Ecology of Polydora Cornuta Bosc , 1802 (Spionidae: Polychaeta)

Journal of Water and Environment Technology, Vol. 9, No.3, 2011 Ecology of Polydora cornuta Bosc , 1802 (Spionidae: Polychaeta) in the Eutrophic Port of Fukuyama, with Special Reference to Life Cycle, Distribution, and Feeding Type

Norimi TAKATA, Hiroyuki TAKAHASHI, Satoshi UKITA, Kyoko YAMASAKI, Hidefumi AWAKIHARA

Nippon Total Science, Inc., 399-46 Minamigaoka, Minoshima, Fukuyama, Hiroshima 721-0957, Japan

ABSTRACT Fukuyama Port is a semi-enclosed harbor located in the central region of the Seto Inland Sea. During June 1998, no dissolved oxygen (DO) was present at the bottom layer of the water column. Low DO conditions continued until October, coinciding with a period of high water temperature. A study of the life cycle, distributive characteristics, and feeding type of Polydora cornuta Bosc, 1802, inhabiting the organically polluted port area, was performed. Polydora cornuta which lives in mud tubes in bottom sediments, was found to be unevenly distributed in the innermost part of the port. Taxonomic characteristics are modifications of setiger 5, which includes major heavy spines. Predicted life-cycle duration, based on the 11.5 - 18.2°C temperature of bottom mud during the normal life period for this species, was 40 to 59 days. Appearance of benthic life-stage individuals was limited to winter and spring (i.e., January to May). However, pelagic larval individuals were present throughout the year. The distribution and density of P. cornuta reflected the DO conditions of the bottom layer and sediment. Stable carbon and nitrogen isotope analysis indicated that this species is a suspension feeder.

Keywords: Fukuyama Port, life cycle, Polydora cornuta, suspension feeder

INTRODUCTION Fukuyama Port (ca. 10 km long, 1 km wide) is located in the Bingo-nada area, the central area of the Seto Inland Sea. The port is a semi-enclosed harbor with a mean tidal range of 2.6 m, poor tidal exchange, and marked eutrophication. Based on the information from studies in other countries, Polydora cornuta Bosc, 1802 along with one or two spionid species should dominate the benthic biomass of the mud of the port basin (Imajima, 1996) and similarly perturbed coastal areas (Anger et al., 1986; Tena et al., 1991; Radashevsky and Hsieh, 2000; Cinar et al., 2005; Surugiu, 2005). Polydora cornuta is a cosmopolitan, opportunistic, and alien species (Grassle and Grassle, 1974; Cinar et al., 2005; Streftaris and Zenetos, 2006; Rice et al., 2008), and little information of its life history in Japanese waters is available. Some species of these genera excavate burrows in the shells of mollusks (Sato-Okoshi and Okoshi, 1993; Sato-Okoshi, 1999; Sato-Okoshi, 2000); however, P. cornuta inhabits mud tubes constructed in the bottom sediment (Takata et al., 1996; Sato-Okoshi, 2000; Yamada et al., 2001). The mature female worms produce egg capsules in the mud tubes, and after emergence and a certain period of pelagic life, the larvae settle onto the sediment. Because P. cornuta is opportunistic, its life cycle is likely to be short (Environment Canada, 2001; Takata, 2011). We studied the animal’s life cycle from laboratory-reared larvae and clarified the distributive characteristics and feeding type using stable isotope analysis.

Address correspondence to Norimi Takata, Nippon Total Science, Inc., Email: [email protected] Received December 12, 2010, Accepted April 30, 2011. - 259 - Journal of Water and Environment Technology, Vol. 9, No.3, 2011

METHODS Sampling and taxonomy We established five monitoring stations for routine assessment of the water and bottom environment and benthic communities in Fukuyama Port (Stn.1 to Stn.5, Fig. 1). Samples taken using water sampler type of Van-Dorn and Smith-McIntyre bottom sampler from all five stations each month from April 1998 to March 1999, and the chemical composition of the water and sediment were analyzed in the laboratory (Nippon Total Science, Inc., Fukuyama, Japan).

Water temperature, salinity, and dissolved oxygen (DO) were measured 0.5 m below the surface, at half the depth, and at 0.5 m above the bottom of the five stations using a salinograph and a DO meter (WQC-20A, DKK-TOA Co., Tokyo, Japan). The oxygen saturation profile was developed with EVS-PRO software (C TECH Development Co., HI, U.S.A).

Ignition loss (IL), chemical oxygen demand (COD), acid volatile sulfide (AVS), and mud content of the sediment samples were measured as described by the Environment Agency, Japan (1988) and Arakawa (1980). Total carbon (T-C) in the sediments was measured using a CHN analyzer (EA-1110, EC Instruments, U.K.) in the laboratory. At the same time as sediment sampling for chemical analysis, benthic fauna were sampled using a 0.05 m2 Smith-McIntyre bottom sampler. These samples were washed through a 1.0 mm mesh sieve and then fixed in 5% (v/v) neutralized formalin. Morphological characteristics of P. cornuta were examined using a stereomicroscope and scanning electron microscope (SEM, JSM-6510LA, JEOL Ltd., Tokyo, Japan). Pelagic larvae were sampled vertically from a depth of 0.5 m from the bottom to the surface using an XX13 (100 μm mesh) plankton net (Rigo Co., Tokyo, Japan). Numbers of collected pelagic larvae were calculated for specific areas (individuals indv./m2) assuming settlement and density of the pelagic larvae. Larvae of P. cornuta and Paraprionospio spp. were separated from the plankton samples. Benthic P. cornuta was identified based on Rice and Simon (1980), Blake and Arnofsky (1999), Blake and Maciolek (1987), and Radashevsky and Hsieh (2000), and benthic Paraprionospio spp. were identified according to Yokoyama (2007) and Yokoyama and Tamai (1981). Identification of larvae of P. cornuta was carried out according to Radashevsky (2005) while identification of larvae of Paraprionospio spp. was carried out according to Yokoyama (1996).

Stn.1 (34° 28’ 46.7”, 133° 23’ 11.6”) Stn.2 (34° 28’ 45.7”, 133° 24’ 06.0”)

Fukuyama Stn.3 (34° 28’ 45.8”, 133° 24’ 31.6”) TheInlandSea Stn.4 (34° 27’ 34.9”, 133° 24’ 39.2”) Stn.5 (34° 26’ 41.7”, 133° 26’ 11.4”)

Fig. 1 - Location of monitoring stations in Fukuyama Port

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Laboratory-reared Polydora cornuta Sediment samples were collected using a 0.05 m2 Smith-McIntyre bottom sampler in the inner part of the port (Stn.1) on April 5, 1999. Samples were taken back to the laboratory, and the sediment containing the organisms’ mud tubes was transferred to a glass tank. Between April 1999 and June 1999 larvae were collected within 24 h of hatching and placed in a 20 L water tank. Tanks were then incubated at three water temperatures (18, 24, or 28°C). Seawater and benthic mud from the field site were used in each tank, and yeast (Nisshin Foods Inc. Tokyo, Japan) was added as a food source at a concentration of 5 mg/L twice a day. Larval settlement in each tank was sampled on a daily basis from day 2 to day 5. After day 5, the worms were treated with MgCl2, and the number of setigers and body length were determined, using a binocular microscope. In addition, the number of days from hatching to settlement on the bottom (pelagic duration) and to hatching of the second generation (generation time) was recorded.

Pelagic duration and generation time were determined from the experiment start date. Because individual settlement on the bottom was relatively easy to observe, mean pelagic duration was calculated to be the mean value of daily settlement. On the other hand, because determining the hatching status of each individual was difficult, generation time was fixed at the starting date of hatching (lowest value).

Stable isotope analysis Sediment samples from the inner part of the port were taken on March 4, 2008 (Stn.1, Fig. 1) using a Smith-McIntyre bottom sampler, and P. cornuta and Capitella sp. were picked out of the bottom sediments. Water samples were collected at 1.0 m above the bottom using a Van Dorn type water sampler, and particulate organic matter (POM) was immediately filtered through a glass-fiber filter (GS-25, Toyo Roshi Kaisha, Ltd., Tokyo, Japan). After collection, all samples were kept at -20°C. Seven randomly selected individuals of P. cornuta and Capitella sp. from the cryopreserved samples and five randomly selected samples of POM and bottom sediment were analyzed for stable isotope ratios of carbon and nitrogen. In the laboratory, seven randomly selected individuals of benthic worms (P. cornuta, Capitella sp.) were carefully extracted, oven dried at 60°C for 24 h or more, and ground with a mortar and pestle. Lipids were then removed using a chloroform : methanol (2 : 1 v/v) solution. The top clear layer was removed, and the residue was dried again for 24 h in a glass desiccator. Particulate organic matter (POM) and sediment were dried for 24 h. Particulate organic matter (POM) samples were removed from the filter after drying and were ground with a mortar and pestle. Samples were then acidified with 1 mol/L HCl overnight at room temperature to remove carbonates and were subsequently washed with Milli-Q water. Stable isotope ratios of carbon and nitrogen were measured using an ANCA-SL mass spectrometer (SerCon Ltd., U.K.).

Isotope ratios for  13 C and 15 N are expressed as deviations from the standard as defined by the following equations:

13C /12C 13 C /12 C  13C(‰)  sample sample PDB PDB 1000 13C /12C PDB PDB

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15 N /14N 15N /14 N  15N(‰)  sample sample N3 N2 1000 15 N /14N N2 N2

Belemnite (PDB) and atmospheric nitrogen were used as isotope standards for carbon and nitrogen, respectively. Differences in the isotope ratios ( 13 C and  15 N) among samples for POM and sediment as well as for P. c o r n ut a and Capitella sp. were analyzed using ANOVA and Tukey’s HSD test.

RESULTS AND DISCUSSION Environmental factors (Water and Sediment) Annual mean values for environmental factors (water and sediment) are presented in Table 1.

Water depth in the port increased from the interior (4.3 m) to the mouth (17.7 m). Monthly water temperatures and salinities at the five monitoring stations are shown in Figs. 2 and 3.

The water temperature increased in March and peaked by August, with the surface layer during this period having the highest temperatures. Little difference in temperature between water depths was apparent between September and March.

Salinity in all layers increased gradually from winter to spring and was lowest in the surface layer. The least difference in salinity between layers was observed at the mouth of the port (Stn.5). Oxygen saturation of the bottom layer was lower in the interior part of the port (Stns.1 and 2), ranging from 34.9% to 74.2%, with marked differences between the monitoring stations (Figs. 4 and 5). Periods of anoxia occurred at Stn.1 in particular, with 0 - 2.5% saturation from June to October. Oxygen-deficient water (oxygen saturation levels of 20% or less) was observed in the surface layer in the interior part of the port in April 1998, had extended to all layers in June, and gradually

Table 1 - Annual mean values of environmental factors in Fukuyama Port (April 1998 to March 1999)

Stn.1 Stn.2 Stn.3 Stn.4 Stn.5 Depth (m) 4.3 4.2 8.2 10.7 17.7 Water Salinity (PSU) Surface layer 25.7(20.7～31.4) 25.3（21.0～29.5） 25.7（19.5～29.9） 27.4（22.0～31.2） 28.1（25.6～31.2） Middle layer 28.1（25.6～31.8） 28.2（25.4～31.3） 29.1（27.2～31.0） 29.5（27.5～31.7） 29.7（26.4～31.8） Bottom layer 28.9（26.9～31.8） 29.2（27.1～31.9） 29.7（27.8～31.8） 30.0（28.0～32.1） 30.2（28.4～32.7） Oxygen Saturation (%) Surface layer 45.2（2.0～128.1） 77.4（25.2～158.2） 98.2（28.4～222.5） 110.6（63.1～205.1）102.2（86.6～140.8） Middle layer 27.9（1.2～92.4） 52.1（13.0～101.7） 62.9（20.3～98.9） 82.2（52.8～109.1） 86.5（58.6～112.4） Bottom layer 34.9（0.0～93.6） 52.8（3.8～100.0） 61.7（10.1～105.2） 64.3（7.4～104.8） 74.2（21.0～116.9） Sediment Temperature (℃） 19.2（11.5～27.4） 18.8（10.0～27.2） 18.6（10.5～27.2） 17.8（11.2～26.4） 17.7（10.5～26.5） Ignition loss (%) 12.8（11.1～14.3） 10.6（8.3～12.6） 8.0（6.2～9.4） 7.9（6.2～10.0） 9.6(6.4～10.7) COD (㎎/g-dry ) 39.8（29.2～46.3） 30.8（20.2～39.4） 11.0（6.8～17.4） 14.5（7.2～24.2） 16.4（10.1～26.4） AVS (㎎/g -dry ) 2.86（2.35～3.61） 1.95（1.28～3.52） 0.33（0.07～1.52） 0.60（0.19～1.01） 0.69（0.28～1.18） Total carbon (㎎/- dry ) 38.2（27.0～45.0） 32.0（19.0～44.0） 18.6（11.0～51.0） 15.4(5.5～29.0） 17.0（10.1～26.0） Mud content (%) 93 83 95 96 98

Numerals in parentheses indicate minimum and maximum values. Surface layer：0.5m under surface. Middle layer：layer of half depth. Bottom layer：0.5m above bottom.

- 262 - Journal of Water and Environment Technology, Vol. 9, No.3, 2011 reached the mouth of the port. In September, an increase in oxygen saturation in water at the mouth of the port was observed, and by October oxygen deficiency was confined to the interior part of the port (Stns.1, 2 and 3). By November oxygen saturation exceeded 60% throughout the port. In March 1999, oxygen deficiency was again observed in the middle layer in the innermost part of the port.

Fig. 2 - Monthly changes in water temperature in Fukuyama Port

Fig. 3 - Monthly changes in salinity in Fukuyama Port

Fig. 4 - Monthly changes in oxygen saturation in Fukuyama Port

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Fig. 5 - Monthly changes in the vertical distribution of dissolved oxygen saturation (%) in Fukuyama Port (April 1998 to March 1999)

The average mud content at each monitoring station varied from 83% to 98%. Ignition loss (IL) in the benthic samples varied from 7.9% to 12.8%. Chemical oxygen demand (COD) varied from 11.0 to 39.8 mg/g dry mud, and T-C varied from 15.4 to 8.2 mg/g dry mud. A high organic composition was apparent in the innermost part of the port (Stn.1). There were no obvious monthly changes in any of the parameters measured (Fig. 6). For the most part, COD, AVS, and T-C were highest throughout the year at Stns.1 and 2 and were lower elsewhere. While IL was higher at Stn.1 than anywhere else, there were no differences among any of the other stations.

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Our results indicate that there is decreased water and sediment quality in the benthic environment of the port. The synchrony of oxygen-deficient water with water temperature and salinity stratification from June to October suggests that oxygen deficiency in the port occurs as an annual cycle.

SEM of Polydora cornuta Figure 7 shows the SEM of P. cornuta. Scanning electron micrographs (SEM) confirmed the previous descriptions of the external morphology of P. cornuta (Rice and Simon, 1980; Blake and Maciolek, 1987; Blake and Arnofsky, 1999; Radashevsky and Hsieh, 2000; Sato-Okoshi, 2000).

15 50 IL (%) 40

10 COD (mg/gdw) 30

20 5

0 0 98'A M J J A S O N D 99'J F M 98'A M J J A S O N D 99'J F M

4 60

50 3 Stn.1

AVS (mg/gdw) 40

T-C (mg/gdw) Stn.2 2 30 Stn.3 20 Stn.4 1 10 Stn.5

0 0 98'A MJJASOND99'J F M 98'A M J J A S O N D 99'J F M

Fig. 6 - Monthly changes in IL, AVS, COD and T-C in sediment collected in Fukuyama Port

A B C D

E F G H

Fig. 7 - Scanning electron micrographs of P. cornuta. Benthic samples from Stn.1 in the inner part of Fukuyama Port were taken on February 1, 1999 and March 4, 2008. Scale bars: (A) 100 μm, (B) (E) (G) 50 μm, (C) (H) 20 μm, (D) (F) 5 μm. (A) Dorsal view of anterior end. (B, C) Major falcate spines and pennoned companion setae of segment 5. (D) Pennoned companion setae of segment 5. (E, F) Bidentate hooded hooks. (G) Dorsal view of posterior end, pygidium. (H) Palp

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Monthly distribution The monthly distribution of P. cornuta and Paraprionospio spp. (benthic and pelagic individuals) at the five monitoring stations in Fukuyama Port are shown in Fig. 8.

The total annual density of P. cornuta and Paraprionospio spp. (benthic and pelagic individuals) at the monitoring stations in Fukuyama Port are shown in Table 2.

Benthic individuals of P. cornuta were, for the most part, found only in the innermost part of the port (Stn.1). Their occurrence was restricted to winter and spring (January to May), with the greatest density in April (1,100 indv./m2). The total annual density of benthic P. cornuta at Stn.1 was approximately 2,400 indv./m2, and pelagic P. cornuta larvae were found throughout the year. High densities of the pelagic form were found

））） 2 2 2 1200 2000 400 1000 800 1500 300 indv./m indv./m indv./m

（ 600 （（ 1000 200

400 200 500 100 0 98’A 0 0 98.AMM 98’A JJ 98.AM 98’98.AA JJ M J M A J J JJ A J J SS Stn.5 A J A S A Stn.5 A OO Stn.4 S O SS Stn.5

NN O Stn.4 Stn.3 DD NN OO Stn.4 Stn.3 Stn.2 D NN 99’99.JJ D Stn.3 Stn.2 DD Stn.1 F 99’99.JJ Stn.2 MM F Stn.1 99’99.JJ M FF Stn.1 M MM

P. cornuta (Benthic) P. Patiens (Benthic) P. Cordifolia (Benthic)

））） 2 2 2 250000 60000

200000 50000

indv./m indv./m indv./m 40000 150000

（（（ 30000

100000 20000 50000 10000 0 0 98’A 98’A 98.A 98.AMM M MJ JJ JJ JJ J Stn.5 AA A A SS Stn.5 Stn.4

S S OO Stn.4

Stn.3 N O N Stn.3 O 99’ F D

Stn.2 D N Stn.2 N M 99.J Stn.1 J Stn.1 D D 99’ FF J99.J M

P. cornuta (Pelagic larvae) Paraprionospio spp. (Pelagic larvae)

Fig. 8 - Monthly distribution of P. cornuta and Paraprionospio spp (benthic and pelagic individuals) at the monitoring stations in Fukuyama Port

Table 2 - Density of benthic and pelagic larvae of P.cornuta, Paraprionospio spp. at five monitoring stations in Fukuyama Port 1998-1999 Stn.1 Stn.2 Stn.3 Stn.4 Stn.5 P. cornuta (Benthic) 2412 54 20 7 0 P. cornuta (Pelagic larvae) 329×104 596×103 348×103 245×102 5189 P. patiens (Benthic) 0 1259 1541 2099 5454 P. cordifolia (Benthic) 0 0 0 28 1259 Paraprionospio spp. (Pelagic larvae) 12193 68514 27568 51797 22143 indv./㎡

- 266 - Journal of Water and Environment Technology, Vol. 9, No.3, 2011 from winter to spring in the innermost and central parts of the port (200  104 indv./m2 at Stn.1 and 240  103 indv./m2 at Stn.2 in March) and were found from June (950  102 indv./m2) to September (130  102 indv./m2) at Stn.2 when water in the innermost part of the port was oxygen deficient. The total annual density was highest at Stn.1 (329  104 indv./m2), with 348  103 and 596  103 indv./m2 at Stns.2 and 3, respectively, and 5,189 to approximately 24,500 indv./m2 at the mouth of the port (Stns.4 and 5). The pelagic larvae of P. cornuta were found at a high density at Stn.1 when high oxygen saturations were characteristic of this station and then were found at Stns.2 and 3 from June to September when oxygen-deficient water dominated Stn.1. This suggests that pelagic larvae migrate to higher levels of oxygen-saturated water.

Two species of benthic individuals of the genus Paraprionospio was collected by us. Paraprionospio patiens was found at all sampling stations except the innermost part of the port (Stn.1) and was found throughout the year but at a low density in July. The occurrence of Paraprionospio cordifolia was, for the most part, limited to the mouth of the port (Stn.5) from autumn to winter (September to February). The total annual density of P. cordifolia was 12% of the density of P. patiens. Pelagic larvae of the genus Paraprionospio were found at all sampling stations but only during the 3 month period from July to September. The number of Paraprionospio larvae ranged from 1,700 to 5,500 indv./m2 at Stn.1; 3,200 to 51,000 indv./m2 at Stn.2; 4,300 to 13,000 indv./m2 at Stn.3; 480 to 46,000 indv./m2 at Stn.4; and 1,600 to 10,000 indv./m2 at Stn.5. The total annual density was approximately 12,000 to 68,000 indv./m2 at each monitoring point. The relationship between water temperature, DO (in each case the average value of three layers), and density of pelagic larvae of Paraprionospio spp. is shown in Fig. 9.

P. cornuta (Pelagic larvae)

200×104 50×104 10×104indv./m

Paraprionospio spp.(Pelagic larvae)

500×102 100×102 50×102indv./m2

Fig. 9 - Relationship between the mean values of water temperature and dissolved oxygen and the density of pelagic larvae of P. cornuta and Paraprionospio spp.

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Pelagic P. cornuta larvae were found at densities of approximately 10  104 to 200  104 indv./m2 throughout the water column with an average temperature ranging from 8.7 to 13.4ºC (range ca. 8 - 27ºC) and DO ranging from 2.6 to 10.0 mg/L (range ca. 0.2 - 12 mg/L). The density of pelagic Paraprionospio spp. larvae, on the other hand, was relatively low (1,000 to 50,000 indv./m2) and was limited to a narrow temperature range (26 - 27ºC) and DO concentration range (1.3 - 7.4 mg/L). The benthic phase of P. cornuta individuals was found only in the innermost part of the port (Stn.1). In contrast, pelagic larvae were found throughout the port, although in lesser numbers near the mouth. These differences in the distribution range during the two phases of the life cycle were due to the differences in response to increasing oxygen deficiency in water.

Life-cycle in the laboratory Laboratory-reared P. cornuta exhibited different growth characteristics at different water temperatures. At 9 days after hatching, the marine animals had approximately 12 setigers and body length of 0.76 mm at 18C, 17 setigers and body length of 1.39 mm at 24C, and 33 setigers and body length of 4.05 mm at 28C. From 17 to 19 days after hatching, setiger numbers were approximately 20, 31, and 46, respectively, and body length was approximately 2.21, 3.20, and 5.28 mm, respectively, at these three temperatures (Tables 3, 4 and 5; Figs. 10 and 11).

Table 3 - Relation between the number of setigers and body length of P. cornuta after hatching during rearing experiment at 18°C

1999.4.13～5.30(18℃） Days No.of Setigers Body Length (mm) n 1 3.0±0.2 0.249±0.01 25 2 3.7±0.6 0.267±0.03 33 3 4.1±0.5 0.301±0.03 34 4 6.0±1.0 0.377±0.05 11 5 8.1±1.2 0.460±0.06 21 6 9.6±1.0 0.519±0.06 26 7 10.8±0.9 0.578±0.07 10 8 11.9±1.1 0.659±0.14 31 9 11.8±1.2 0.761±0.11 15 11 13.0±1.0 0.970±0.20 4 14 15.0±2.1 1.364±0.33 7 18 20.0±3.2 2.211±0.37 5 23 29.2±5.2 4.670±1.74 5 28 35.4±6.9 6.266±2.83 12 33 47.4±5.3 9.617±1.94 7 38 58.3±8.9 13.146±2.20 8 43 65.0±8.4 14.671±4.14 8 48 70.8±9.2 13.953±2.57 6 Data are given as mean ± SD

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Table 4 - Relation between the number of setigers and body length of P. cornuta after hatching during rearing experiment at 24°C

1999.6.8～7.14(24℃） Days No.of Setigers Body Length (mm) n 1 3.0±0.2 0.236±0.02 45 2 4.1±08 0.319±0.04 28 3 6.4±1.3 0.385±0.05 13 4 10.8±1.6 0.544±0.11 31 5 13.6±1.4 0.771±0.13 77 6 14.6±0.9 0.984±0.16 21 7 16.1±1.8 1.106±0.26 87 9 17.1±1.8 1.392±0.30 24 11 22.8±5.4 2.234±0.94 63 14 28.6±6.9 3.019±1.22 52 17 30.6±6.2 3.195±0.96 74 20 32.7±5.7 4.246±0.91 28 23 36.2±5.1 4.242±1.10 38 26 34.3±5.4 4.125±0.91 28 29 37.9±6.3 4.764±0.82 23 32 36.5±6.7 4.104±0.95 31 37 39.3±5.4 4.709±1.02 22 Data are given as mean ± SD

Table 5 - Relation between the number of setigers and body length of P. cornuta after hatching during rearing experiment at 28°C

1999.7.1～7.26(28℃） Days No.of Setigers Body Length (mm) n 1 3.7±0.5 0.258±0.03 12 2 6.8±1.5 0.389±0.07 14 3 10.9±1.6 0.614±0.10 23 4 13.7±1.2 1.000±0.19 29 5 17.2±1.6 1.466±0.29 40 7 28.0±3.3 2.981±0.60 21 9 33.7±4.7 4.052±0.93 29 12 40.7±5.1 5.525±0.94 28 14 43.2±6.5 5.400±1.04 22 19 45.6±6.6 5.278±1.19 20 26 50.2±6.3 6.022±1.27 55 Data are given as mean ± SD

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20 ○ ℃ 18 18 米 24℃ 16 ● 28℃ (mm)

12 length

8 Body 6

0 0 1020304050 Days

Fig. 10 - Changes in the setigers number of P. cornuta during experimental rearing at different temperatures. F2 Hatching ( ), Settlement ( );(mean ± SD)

80 ○ 18℃ 米 24℃ 70 ● 28℃

60 setigers

of 50

40 No.

0 0 1020304050 Days

Fig. 11 - Changes in the body length of P. cornuta during experimental rearing at different temperatures. F2 Hatching ( ), Settlemen t( ); (mean ± SD)

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Under all three temperature regimes (18, 24, and 28ºC), larvae were pelagic immediately after hatching. At 18ºC, the larvae began to settle at the bottom (pelagic duration) 8 days after hatching, and all had settled by 11 days. Tubes with attached egg sacs were found after 38 days, and the second generation of larvae (F2) began to hatch (generation time) after 41 days. The life cycle was thus completed in approximately 40 days under laboratory conditions at 18ºC. At 24ºC, the mean pelagic duration was 9.5 days and the shortest generation time was 22 days. At 28ºC, the mean pelagic duration was 6 days and the shortest generation time was 13 days. The mean pelagic duration and the generation time (lowest value) for the three temperature conditions are shown in Fig. 12.

During the benthic phase of P. cornuta (January to May), the temperature of the bottom sediment in the port ranged from 11.5 to 18.2ºC. Using the regression equation, the time to hatching of the second generation was estimated to be 59 and 40 days at 11.5 and 18.2ºC, respectively, and the time to settlement was estimated to be 13 and 9 days, respectively. At 10 days after hatching, growth rate and the number of setigers were approximately four times greater at 18ºC, seven (number of setigers) to nine (body length) times greater at 24ºC, and 11 (number of setigers) to 15 (body length) times greater at 28ºC. Despite the faster growth rate at higher temperatures, the life cycle at all temperatures was completed within a relatively short period of 13 to 41 days. The results of our study are in agreement with those of Anger et al., (1986) who reported that larval development of three species of Polydora is affected by temperature. Our results are also in agreement with the reported generation times for this species of 28 days or less at 22 ± 2ºC in the laboratory (Environment Canada, 2001). The life cycle of P. cornuta in Fukuyama Port was estimated to be 40 to 59 days at 11.5 and 18.2ºC, respectively. Therefore, roughly three generations can be completed during the approximately 5 month benthic phase, suggesting that P. cornuta has an extremely high fecundity. Our results show that P. cornuta is short-lived, has high fecundity, has the ability to disperse over a wide area, has good evasive ability, and is capable of rapidly finding new unoccupied habitat. However, if the habitat becomes unsuitable, the

y = -2.8289x + 91.342 Ｄａｙｓ R² = 0.9922 40

20 y = -0.5526x + 19.395 R² = 0.9983

0 10 15 20 25 30 Temperature (℃)

Fig. 12 - Relationship between water temperature and generation time under laboratory conditions. F2 Hatching (▲), Settlement (●)

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Feeding type Isotope values of the sediment, POM, P. c o r n ut a and Capitella sp. are presented in Table 6.

Results from ANOVA and Tukey’s HSD test indicated that the  13 C and  15 N values for the two food sources (POM and sediment) and for P. cornuta and Capitella sp. were significantly different (both at P < 0.05) from each other (Fig. 13). The  13 C value for P. cornuta was very similar to those for POM and sediment, whereas the  15 N value for this species was lower than that for POM.

The carbon isotope ratios for P. cornuta, Capitella sp., POM, and sediment were in the range of the average value for oceanic phytoplankton (Minagawa and Yoshioka ,2006;

Table 6 - The 13 C and 15 N values of P. cornuta, Capitella sp. and two food sources (POM and sediment) in Fukuyama Port. Data are given as mean ± SD

δ13C(‰） δ15N(‰） Samples n Range Mean±SD Range Mean±SD P. cornuta -19.7 to -17.1 -18.2±0.7 20.5 to 23.1 21.5±0.8 7 Capitella sp. -21.7 to -20.3 -20.8±0.6 9.9 to 12.0 10.7±0.8 7 POM -22.6 to -20.4 -21.1±0.9 22.7 to 23.2 22.9±0.2 5 Sediment -25.7 to -23.5 -24.7±0.8 6.3 to 8.2 7.2±0.7 5

25 N (‰）N 15 20 δ

5 -30 -25 -20 -15 -10 δ 13C(‰）

Fig. 13 - Relationship between  13 C and 15 N values of P. cornuta (■), Capitella sp. (□) and two food sources (POM (▲), Sediment (△)) in Fukuyama Port. Data are given as mean ± SD.

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Santschi et al.,1995). However, the nitrogen stable isotope ratio had a wider range. Correlation plots indicated two distinct groups: P. cornuta with POM and Capitella sp. with sediment. An animal is on the average enriched in  13 C by about 1‰ relative to the diet (DeNiro and Epstein, 1978; Fry and Sherr, 1984). Nitrogen isotope enrichment of +3.4‰ occurs independently of habitat, from nitrogen excreted and growth rate (DeNiro and Epstein, 1981; Minagawa and Wada, 1984). Assuming two kinds of food resources (i.e., POM, sediment), the ratio of the isotope of the animal (in this case P. cornuta) is: δs = f1 × δ1 + (1 - f1) × δ2. The availability of a food resource (1) is f1 = (δs - δ2) / (δ1 - δ2) (Minagawa and Yoshioka, 2006)) This equation was used to derive a ratio of 9 : 1 (POM to sediment) from our data.

Spionids have been reported to have a variety of feeding modes (Dauer et al., 1981; Hayashi, 1998). In general, these polychaetes feed mainly on bottom sediment rather than on suspended material, and the latter appears to be taken in as a supplement. Spionids usually burrow vertically into the sediment and live in a burrow with the head pointing upward. The palps extend out of the burrow into the water and are whipped around. Suspended particles may be captured in this way, depending on the conditions, but the animal lays the palps on the surface of the sediment and feeds by repeatedly sweeping the surface of the sediment to capture food particles. Our results, however, indicate that the feeding method of P. cornuta in Fukuyama Port is suspension feeding.

CONCLUSIONS Polydora cornuta was found living in tubes in an uneven distribution pattern in Fukuyama Port. A morphological characteristic of this species is the presence of large modified setae on the fifth setiger. At the start of its life history, P. cornuta larvae are pelagic but subsequently settle at the bottom and enter a benthic phase, which lasts approximately 5 months, from January to May. Benthic individuals of P. cornuta occurred disproportionately only in the innermost part of the port, whereas pelagic larvae were found across the entire port, although in smaller numbers near the mouth. The pelagic phase of P. cornuta was found throughout the year, in contrast to the benthic phase, which showed intermittent temporal distributions.

The laboratory rearing experiment showed that the life cycle of P. cornuta was completed in about 40 to 59 days. Therefore, about three generations can be completed in the approximately 5 month benthic phase, suggesting that P. cornuta has a high fecundity.

Stable carbon and nitrogen isotope analysis demonstrated that P. cornuta is a suspension feeder that ingests POM.

Our results showed that P. cornuta in Fukuyama Port has a high reproductive capacity and is capable of rapidly finding new, unoccupied places to inhabit, although numbers decline drastically if the habitat becomes unsuitable. These patterns are partly characteristic of opportunism, indicating that P. cornuta is most likely an opportunistic species.

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