116-122.

Takayanagi, H., Y. Yamamoto, and N, Takeda,1986. An ovary-stimulating factor in theshrimp, Pararya carnpressa. J. Exp. Zool.240 203-209.

Tsukimura, B. and D.W. Burst. 1992. Regulationof methyl farnesoate in the hernolyinph andmandibular organ of the lobster, Hvrnarusantericanus. Gen. Comp. Endocrinol. 86:297-303.

Wilder, M.N. and K, Aida, 1995, Crustacean

ecdysteroids and juvenoids: chemistry andphysiological roles in two species of prawn,Macrvbrachi urn rvsenbergii and Penaeus

japvnicus. Isr. J. Aquacult, Bamidgch, 47:129-136.

Wilder, M.N., T. Okurnura, K. Aida, and I. Hanyu.1990. Ecdy steroid fluctuations duringembryogcnesis in the giant freshwaterprawn, hfacrobrachiurn rvsenbergii, Gen,Comp. Endocrinol. 80:93-100.

Wilder, M.N., T. Okumura, and K. Aida. 1991.Accumulation of ovarian ecdysteroids insynchronization with gonadal developmentin the giant fresh water prawn,Macrvbrachiurn rvsenbergii. Zoo]. Sci. 8:919-927,

Wilder, IVI.N., T. Okurnura, Y. Suzuki, N. Fusetani,

and K. Aida, 1994. Vite]iogeniit productioninduced by eyestalk ablation in the giantfreshwater prawn Macrobrachiuntrosenbergii and trial methy] farnesoateadministratio~. Zoo], Sci. 11: 45-53

Wilder isa

Wi!der, M,N�S. Okada, N. Fusetani, and K, Aida,1995. Hemolyrnph profiles of juvenoidsubstances in the giant freshwater prawnMacrobrachiurn rosenbergii in relation toreproduction and molting, Fish. Sci. 61: 175-1 76,

Yang, W-J., K, Aida, A. Tcrauchi, H. Sonobc, andH. Nagasawa, 1996. Amino acid sequenceof a peptide with molt-inhibiting activity fromthe kuruma prawn Penaeus japnnicus,Peptides 17; 197-202,

Adachi et al. 137

PRIMARY PRODUCTIVITY OF SANDY SHORES

K urn iko Adachi

National Research Institute of Fisheries EngineeringEbidai, Hasaki. Ibaraki 314-0421, Japan

e-mail: kin C~'nrife.affrc,go.jpKatsunori Kimoto

Seikai national Fisheries Research Institute49 Kokubu, Nagasaki 850-095 l. Japan

c-mail: kimotoOsnf.affrc.go.jpJunya Higano

Japan International Research Center for Agriculture Science1-2 Ohwashi, Tsukuba, Ibaraki 305-0851, Japan

e-mail; higa@'jircas.affrc,go.jp

ABSTRACT

Many kinds of aquatic organisms «rc found inhabiting the surf rxinc around exposed sandy shores and, inparticular, plankton feeders such as the sandy beach clam is an important species contributing to fisheryresources. 1'his fact shows that thc biontass of phytoplankton in these areas is abundant. which comes fromprimary producuon. Thus, it inay be possible to establish no-feed aquaculture and nursery culture of bivaJvesby using abundant, natural phytoplankton as feed in such areas. However, because sandy shores are oftenutilized for various human activities, the beach shape is sometimes artificially modified � It is therefore necessaryto clarify the mechanisms which support primary production in exposed sandy shores in order to mainuun andimprove biological production m harmony with a variety of coastal uses. In this context, wc have investigatedprimary production and characteristics of nutrients in the surf and outer turbulent zones of an exposed sandyshnre located in lbaraki prefecture, Japan. In genera!, it has been shown that the primary production rate in theotxan strongly depends on light intensity and water temperature. However. the results of the present investiganonon exposed sandy shores euggcst that the most important factor regulating primary production is nutrientconcentration. Therefore, understanding the dynamics and mechanisms of nutrieni supply is considered animportant step in evsJuating primary production. Moreover, it has been shown that productivity is high in thesurf zoiie as well as ol'fshore. It is thought that the physical characteristics of the surf zone, i.c., turbulence ofwater caused by waves, run-up of seawater, infiltration of run-up water in sand. and exudation of undergroundwater. are related to high primary production,

INTRODUCTION

It is thought that biota are poor in the surfzone on exposed sandy shores because theturbulence of seawater is violent, and there are nosteady adhesion bases. However, many kinds ofaquatic organisms inhabit such areas, and the sandybeach clams are itnportant fishery resources onsandy shores. The fact that there is an abundanceof organisms of low trophic levels, such as planktonfeeder, are inhabited shows that the phytoplanktonbiomass is enough to support these organisms,

However, primary production researcharound the surf zone on exposed sandy shores hasbeen rare Brown and McLachlan, 1990!,

Moreover, the supply mechanism of nutrients whichis an important factor to measure the priinaryproduction is not well known. The maincharacteristics of exposed sandy shores are asfollows. First, seawaler and sediments are alwaysturbulent because of waves breaking against thcbeach. Second, seawater infiltrates the sandthrough run-up on the beach. Therefore, it isthought that the substance exchange between threespheres land, hydrosphere, and atinosphere! isactive. It is very interesting to know how nutrientsare supplied in such enviroiunental conditions.

Sandy shores are utilized for varioushuman activities, From the fishery point of view,sandy shores tnay be utilized as fishing grounds.

ttJNR Technical Report No. 26

METHODS

Sea of Japan

ic ocean

Figttre 1. Location of Kashittsa-oada attest the Hasaki Oceanogtaphical Research Stattott.

no feed aguaculture grounds and as bivalvenursery culture grounds which take adavantage ofabundant phytoplankton as feed. Moreover, inreference to land development and coastalprotection, beach shape is inodified artificial!y. Itis therefore necessary to clarify the mechanismsof substance cycling which supports primaryproduction on exposed sandy shores in order tomaintain and itnprove biological production inharmony with a variety of coastal uses.

We have been studying these mechani smssince 1992. The temporal and spatial variation ofboth phytoplankton biomass and nutrierltconcentration in the surf zone were rescarchedfroin 1992 to 1994, Primary production has beenmeasured in the surf zone and of'fshore area everyseason since 1995. Moreover, research concerningthe behavior of the underground water around thebeach began in 1996. Here, the research resultsof the variation of phytoplankton biomass, nutrients,and primary production in the surf zone and offshorearea are introduced.

The research area is Kashiina-nada,located in the southern part of Ibaraki Prefecture,Japan, on the Pacific Ocean as shown in Figurc I,

Kashima-nada is an exposed and shallow sandyshore, and its total length along the shoreline fromthe Oharai beach at the northern end to the Hasakibeach in the southern end is about 80 km, It is oneof the major sandy beaches in Japan. The beachis divided into two parts, the north and the southsides, with the Kashima Port in the middle, Thebeach is flat and wide, but recently the coastalerosion has occured in places, especially in thecenteral part.

Field research concerning variation ofchlorophyll a and nutricnts around the surf zonewas cried out at the research pier near the HasakiOceanographical Research Station HORS!, Portand Harbor Research Institute, Ministry ofTransport, as shown in Figure 2. The sandy beachthere is very flat and wide. Thc nearshorc alsohas a gentle bottom slope and wide surf zone.Shoreline water and bo h sea surface and bottomwater at the offshore end of the pier, 380 m offshorefrom the shoteIine, 5 m in depth, was sampled inorder to dcterinine chlorophyll a concentration andsize distribution of phytoplankton. Because wewanted to obtain detailed knowledge about thetemporal variation of phytoplankton concentrationin this research, the seawater was sampledapproximately 500 times in 3 yr from 1992 to 1994.Nutrient concentration was deterinined from 1993

k4aetit et an }39

Figure 2. Photograph of the the Hasaki OceanographicalResearch Station.

on. The size distribution of phytoplankton wasmeasured once a week from 1993 to 1994.

Chlorophyll a distribution was examinedalong thc beach from Oharai beach to Hasaki beachto characterize phytoplankton biomass in the surfzone on Kashima-nada. This research was carried

out eight times from 1992 to 1994.Primary production was measured by the

site tncthod pseudosite method in stormy weather!at. Sta,3 �.7 miles offshore from HORS, in 10-mdepths! and Sta. 6 �.7 miles offshore from HORS,in 40-m depths! as shown in Figure 3, First, verticaldistribution of light quantum in the sea wasmeasured. Next, sea surface water and seawaterof each depth of quantum number at 50, 25, 10,and 1% in comparison with the surface water weresampled, and these water samples were dividedinto I-L transparent polycarbonatc bottles. Thesebottles were hung at original depths after carbon-13 "C! reagent was added and phytoplankton inthe bottles was cultured for 3 or 4 h. Finally, thephotosynthetic rate was estimated by measuringthe quantity of "C uptake by phytoplankton whileculturing. Additionally, water tetnperatttre, sahnity,and the concentration of both chlorophyll a andnutrients were measured at many points in this areainduding rhe research position.

On the pier of HORS, the photosyntheticrate of the surface water at the shoreline part I min depth! and both surface water and bottom waterat the offshore end of the pier � m in depth! were

It t'air' Kashrna Port

t

RS ., St.3

i C

Rival Ton&

II I! !

!I

2Xm ~

St 6

I 35 45

r 2am

14D' 40 140' 5Q

Figure 3. Location of the field research on offshore area.

similarly measured by the site method, The watertemperature, salinity, and concentration of bothchlorophyll a and nvtrients were also measured.

In the offshore area, primary productionwas measured eight times around noon ort a day injuly, August, and November 1995; February, May,july, and November l 996; and May 1997. In thesurf zone, the research was carried out five timeson the same day or one of the same days asoffshore research,

Chlorophy]l a was analyzed as follosvs,After water samples were filtcrcd through a I -Itinglass fiber filter and the pigments in the particlewhich had been caught on the filter were extractedwith acetone, concentration of the pigment wasdetermined by thc spectrum method Lorenzen1967!

Five kinds of nutrients nitrate, nitrite,ammonium, phosphate, and silicate! weredetermined with an autoanalyzer TRAACS-800,Bran+Ruebbe Co.! by absorption photometry.Stable isotope "C was determined with "Canalyzer Nippon-Bunkho Co.! located at theNational Research Institute of Fisherics Science.

14tt U3'.ta Technical Report >o. 2a

~ el4 ~

~ ~ Ie g~ Il

~ W ~~ ~

~ ~ p aI ~

pl iy+e8 30

~, ~

o 25

50

40

l~ g ' ~I~ - 4

Fttptre 4. The tetnporal vanatlon of tcmperatttre sahnt ty and chlorophyll a at the nhoreltne of the Hasaltt Oceanopaphtcal ResearchStation.

� 20~

O '. 1510

35

~tat 30

20

I rAg

' s'

ei; pa~ ~ ~

P~

4 7 10 ] 4 7 10 1 4 7 10

] 992 1993 1994

Adachi et al. tent

«Terrp, ~cal ~ t"hla � ~I'U4-f' ~ N03-hl

r. Disoharw from Rtvor Tono- ~ <i02-Sl I Aainfall35 I

~s'I30 12

10

l! 4L.0z1

0 Z

25-

20 t- r

15

/ i,", I i /'$5

600 ' - '~'*

a 500

400�

300

200'

100-

0 ,I19941993

Figsrra 5. The venation of tetnperantre, salinity, chlorophyll a. nitrate, phosphate and silicate at the shoreline of the HasaktOceanographical Research Statton; discharge from River Tone; and rainfall at the Choshi Weather Station. Rainfall is showed assttm, and others are showed as mean value,

Tetnporal and spatia! variation ofphytoplankton biomass and nutrientconcentration in the surf zone

Figure 4 shows the temporal variation ofch!orophyll a at the shoreline of HORS from 1992to !994, Monthly tnean values of the watertemperature, salinity, chlorophyll a, and nutrientconcentration at the shoreline of HORS are shown

in the upper part of Figure 5; total rainfall at Choshiand mean va!ue of discharge from the River Tone Ministry of Construction !994! observed al. 76km above the river mouth are in the lower part ofFigure 5.

Chlorophyll a varied from about 1 to 20!tg/L. The atmua! mean value of chlorophyB a at

the shore! ine frotn 1992 to 1994 was 9.5, 4.6, and4.8 !tg/L, respectively, and the biomass from 1993to 1994 was low compared with that in 1992.Bivalve juveniles such as the Japanese surf clamPseudocardium sachaltnensis appearedabundant! y in this area in 1993 where it has grownwe! l. There is a possibility that the ch!orophyll adecrease after 1993 was caused by ingestionpressure by these clams.

Chlorophyll a concentration showed atendency to be high at the shoreline compared withthe offshore end of the pier, Chlorophyll aconcentration was very high during one tnonth fromthe end of April to the end of May, and it wasthought that this phenomenon was due to a spring

t42 VJvR Tcchnical Report Na. zs

20

- 10

2 3 4 5 6 7 8 9 '0 1112 1 2 3 4 5 6 7 8 9 10 I I 1?

25

20

rye 15

10

O

1 2 3 4 5 6 7 8 9 10 II '2 1 2 3 4 5 6 7 8 9 10 ll 12

1993 MOnth 1994

Figure 6. Thc variation of chlorophyll a concentration which is classified according to the size at the shoreline of the HastdttOceanographical Research Station and the bottom water of the offshore end of the pter.

phytoplankton bloom. Chlorophyll a concentrationchanged drastically over a short term. Such achange occurred because of a sudden change inweather or oceanographic phenomena such aswave, current, wind, or discharge from the RiverTone,

Though floating diatoms were dominant inthe suspended matter, many fecal pellets anddetritus were seen.

The variation of the nutrient concentration

at the shoreline is described as fo]lows, Usually,concentration of nitrate, silicate, and phosphate wasless than 10, 20 and 0,4 IsM, respectively.

However, a very high value was often seen indiurnal variation, and it was thought that thisdepended on sudden changes in weather as wellas the chlorophyll a variation, The nutrientconcentration decreased during the spring bloombecause of uptake by phytoplankton. That wasexhausted in the surnrner and recovered graduallyin the autumn. At HORS, nutrient concentrationat the shoreline was a little higher than at theoffshore end of the pier.

It is believed that the main supply sourcesof nutrients are the offshore bottom water, theinland waters such as the river water, and the beach

Adacbi et ab 143

Io IQharai ~ mgam yA

I

I,a

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e

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l9 l' *" 'li 't " 3s 'l 'I ' i i 6 ' ' r ' i r i';t

Temp. 'C! Sal. psu! chl.a >9!'l.! NO3-N ~M! Si02-S; ~14!

3/26/92 7/3/92 8/26/92 3/6/93 B/24/93 3,'3l/94 6/29/94 7/22/94

underground water, in addition to the regeneratednutrients in the ecosystem. A correlation was seenbetween salinity and both the concentration ofnitrate and silicate; therefore, it was thought thatthe influence of the inland water was a strong supplysource of nutrients for the surf zone.

Figure 6 shows the annual variation of'chlorophyll a concentration that is classifiedaccording to phytoplankton size at both theshoreline and the bottom at the offshore end of

the pier. Phytoplankton size was large duringthe winter and spring months, especially thespring bloom, and dominant size was 20-60 pm.It was shown that the size was smaller in

summer. At the offshore end of the pier, themean size of phytoplankton was larger than atthe shoreline and it was thought that largesuspended particles were disposed to sinkalthough turbulence of the water was violent.

Figure 7 shows the chlorophyll a distributionof the shoreline water along Kashima-nada,Chlorophyll a concentration was high in both thenorth and south, but low in the central part. Thebeach at both ends is flat and wide where sand is

f tne, while he beach in the center part has a steepincline where sand is coarse. The River tlaka

flows into the north end of the Kashima-nada and

the River Tone flows into its south end. There is a

tendency for river water to go southward afterflowing into the sea. Therefore, in the southernpart with few influences of the river water, salinitywas high and nutrient concentration was low.However, the level of chlorophyll a was high atHORS. This suggests that primary production isbeing influenced by the shape of the sand, inaddition to the influence of river water,

Estimates of primary productionFigure 8 shows the optical quantum vertical

distribution at the observation points Sta 3 andSta. 6! in the offshore area of Kashima-nada Lighttransmittance short in winter and spring and longin summer and autunm, During the entire researchperiod, light reached the seabed and the value wasfrom 2.5 to 12% compared with the surface atSta. 3. Therefore, it can be said that all layerswere productive, euphotic zones. At Sta. 6, thecompensation depth, depths at 1% of light intensity

Figure 7 Thc variation of temperature, salinity, chlorophyll u, nitrate and silicate at some shoreline points along the Kashima-narfa.

ttgga Tecbtucal Report Xa, 2$

Quartturtt Number lt6!0.10

4E

6i

0.1 0.5

10

E

+ 20

a

30

40

Ftgure 8. The vertical distribution uf the optical quantum number at Sta. 3 and Sta. 6.

1995

.1995

1995.1996

yl996� Jul. 1996

Nov.1996.-- � M ay1997

at the sea surface, varied fiom 15 to 35 m,ChloroPhyll a, measured when primary

production was measured, showed the sametendency as the previous research on the surf zone.ln summary, concentration was high from earlysprirtg to around May and low, around l p.g/L, from

e rainy season, June and July, to Augustin both the surf zone and the offshore. A highconcentration layer, from 1 G to 40 lt~, existed

widely from 0 to 1G m in depth, and the seawaterwas brown, in May 1997,

Figure 9 shows an example of the verticaldistribution of the nutrient concentration at Sta. 3and Sta, 6. The nutrient concentration in the offshorewas vertically the same in autumn and winter. Onthe other hand, it was exhausted during July andAugust in the upper part of the pycnocline becausethe supply of nutrients was cut off with stratification

Adachi et ai. 145

1996 11.23NG3-N, NH4-N StO2-Si< p iM! NO3-N, NH4-N 'Sl02-St y M,l

0 IO0 w: e

tI ~

200

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<995NG3"N NH4-N, Si02-St p M!

0 2 4 6 8 107

Y

0 1 6.2 0.3 0.4 0 5NQZ-N, PO4-P] p M!

Figure 9. The vertical distribution of the nutrients at Sta. 3 and Sta. 6 in August ! 995 and November 1996.

after large nutrient consumption during the springbloom,

Figure 10 shows an example of verticaldistribution of the primary production rate. At Sta.6, the production rate per day was high at thesurface with a tendency to rapidly decrease in thedepth of stratification and to gradually decrease inthe depth of vertical mixing. Sta. 3 showed roughlythe same verticality. Moreover, the productionmaximum layer was seen where the chlorophyll amaximum layer existed in the spring, In order tomake a clear temporal and spatial difference inprimary production, the values on each surface ofthe observation station are shown in Figure 11. Inthe summer, the pnmary production rates were verysmall in the offshore surface, ranging from 7 to 12p.g-C/L/day. In the other season, the values rangedfrom 15 to 100 p,g-C/Uday. Values from 11 to 245ling-C/L/day were obtained at the shoreline. This

8 8NO3-N, NH4-N, Si02 St p M,'t

0 2 4 6 8 10

� tO~-

!

Q 30-f l St,6

0 01 62 03 04 05NG2-N, PO4 � P p M!

result can be called equal or a higher valuecompared with the value of the offshore station.However, there is no winter measureinent yet.

Assimilation number pg-C/ttrg-chl.a/h}was compared as an index of the photosyntheticactivity, Assimilation number ranged from 0.4 to5.0 in the offshore surface, and from 0.7 to 3.1 inthe surf zone. Because vertical distribution of

chlorophyll a was roughly the same except in May,vertical distribution of the assimilation number

showed the same tendency as vertical distributionof production. A clear correlation was not obtainedbetween assimilation number and water

temperature.In estimating primary production vertically

for the whole water column, though the productionper unit area at Sta. 6 was naturally large comparedwith the onshore area where the depth wasshallower than the compensation depth, there was

146 UJNR Technical Report 'No. 26

Quantum %%u!, Producti on . ate ~g-C/L/day!0 20 40 60 80 100

10 0 2 4 6 8 10Chl.a erg/L!

Quantum Fo!, Producti or. rate ~g/L/day!0 20 40 60 80 100

10 0 2 4 6 8 10Chl.a ag/L!

Quantum /!, P roducti on rate ~ g-C/L/day!0 0 20 40 60 80 100

0 2 4 6 8 10

Chl.a ~g/L!0 2 4 6 8

Chl, a ~g/1 !

Quantum�!,Production rate <g � C/L/day!0 20 40 60 80 100

IO2 4 6 8 10

Chl.a >g/L!

~uantum o Chl.a c production rate

Figure 10. The vertical distribution of the primary production rate per day at Sta, 3 and Sta. 6 in 1996.

4

IR8

4

to

4 6 8

4

cx 6pR

8

Quantum %%d!, Producti on rate ~g-C/L /day!

0 20 40 60 80 100

10

~ 20

30

40 0 2 4 6 8 10Chl.a p.g/L!

Quantum %%u!, Producti on rate wg-C/L /day!0 20 40 60 80 100

10

~ 20

~ 30

40 0 2 4 6 8 10Chl, a ~g/I. !

Quan92.um X!,Production rate w g-C/L/day!

00 20 40 60 80 ' 00

~ 10

20

~ 30

Quantum %!,Production rate ug-C/L/day!0 20 40 60 80 100

10

20

~ 30

40 0 2 4 6 & 10Chl.a ~g/L!

Adachi et ah 147

10

a1995

t

1997]996 >995 1996 1997

10

1995 1996 1995 1996 1997

l996 1995 1996 1997

Figure 11. The values of nutxients, chlorophyll a, assimilation number and primary production at sea surface and production pereach unit area of water column on the surf zone along the Hasaki Oceanographical Research Station and offshore area.

8 6C!

2

8

m 4

C3

~100

5O

D

o 10

0.6

0.2

5

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2

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Z Q

1997

~ 5000

E EOOO

500

g 10050

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1997

~ Shoreline > End of pier + St. 3 < St. 6

148 UJNR Tecaatcai Report Yio. 26

no marked difference as the depth changed, Itwas supposed that the reason for this was thetendency for a large chlorophyll a concentration inthe surf zone compared to the offshore area.Results from Sta. 6 were about the same asreported as the mean value in the whole area ofthe Seto Inland Sea of Japan �.38g-C/rn'/day! Coastal Oceanography Research Committee1985!.

Primary production on the sandy shore inKashima-nada was roughly the same as that onsemi-sheltered areas observed in other research.Moreover, it was shown to bc very high in veryshallow areas such as near the surf zone, Butchlorophyll a decreases in summer and so followsprimary production. In general, the assimilationnumber in the ocean strongly depends on watertemperature and light intensity under water Harrison and Platt 1980!. However, our researchresults on exposed sandy shores showed that theassimilation nutnber was not so high in the summeralthough conditions of both light and temperaturewere ideal. This suggests that nutrientconcentration is also an itnportant factor in primaryproduction.

Abundant bivalves inhabit the surf zone.Because bivalve irigestion rises with watertemperature, estimates of primary production maybe low in sununer because phytoplankton is beingconsumed. We believe that nutrients supp!ied arelarger than the quantity estimated, and are usedpromptly. It is important to understand the behaviorand supply mechanism of nutrients in order toevaluate the biological productivity on exposedsandy shores more closely.

ACKNOWLEDGMENT

We thank thc staffs of the Port and HarborResearch Institute, the R/V Taka-maru, and theMarine Production Division of the NationalResearch Institute of Fisheries Science for the fieldstudies in Kashima-nada and carbon analysis,

LITERATURE CITED

Brown, A. C. and A McLachlan. 1990. Ecologyof Sandy Shores. Elsevier, Amsterdam,Netherlands. 328 p.

Coastal Oceanography Research Committee,Thc Oceanographical Society of Japan,Editors. 1985. Coastal Oceanography ofJapanese Islands. Tokai Univ. Press, Tokyo.1106 p.

Harrison, W. G. and T. Platt. 1980. Variations inassimilation number of coastal marinephytoplankton: effects of environmental co-variates. J. Plankton Res. 2 249-259.

Lorenzen, C.J. 1967. Detertnination of chlorophylland pheo-pigments: spectrophototnetricequation. Limnol. Oceanogr. 12. 343-346.

River Bureau, Ministry of Construction, Editors.1992-1994. Annual Report of Discharge.Vol. 43-45. Japan Ri ver Association, Tokyo.[In Japanese J,

stakasone ru al. t49

NUTRIENT CONCENTRATIONS IN CROUNDWATER THROUGHSANDY BEACHES

Takuma Nakasone, Kumiko Adachi, Tomoyuki TakeuchiNational Research Institute of Fisheries Engineering

Ebidai, Hasaki, Iharaki, 314-0421 Japane-mail: takumaCnrife.affrc.go jp; kinOnrife.affrc.go jp; takeucbi@nrife.affrc.go jp

J un ya HiganoJapan International Research Center for Agricultural Sciences

1-2 Ohwashi, Tsukuba, Ibaraki, 305-8686 Japane-maik higa@jircas, affrc.go.jp

and

Hiroshi YagiTokyo Institute of Technology

2-12-1 Oookayama, Meguro, Tokyo, 152-8552 Japane-mail: hyagi@cv.titech,ac.jp

ABSTRACT

The ecological functions of sandy beaches are mruntaimng biological productivity and purifying coastal waterquality. Quanti tati ve estimation of these functions is accessary for con servauon of sandy beaches and maintenanceof biological producti vity. We studied the f'unction of maintaining biological productivity. Generally, groundwaterwhich flows through sandy beaches to coastal waters is considered to be the source of outrients that supports thebiological productivity of coastal waters. Samples of groundwater were collected in an exposed sandy beach atXasaki, Ibaraki Prefecture. J npan, and the concentrations of nitrate, nitrite, ammonium, phosphate, silicate. andsalinity contained in the groundwater were analyzed. Eight sample pipes at different locations and at variousdepths from the shoreline to the backbeach were sct. The sampling period was from July 1996 to January l 997The quantity of nutrients into coastal water was estimated, considering the moving volume of water caused bythe change of groundwater level following tidal change From this experiruent, it vas found that the nutrieuts offreshwater in the backbeach flowed into coastal water mixing with seawater aud decreasing the concentrauon.

INTRODUCTION

In Japan, there used to be 10,000 km ofsandy beaches out of 30,000 km of coastline.Presently, natural sandy beaches have beenreduced to only 4000 krn long due to variousconstructions for disaster prevention, ports, andainenity facilities, On the other hand, manyresearchers have reported the importance of theecological functions of sandy beaches Brown andMcLachlan 1990, Morimoto 1993, Adachi et al.1994!. The ecological functions of sandy beachesare maintaining biological productivity and purifyingcoastal water quality. Quantitative estimation ofthese functions is necessary for conservation ofsandy beaches and maintenance of biological

productivity of coastal waters. Generally, biologicalproductivity of the nearshore ocean is very high.This high productivity is supported by the highconcentration of nutrients in coastal waters, The

main source of nutrients is considered to bc

freshwater from rivers, upwelled waters from thedeep sca, and groundwater through sandy beaches.Now, aquacultuie without artificial feeding has beenproposed to relieve the organic load to offshore,and the knowledge on the dynainics of nutricnts ingroundwater through sandy beaches will helpdevelop the technology of aquaculture withoutartificial feedings.

In this paper, we investigated the functionof maintaining biological productivity. We ~suredthe nutrient concentratiOnS iil grOund Water thrOugh

aki

anographicalearch Station

HORS!

ibaraki PrefectureFigure I. Sampling site.

sandy beaches to understand the role of nutrientsin groundwater.

SAMPLING SITE

We performed sampling in the sandy beacharound the research pier at the HasakiOceanographical Research Station HORS! of thePort and Harbor Research Institute, Ministry ofTransport, Japan Fig. 1!. The length of theresearch pier is 427 m to offshore. This coastalarea is an exposed sandy beach facing the PacificOcean. The mouth of Tone-gawa gawa meansriver in Japan! which has the largest river basin inJapan is located 16 kin south. The fish and thec]ams which live in this nearshore ocean are veryva]uablc resources for fisheries,

SAMPLING DESIGNW«ank eight sample pipes at different

loca«ons and at various depths from the shore]ine«dtc b«kbeach. Sampling locations were 0 m Pl!, 25 m P2! and 65 m p3! from the base of

p'e t «backbeach Fig. 2!. Consideringthe»fluence of the drain to the ocean which is

"of the pier, we estabIished P4 at theof P2 Fig, 2!. At each point, we sank

'"ree p~pes at various depths Fig, 3'I.w«oil ected seawater sainp]es fromthe shore]in and surface area at 200 m water

m! and 380 m water depth of 5 m!e of the pier �00-0, 380-0! to the

~yR technical Report No. 26iso

MATERIALS AND METHODS

Figure 2. Hasaki Oceanographic Research Station HORS!.

offshore, and froin the bottom layer at 380 m�80-B!. Groundwater satnples were collectedthrough each pipe using a pump from July 1996 toJanuary ]997. From 31 July to 1 August 1996, wesampled continua]ly every 2 or 3 h.

Water samples were collected in 300-inlbottles. Subsamples for determining concentrationof dissolved nutrients were filtered through0.45-mm membrane filters to rcmove suspendedsolids and were kept frozen until analysis to avoidbio]ogica] change of the nutrients. We used anautoanalyzer for analysis of nutrients, Theretnaining water samples were used to detertninc

inity with an inductively coup]ed sa]inotnete. AtHORS, the basic characteristics data of the coasta]enviromnent has been collected periodically. Wcused this data for analysis.

Nakasoae et, al. 151

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Figure 4. Nutrient concentrations in surnrner.

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Nutrient concentrations m winter.

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shoreline 34.2

33.12004

3804 33.2 34.3380-8 33.2

Table 1. Salinity concentration ppt!.

The groundwater level at P2 varied withthe influence of tide but at P3 it was uniform withoutany tidal influence. The salinity at P3 was usuallyless than 1 ppt, and is considered to be near yfreshwater. In the sumtner season, salinity at P2was 2-5 and at P 1 was 20-30 ppt, respectively. Inthe winter season, due to the increase of seawatersurges inshore following decrease of groundelevation, salinity increased to approximately 25ppt. We found that the salinity at shallow pointswas higher than at deep poi nts Table I !. Nutrientconcentrations of coastal seawater exceptphosphate were low in sutntner and high in winter.However, this tendency was not distinctlyrecognized in the case of groundwater. Nitrateconcentration of groundwater varied between 150and 250 ItM, and silicate concentration variedbetween 80 and 150 ItM, which is very highcotnpared with the nutrients of seawater Figs. 4,5!. In most cases, the concentration of ammoniumin groundwater was less than 2 ItM, and theconcentration of nitrite was less than I pM.However, the concentration at P3 and P4 increasedoccasionally. This phenomenon is thought to occurby the inflow of freshwater frotn the backbeach

l52 ugtstt Tecitnieat RePnrt No, 26

RESULTS AND DISCUSSIONS and from the drain.

At P4, nitrate concentration variedbetween 100 and 120 pM; silicate varied between130 and 170 pM; and phosphate varied between0.5 and 2 ItlVI, Usually, the nutrient concentrationsat the location of the upland side were higher thanthat of the sea side, This suggests that the nutrientswere supplied by freshwater in the backbeach andflowed into coastal water mixing with seawater.There was no distinct feature for verticaldi stribution of nutrients.

The nutrient concentrations at shorelinebecaine high when the discharge of groundwaterincreased, noticeable by observing tide andgroundwater level fluctuations Fig. 6!. Thegroundwater level was consistently higher thanseawater IeveL This suggests the contribution ofgroundwater to the nutrients of coastal water.However, this phenomenon was observed only inthc summer season.

Mixing of groundwater with seawatercreates gradients of nutrients and salinity. Whensalinity is considered as a conservative tracer ofmixing, then it is possible to quantify the uptakeand release of biologically and chemicaHy-reactivecompounds within the mixing zone. If thecorrelation between salinity and the nutrient

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Figure 6. Time varianon of nutrient concentration ai shoreline.

350$00

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concentration is linear, nutrients tnay be consideredconservative. Therefore, we used the relation

between salinity and nutrients Fig, 7!. This figureis called the 'mixing diagram.' Nitrate and nitriteconcentrations became very high in thc case ofsotne concentrations of salinity. The nitrateconcentration becatne especially high when thcsalinity was 3-5 ppt which mainly occurred at P2,and the nitrite concentration became high whenthe salinity was approximately 20 ppt whichoccurred mainly at Pl, This phenomenon i»considered to bc nitrification by nitrifying bacteria.Reportedly, the activity of bacteria which oxidizcsnitrite is maximutn when the salinity concentrationis 3-5 ppt Kurihara 1988!. Our results correspondto that report. Furthertnore, from our results wecan infer the presence of dissolved oxygen in thegroundwater, Frotn Figure 7 c!, there was a linearrelation between salinity and silicate. Therefore,silicate seemed to be supplied by freshwater in thebackbeach and flowed into the coastal water tnixingwith seawater.

The fluctuation of thc concentration of

nitrate at P2 seemed to correlate with nitritc at Pl,

ammonium at P2, and nitrite at P2 Fig. 8!, Weconsider that it was the facilitation of nitrification

due to the in~ of amrnoniurn,

10 20Salinity

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Ftgare 7. Mixing diagram,

l54 UJVR Technical Report Co. 26

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Figure 8. Titne variation of mtrate, nitrite anti ammonium.

Figure 9. %e variatinn Of nitrate conCentratiOn.

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Quantity rnol.!Nutrient

Ammonium NHe!

Nitrite NO2!

4.3

10

Nitrate NOs!

Phosphate POa!

Silicate Si02! 260

'table 2. Estimation of quantity of nutrients for l km coastaline for a tidal cycle!.

The concentrations of freshwater nutrients

which were diluted by seawater were calculatedby estimating the rate of mixing of freshwater andseawater. Hy these calculated values, we canestimate the quantity of nutrients which wercbiologically or chemically released or uptaken. Thcmixing rate was calculated from salinityconcentration which was considered to be notreactive biologically or chemically. The tneasupxlconcentration of nitrate was higher than thecalculated concentration at P2 and the measuredconcentration of nitrite was higher than thecalculated one at Pl Figs. 9, 10!. This indicatesthat nitrification occurred in the sandy beach. In

35

~ 30

v 25

20

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the case of phosphate and silicate, the measuredconcentration corresponds to the calculated one,hut there were a few cases which did not

correspond Figs. 11, 12!. Noncorrespondencewas considered to be the release following thedecotnposition of organic compounds bymicroorganisms or adsorption to sand particles orsuspended solids Sewell 1982, Johannes and Hearn1985!.

We estitnated the quantity of nuuientswhich flow into the coastal sca. We calculated

the discharge of groundwater into thc coastal scaby the Sakatnoto method Sakarnoto 1991!.Sakamoto proposed that the discharge of

t;JNR Technical Report No, 26

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Figure 11 The variatron of phosphate concentration.

Figure 12. The variation ot silicate concentration.

158 p3ga Techntca} Repor

Ch s of grain distribution of bed materialangcs 0

in me su z«rf zone � field observation at Hasak iphicaI Research Facility. Rep.

P«Harbor Res. Inst. 29 Z!: 38-61, [InJapanese with English summary],

Kurthara, Y., Editor. 1988, FcologyEcotechnology in Fstuarine-Coastal Area.Univ. Tokai Press. 335 p. [In Japanese].

Morimoto, K. 1993, Nutrients budget and waterctrculation at intertidal zone. Bull Coacta]Oceanogr. 30�!; 208-223- [In Japanesewith English summary].

Sakamoto, I. I 991. Use of respiration in the sandybeach or on the t>da] Hat. ] Perm able ~ndybeach. Mar. Potlut. Bull. 23; 123-13P,

Sewell, P. L. 1982. Urban groundwater as apossible nutrient source for an estuarinebenthic algal bloom, Fstuarine Coastal ShelfSci. 15: 569-576.