631

Journal of Oceanography, Vol. 60, pp. 631 to 636, 2004

Short Contribution

Keywords:⋅⋅⋅⋅⋅ Bivalves,⋅⋅⋅⋅⋅ Ruditapesphilippinarum,

⋅⋅⋅⋅⋅ plankton,⋅⋅⋅⋅⋅ larva,⋅⋅⋅⋅⋅ reproduction,⋅⋅⋅⋅⋅ Tokyo Bay.

* Corresponding author. E-mail: furukawa-k92y2@ysk.nilim.go.jp

† Present address: National Institute for Land and Infrastructure Man-

agement, Nagase, Yokosuka, Kanagawa 239-0826, Japan.

Copyright © The Oceanographic Society of Japan.

Detailed Observation of Spatial Abundance of ClamLarva Ruditapes philippinarum in Tokyo Bay, CentralJapan

TOMOYUKI KASUYA1†, MASAMI HAMAGUCHI2 and KEITA FURUKAWA3*

1Corporation for Advanced Transport & Technology, Uchisaiwai-cho, Chiyoda-ku, Tokyo 100-0011, Japan2National Research Institute of Fisheries and Environment of Inland Sea, Maruishi, Ohno-cho, Saeki-gun, Hiroshima 739-0452, Japan3National Institute for Land and Infrastructure Management, Nagase, Yokosuka-shi, Kanagawa 239-0826, Japan

(Received 2 September 2002; in revised form 26 June 2003; accepted 26 June 2003)

The spatial distribution of the larval abundance of the clam Ruditapes philippinarumhas been investigated at 65 stations throughout Tokyo Bay on August 2, 2001. Thelarge number of small D-shaped larvae that were found shortly after hatching in thewaters around the Banzu, Futtu, and Sanmaizu-Haneda areas indicates that spawn-ing populations in these areas probably contribute greatly to the larval supply in thebay. Small larvae also occurred abundantly around the Yokohama and Ichihara portareas, suggesting that these port regions play a role in the transport of larvae intoTokyo Bay.

do they go?” Because the identification of larval bivalvesis rather difficult, however, little is known about the lar-val abundance and advection of the clam (Matsumura etal., 2001). As a first step in clarifying the ecology of theearly life stages of R. philippinarum in Tokyo Bay, thispaper briefly describes the spatial distribution and sizecomposition of the larvae, and then discusses the spawn-ing areas of R. philippinarum in the bay. We also discussthe validity of estimating larval abundance using a 100-µm mesh net.

2. Materials and Methods

2.1 SamplingTokyo Bay is located along the Pacific coast of

Honshu, central Japan. The bay is divided into inner andouter areas by a boundary line connecting Futtu withKannonzaki (Fig. 1). In this paper, Tokyo Bay means theinner part of the bay, covering a 960-km2 surface areawith a 15-m mean depth.

In Tokyo Bay, R. philippinarum spawns twice a year,with peak periods from April to June and August to Octo-ber (Toba et al., 1993). Samples were collected at 65 sta-tions spaced at intervals of ca. 3.5 km in Tokyo Bay onAugust 2, 2001 (Fig. 1). We collected the samples within

1. IntroductionThe clam Ruditapes philippinarum is one of the most

commercially important food bivalves for the Japanese.It is abundant on the sand-mud sediments of tidal flatsand shallows in regions stretching from Hokkaido toKyushu. Recently, however, clam catches have decreasedin Tokyo Bay as well as in other coastal and inlet watersareas in Japan and this decrease may be due to the de-struction of habitats as coastal areas become further de-veloped (Kakino, 1992). As most of the tidal flats andshallows in Tokyo Bay have disappeared, restoration of ahabitat for R. philippinarum in the bay is being investi-gated. This could include the construction of artificialshallows or tidal flats, and protecting the clam’s larvalsupply areas, but to do this successfully it is necessary tounderstand R. philippinarum larval advection during theplanktonic stage, i.e. “where do they come from and where

632 T. Kasuya et al.

a five-hour period during daylight using eight boats. Thestations were grouped into eight groups, and each groupwas covered by one boat. As hypoxia/anoxia occurswidely in the bottom layer of Tokyo Bay during summer(Furota, 1988), no sampling was conducted at depths be-low 12 m. We were able to collect the samples evenly atspecific depth layers (0–4 m, 4–8 m, and 8–12 m) by re-peatedly raising and lowering a weighted flexible hoseattached to a water pump. We collected planktonic larvaeof R. philippinarum by pumping up and filtering 200 litersof seawater from the three depth layers, first through a100-µm mesh net and then through a 50-µm one to col-lect the smaller larvae. The number of depth layers sam-pled varied depending on the depth of each station (Table1).

After all the sampling had been completed, the sam-ples collected using the 100-µm net were immediatelycooled and divided into four sub-samples on land using aplankton splitter. Two of the four sub-samples were pre-served in 5% buffered formalin, and the others werecooled before storage in a freezer (<–45°C) in the labo-ratory. The 50-µm net samples were also cooled immedi-ately and stored in the same freezer.

Temperatures and salinities were determined at eachstation using an STD recorder (Alec Electronics Co. Ltd.,

AST-500) and water density (σt) was calculated fromthose data.

2.2 Identification of larval Ruditapes philippinarumMost bivalves develop into a trochophore and then a

veliger larva with a shell after hatching. Veliger larvaeare classified into D-shaped larvae with a straight hingeline, umbo larva, or fully grown larva according to thestage of development. A fully grown larva grows into ajuvenile clam and settles into a habitat. In this paper weidentified R. philippinarum planktonic larvae using theveliger larvae because veliger larvae with shells are firmerthan trochophore larvae and seem to maintain their shapeafter filtering.

We used the monoclonal antibody method (cf.Hamaguchi et al., 1997) to identify R. philippinarumplanktonic larvae in the frozen 50-µm and 100-µm netsub-samples. After exposing the thawed specimens toantibodies, they were observed using a fluorescence mi-croscope. We counted the larvae, recorded their develop-mental stage (i.e., D-shaped or umbo larvae) and mea-sured the shell length (SL) of up to a maximum of 100specimens using an eyepiece micrometer accurate to thenearest 10 µm. Since fully grown larvae are morphologi-cally similar to umbo larvae, we counted them as umbolarvae. The monoclonal antibody method achieved 95%accuracy in distinguishing larval R. philippinarum fromother larvae in the Seto Inland Sea (Hamaguchi, pers.obs.). The clam Paphia undulata, which is closely relatedto R. philippinarum, was found in Tokyo Bay (Kuwabara,1990) and the antibodies also crossreact with this clam(Hamaguchi, pers. obs.). In the present study, larvae thatwere difficult to identify were finally identified using thefluorescence microscope as R. philippinarum larva byconducting a polymerase chain reaction (PCR) test forD-shaped larvae and by morphologic observations accord-ing to Tanaka (1982) for umbo larvae. We then calcu-lated the density of the R. philippinarum planktonic lar-vae (ind. m–3).

3. Results and Discussion

3.1 Abundance and size distribution of Ruditapesphilippinarum planktonic larvaeWe obtained a large number of R. philippinarum lar-

vae. Of all the bivalve larvae collected, 0.1 to 24.2% be-longed to R. philippinarum. The SL of the collected plank-tonic larvae of R. philippinarum was in the range of 90–130 µm for D-shaped larvae and 130–230 µm for umbolarvae. Larvae with a SL of 90–210 µm, mainly 100–120µm, were found in the 50-µm net sample and those withan SL of 130–230 µm, mainly 150–180 µm, were in the100-µm net sub-sample (Fig. 2). The size frequency dis-tribution of R. philippinarum larvae of the 50- and the

Fig. 1. Tokyo Bay and locations of sampling stations (solidcircles), where stations surrounded by a dashed line wereinvestigated with one boat. The main habitats of benthicRuditapes philippinarum in the bay are shaded and the dot-ted line at the mouth of Tokyo Bay defines the inner part ofthe bay.

Distribution of Ruditapes philippinarum Planktonic Larvae 633

100-µm-net specimens indicated that <140-µm-SL larvaemight pass through a 100-µm mesh, i.e. 70% of a SL. D-shaped larvae of R. philippinarum generally exceed 100-µm SL (Toba, 1992), indicating that we could accuratelyestimate the abundance of D-shaped and umbo larvae byusing a 50-µm mesh net after water sampling.

R. philippinarum planktonic larvae were found atalmost all stations (Fig. 3), indicating that they dispersewidely during the planktonic stage. D-shaped larvae wereabundant in the Sanmaizu-Haneda, Banzu, and Futtu ar-eas with a maximum density of 2510 ind. m–3, and umbolarvae were abundant in the Ichihara, Banzu, and Futtuareas with a maximum density of 1525 ind. m–3 (Table 1,Fig. 3). R. philippinarum planktonic larvae were fairlyevenly distributed vertically at 0 to 12 m in Tokyo Bay,and no relationship was found between the horizontal dis-

tribution of the larvae and water temperature, salinity, andσt at three depths (Figs. 3 and 4). R. philippinarum de-velops from a fertilized egg to a 100-µm SL larva withintwo days at 20°C in the laboratory (Toba, 1992), and set-tles within about 2–3 weeks after hatching at an SL ofaround 200 µm (cf. Toba, 1987). Because the D-shapedlarvae, mainly in the 100–120-µm SL size class, appearto have been collected shortly after hatching, their distri-bution indicates a R. philippinarum spawning area in To-kyo Bay. In contrast, the umbo larvae collected, mainlyin the 150–180-µm SL size class, probably drifted forseveral days after hatching and remained in a planktonicstage for a while. As R. philippinarum planktonic larvaecertainly disperse widely during the planktonic stage inTokyo Bay (see above), it remains unclear whether lar-vae in the 150–180-µm SL size class were spawned in

Table 1. Densities of D-shaped (bold numbers) and umbo larvae (numbers in parentheses) of Ruditapes philippinarum calculatedfrom 50-µm net samples and 100-µm net sub-samples. The symbol (—) means no sampling.

Stn. Depth

(m)

Density (ind. m–3) Stn. Depth

(m)

Density (ind. m–3)

0–4 m 4–8 m 8–12 m 0–4 m 4–8 m 8–12 m

1 4 320 (150) — — 34 23 360 (225) 310 (170) 200 (85)

2 6 290 (225) — — 35 10 200 (105) 515 (35) —

3 7 60 (0) — — 36 5 955 (890) — —

4 6 20 (185) — — 37 18 430 (315) 290 (280) 550 (115)

5 13 420 (115) 255 (115) 175 (35) 38 25 260 (165) 380 (205) 750 (300)

6 11 145 (185) 410 (70) — 39 28 5 (45) 15 (0) 210 (5)

7 9 55 (360) 145 (270) — 40 23 225 (105) 160 (60) 670 (335)

8 5 50 (100) — — 41 18 155 (85) 240 (45) 160 (125)

9 9 65 (120) 230 (300) — 42 30 145 (25) 130 (40) 250 (75)

10 11 250 (20) 345 (75) — 43 25 235 (50) 200 (30) 260 (20)

11 13 160 (160) 115 (165) 390 (5) 44 16 825 (55) 390 (45) 575 (120)

12 14 510 (325) 845 (215) 190 (0) 45 14 1725 (365) 2510 (1100) —

13 13 285 (210) 325 (70) 55 (70) 46 6 105 (350) — —

14 9 165 (65) 195 (0) — 47 10 170 (260) 295 (110) —

15 17 30 (40) 620 (435) 835 (60) 48 20 210 (125) 225 (65) 530 (65)

16 16 165 (80) 285 (110) 545 (365) 49 30 90 (25) 295 (5) 510 (140)

17 14 230 (25) 465 (105) 940 (110) 50 22 165 (45) 165 (45) 360 (270)

18 11 10 (80) 185 (40) — 51 27 570 (40) 930 (5) 560 (30)

19 6 1710 (70) — — 52 25 55 (40) 220 (70) 215 (300)

20 11 2180 (35) 1085 (170) — 53 20 90 (25) 80 (0) 105 (25)

21 14 330 (110) 775 (385) 375 (90) 54 10 710 (90) 260 (65) —

22 17 95 (120) 140 (180) 680 (275) 55 11 805 (435) 830 (60) —

23 19 195 (15) 460 (40) 110 (40) 56 4 920 (325) — —

24 18 70 (245) 190 (335) 210 (775) 57 15 715 (200) 1030 (110) 1010 (20)

25 14 85 (45) 510 (110) 285 (520) 58 15 165 (0) 115 (20) 235 (45)

26 15 245 (340) 785 (1525) 455 (125) 59 34 770 (85) 320 (25) 200 (150)

27 14 150 (775) 385 (465) 90 (275) 60 43 125 (20) 125 (60) 265 (55)

28 21 120 (20) 210 (145) 230 (225) 61 43 80 (60) 100 (25) 110 (25)

29 21 95 (20) 60 (20) 60 (40) 62 17 375 (240) 225 (90) 940 (115)

30 17 275 (5) 215 (180) 285 (310) 63 4 370 (565) — —

31 13 1165 (595) 510 (275) 260 (30) 64 28 190 (75) 335 (75) 350 (80)

32 21 345 (285) 570 (200) 385 (390) 65 19 390 (50) 315 (50) 390 (35)

33 24 75 (65) 170 (70) 265 (285)

634 T. Kasuya et al.

the Ichihara, Banzu, and Futtu areas or were transportedthere from other spawning areas.

In Mikawa Bay, Japan, R. philippinarum planktoniclarvae are mainly found at a 3-m depth (Suzuki et al.,2002). In the Seto Inland Sea, Japan, R. philippinarumplanktonic larvae were concentrated at a depth layer of30 of salinity in the water column when the salinity at thesurface fell due to heavy rain (Hamaguchi, pers. obs.). InTokyo Bay, as pycnoclines mostly existed at depths rang-ing from 8 to 15 m in August 2, 2001, R. philippinarumplanktonic larvae may possibly be transported passivelyin a mixing layer in Tokyo Bay. To add to the data gath-ered from these field observations, the larval transportprocesses of R. philippinarum should be analyzed usinga hydrodynamic numeric model in a future study.

3.2 Spawning area of Ruditapes philippinarumThe horizontal distribution of D-shaped larvae with

a ≤100-µm SL, obtained by multiplying the density (Ta-ble 1) by the size frequency of the respective samples,showed that densities of >500 ind. m–3 occurred aroundthe Sanmaizu-Haneda and Banzu areas where there arehabitats of benthic R. philippinarum in Tokyo Bay (Fig.5). Larvae with a ≤100-µm SL also occurred at a densityof 200–300 ind. m–3 around the Yokohama, Futtu, andIchihara areas. As R. philippinarum develops from a fer-tilized egg to a 100-µm SL larva within two days at 20°Cin the laboratory (Toba, 1992), larvae with a ≤100-µmSL may remain within the area (Banzu, Futtu, Sanmaizu-Haneda, Ichihara, and Yokohama) where they arespawned.

The Banzu and Futtu areas are shallows and tidalflats, in an almost natural condition, and R. philippinarumoccurs densely in these areas (Kakino, 1992). Their re-production might lead to a high abundance of planktoniclarva in these areas. In the Sanmaizu-Haneda area, some

natural tidal flats remain, and some artificial shallowshave been constructed. R. philippinarum occurs abun-dantly around Sanmaizu (Furota, pers. comm.) and aroundthe mouth of the Tamagawa River (Kuwabara, 1990).Although there have been few quantitative studies of theabundance of R. philippinarum in the Sanmaizu-Hanedaarea, the density of small larvae in this area is almostequal to that in the Banzu area. The spawning populationsin the Sanmaizu-Haneda area, as well as in the Banzuarea, probably contribute greatly to the larval supply inTokyo Bay.

The Yokohama and Ichihara areas are ports protectedalmost completely by a vertical sea wall. In the Ichiharaarea, R. philippinarum appears in the sandy bottom adja-cent to the sea wall (Toba, pers. comm.). R. philippinarumhas also been collected from the port region aroundYokohama (Kuwabara, 1990); these port regions mightalso play a role in the larval transport of R. philippinaruminto Tokyo Bay.

4. ConclusionIn this study we have shown the validity of our

method of estimating the abundance of R. philippinarum

Fig. 2. Size frequency distributions of Ruditapes philippinarumlarvae collected using a 50-µm and 100-µm nets on August2, 2001. The data are pooled from all stations, where n isthe number of measured larvae.

Fig. 3. Horizontal density distributions of Ruditapesphilippinarum D-shaped and umbo larvae from three depthlayers on August 2, 2001. Densities are proportional to thearea of the circle.

Distribution of Ruditapes philippinarum Planktonic Larvae 635

Fig. 5. Horizontal density distributions of Ruditapesphilippinarum D-shaped larvae with a ≤100-µm SL fromthree depth layers on August 2, 2001. The density of ≤100-µm-SL larvae was obtained by multiplying the density (Ta-ble 1) by the size-frequency of respective samples.

Fig. 4. Horizontal distributions of water temperature, salinity,and σt at 0-, 4-, and 8-m depths.

AcknowledgementsWe thank the staff of the Marine Environment Divi-

sion at the National Institute for Land and InfrastructureManagement for their collaboration in collecting samples,and we greatly appreciate the advice given by Dr.Mitsuharu Toba. We also thank two anonymous review-ers for their helpful comments on the manuscript. Thisstudy was partly supported by the Program for Promot-ing Fundamental Transport Technology Research from theCorporation for Advanced Transport & Technology(CATT).

ReferencesFurota, T. (1988): Effects of low-oxygen water on benthic and

sessile animal communities in Tokyo Bay. Bull. CoastalOceanogr., 25, 104–113 (in Japanese).

Hamaguchi, M., H. Usuki and H. Ishioka (1997): Interrelation-ship of Japanese littleneck clam, Ruditapes philippinarum,and other animals on tidal flat. Fisheries Eng., 33, 201–211(in Japanese).

larvae using a 50-µm mesh net to filter samples taken ata range of depths. Our observations of the horizontaldistribution of small D-shaped R. philippinarum larvaehave revealed some spawning areas of the clam, includ-ing a port region in Tokyo Bay. Recently, it has been re-alized that shallows and tidal flats have a water purifica-tion function, in addition to their role as a nursery forjuveniles, and the construction of artificial shallows ortidal flats around ports has been planned. Artificial shal-lows and tidal flats may increase the habitat of R.philippinarum, and could potentially contribute to restor-ing the R. philippinarum resource. Nevertheless, althoughport regions have not been recognized as a habitat andspawning area for fishery organisms, the abundance anddistribution of organisms in the port area should be closelyinvestigated before constructing shallows or tidal flats inTokyo Bay.

In this experiment, other zooplankters found in theformalin-fixed 100-µm net sub-samples, including theheterotrophic dinoflagellate Noctiluca scintillans, werealso identified and counted. In addition, we made similarobservations, including measuring the dissolved oxygencontent, on August 6 and 10, 2001, in Tokyo Bay. Weintend to combine these data to clarify the factors affect-ing the distribution and abundance of R. philippinarumplanktonic larvae, i.e. hydrography, hypoxic/anoxic wa-ter mass, predation by carnivorous plankters, and larvaltransport processes in Tokyo Bay. Other spawning areas,e.g. the Sanbanse and Kanazawa areas, where benthic R.philippinarum occurs abundantly (Fig. 1), will also beevaluated.

636 T. Kasuya et al.

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