The settlement cues of an articulated coralline alga Marginisporum crassissima for the Japanese top shell Turbo cornutus.
KEY WORDS: articulated coralline algae, biofilms, Marginisporum crassissima, settlement, Turbo cornutus
For marine invertebrates that have planktonic larval periods, environmental factors that function at a variety of spatial and temporal scales during the pre and postsettlement stages determine the successful recruitment of juveniles (Underwood & Keough 2001). Because of low tolerances to various negative factors, such as predation, starvation, and physical disturbance, mortality during the juvenile phase shortly after settlement can be extremely high (Keough & Downes 1982). However, juvenile survivorship varies considerably among habitats with their own unique biological/physiological environments (Naylor & McShane 1997, Herbert & Hawkins 2006, Walker 2007). Settlement on optimal substrata by larvae enhances their survival by enabling the settled individuals to obtain adequate food and shelter from predators and physical disturbances. The settlement of planktonic larvae is often influenced by chemical, biological, and physical factors that are associated with environments suitable for settled juveniles. Therefore, the identification of factors affecting larval settlement is important for our understanding of settlement success and population levels at recruitment.
The Japanese top shell, Turbo cornutus, is one of the most important fishery resources in the coastal waters of Japan. Despite its commercial importance, the ecological study on the top shell is limited, especially during the early life stages. The top shell species has planktonic larval stages of several days before transforming into benthic stages (Ai 1965). The larvae are lecithotrophic and carry a yolk supply derived from the egg. Newly settled top shell juveniles have been found only in algal turfs of articulated coralline algae (ACA) (Sasaki 2003). Marginisporum crassissima and four other ACA species, common species in the Pacific coast of Japan, strongly induced the larval settlement of the top shell in the laboratory experiments (Hayakawa et al. 2007, Hayakawa et al. 2008). The top shell juvenile's distribution specific to ACA turfs is primarily attributed to the selective larval settlement, which is induced by the ACA species (Hayakawa et al. 2007). However, it remains unclear which specific features of the ACA species induce the settlement of top shell larvae.
Selective larval settlement has been intensively studied in abalone species belonging to Vetigastropoda with top shell species. Settlement is induced by cues from a wide range of sources, including crustose coralline algae (CCA), films of diatoms, some artificial inducers (reviewed by Roberts 2001), and ACA species (Huggett et al. 2005, Williams et al. 2008) although abalone seem to be quite species-specific with respect to their agent of induction. CCA species, which belong to the Corallinaceae along with ACA, are inducers of abalone larval settlement and their settlement cues have been well studied. The species, morphology, developmental phase, and associated biofilms consisting of benthic diatoms and bacteria of CCA affect the settlement of abalone larvae (reviewed by Roberts 2001). Chemical substances, such as compounds associated with phycobiliproteins, are believed to be the most likely settlement cues offered by CCA, but the actual settlement cues remain unknown, even for abalone larvae.
The biofilms that grow on macroalgal surface are believed to affect the settlement of many invertebrate larvae (Johnson & Sutton 1994, Negri et al. 2001). If these biofilms, which mainly consist of benthic diatoms and bacteria, play a key role in settlement induction of top shell larvae, the spatial or temporal changes of the biofilms on ACA may influence the choice of settlement substrata and the distribution of the postlarvae. The role of ACA-associate biofilms as settlement inducers must be studied to understand selective settlement by larval top shell on ACA species.
In this study, we conducted a series of laboratory experiments to identify the sources of cues for larval settlement of top shell T. cornutus on the ACA species M. crassissima. The influences of ACA morphology, ACA-derived chemicals, and ACA-associate biofilms (diatoms and bacteria) on larval settlement were experimentally investigated.
MATERIAL AND METHODS
Culture of Top Shell Larvae
Larval top shell were hatched and reared at the Nagasaki Prefectural Institute of Fisheries (NPIF), Nagasaki, Japan, during June 2006 and June 2007. The larvae were derived from a brood stock consisting of more than 50 individuals. Gametes from all animals were mixed for fertilization to produce a single batch of larvae. Fertilized eggs were gently washed with filtered seawater to remove excessive sperm and raised to the veliger stage at 24 [+ or -] 1[degrees]C in 1-[micro]m filtered seawater (32.2 [per thousand] to 34.6 [per thousand]). Larvae were held at concentrations of 1 to 2 larvae per mL in a 1,000-L water tank with continuous flow of filtered seawater (4.8-L per minute); three batches of top shell larvae from the same brood stock were produced in this way (two batches in 2006 and one in 2007). The spawning dates of each batch were June 13, 2006 (the first batch in 2006), June 19, 2006 (the second batch in 2006) and June 15, 2007. Competent larvae (2-3 days old) only from the same batch were used in each settlement experiment. As early development and behaviors in larval stages are very similar between this top shell species and abalone species (Ai 1965), competence of settlement was judged by the appearance of the tubule of the cephalic tentacle and crawling behavior on the substrata, following the definition for abalone larvae (Hahn 1989).
Design of Settlement Experiments
Whole live algae of the ACA species Marginisporum crassissima were collected by SCUBA from the subtidal zone (water depth 1-2 m, water temperature 20[degrees]C to 23[degrees]C and salinity 32.8 [per thousand] to 34.4 [per thousand] in June 2006 and 2007) on the east coast of Sagami Bay, Kanagawa, Japan. Specimens were transported to NPIF immediately after collection (within 6 h) and maintained in running ambient seawater (20[degrees]C to 24[degrees]C, 32.2 [per thousand] to 34.6 [per thousand]) in large plastic tanks for less than five days before use. Illumination was done only by natural sunlight from roof light windows. Algal fragments (2 cm long) were prepared by cutting the thalli immediately before the experiments. All fragments used in an experiment were prepared from separate individuals to ensure independence. The algal fragments were gently washed in filtered seawater (FSW: 0.45-[micro]m HA; Millipore, Billerica, MA) and held in FSW at 24.5[degrees]C for 1 h before being transferred to wells of six-well plates (Corning 3516).
For each experiment, approximately 50 competent larvae were introduced into a well of a six-well plate containing a settlement substratum and 10 mL of FSW. The well plates were wrapped in clear cellophane film and kept at 24.5[degrees]C, under a 12L:12D light regimen. Each experimental treatment contained six replicates. Six wells containing the ACA fragments that were not processed (see methods for each experiment) were used as positive controls and another six wells that lacked substrata were used as negative controls for each experiment.
Larval behavior was observed under a dissecting microscope. We use the term "settled individual" for individuals that attached and successfully metamorphosed. Metamorphosed larvae lost their velum and began formation of the juvenile , shell. The number of settled individuals was counted after 24 and/or 48 h. The top shell juvenile does not show feeding activities for several days after settlement (Ai 1965), no additional feeding was done in each experiment.
Settlement in Response to ACA Morphology
The larval settlement response to the ACA (M. crassissima) morphology was tested, using larvae from the first batch in 2006. Algal morphology refers to the algal macrostructure, which can be distinguished under a dissecting microscope, and does not include the microstructure of the algal surface in this study. In this experiment, dead ACA fragments were prepared by soaking live ACA fragments in a 90% ethanol solution for 72 h followed by heating in an oven at 40[degrees]C for 48 h. This treatment was believed to remove any other possible cues for larval settlement, such as chemicals and surface-associated diatoms and bacteria, without modifying the morphology of the ACA fragments. The dead ACA fragments were gently rinsed with FSW and held in FSW for I h before being placed into the experimental wells. In addition, a fruticose artificial alga ("new aqua plant," Sudo Co. Ltd, Nagoya Japan) was used to test the influence of substratum morphology on larval settlement. The artificial algae were cut to create fragments that were the same size as the ACA fragments, i.e., 2 cm in length. The percentages of individuals that settled on fragments of live ACA (positive control), dead ACA, artificial algae, and the negative control (lacking a substratum) were measured 48 h after the larvae were introduced.
Settlement in Response to Diatoms Growing on ACA Surfaces
The influence of epiphytic diatoms growing on the ACA on top shell larval settlement was assessed by modifying diatom density, using larvae from the first batch in 2006. This experiment included substrates created according to the following five treatments.
1. Inhibiting diatom growth treatment: ACA fragments were held in an aerated 2.5 mg/L germanium dioxide solution for 3 wk. The germanium dioxide solution was changed every 3-4 days, and the fragments were maintained at 24.5[degrees]C with a 12L:12D light:dark cycle. Germanium dioxide inhibits diatom growth, but does not affect the growth of any other algae (Chapman 1973).
2. Accelerating diatom growth treatment: The ACA fragments were soaked in modified Jorgensen medium (Jorgensen 1962) containing an addition of 0.05 [micro]g/L of vitamin [B.sub.12]. The ACA fragments were maintained under the same conditions and for the same duration as in the Inhibiting diatom growth treatment. The modified Jorgensen medium is a culture solution that accelerates the growth of diatoms (Kawamura & Takami 1995).
3. Control treatment for soaking: Algal pieces of M. crassissima were held in FSW for 3 wk; the FSW was changed every 3-4 days and the ACA fragments were kept under the same conditions and for the same duration as in the above treatments.
4. Positive control: Fragments of the unprocessed ACA.
5. Negative control: Wells without algal fragments.
The percentage of individuals that settled in each treatment was measured at 24 and 48 h after the addition of larvae. To determine the success of the above treatments in inhibiting or promoting the growth of diatoms on the surface of M. crassissima, the density of epiphytic diatoms was measured under a scanning electron microscope (SEM). Four pieces of algae from each treatment were fixed in a 4% seawater-formalin solution, air-dried, and sputter coated for the SEM observations (HITACHI-4500; Hitachi High-Technologies, Tokyo, Japan). On each sample, the number of diatoms was counted for 15 haphazardly selected sections at x15,000 magnification.
Settlement in Response to Bacteria Growing on ACA Surfaces
To determine if settlement cues were associated with bacteria growing on the algal surface, we presented ACA fragments that had been processed with antibiotics to the top shell larvae. Larvae were from the second batch in 2006. This experiment included substrates created according to the following 4 treatments.
1. Antibiotic treatment: Algal fragments were soaked in a 10% povidone iodine solution that was diluted in FSW for 10 min, rinsed in FSW, and held in an antibiotic solution containing streptomycin (20 mg/L), penicillin (10 mg/L), neomycin (2 mg/L), and kanamycin (10 mg/L) for 48 h at 24.5[degrees]C with a 12L:12D light:dark cycle (modified from Huggett et al. 2005).
2. Control treatment for soaking: Algal pieces were held in FSW for 48 h under the same conditions in the Antibiotic treatment.
3. Positive control: Fragments of the unprocessed ACA.
4. Negative control: Wells without algal fragments.
The percentage of individuals that settled in each treatment was measured 24 and 48 h after larvae were placed. To assess the effectiveness of the above treatments in reducing the density of bacteria on algal surfaces, the density of bacteria was determined under SEM both before and after the experiment. The bacteria were enumerated by the same method as in the diatom experiment, but at a magnification of x25,000.
Settlement in Response to Algal Extracts
To examine the effects of natural ACA products on the settlement of top shell larvae, two types of extracts were tested using the following 5 treatments. Larvae used in this experiment were from the batch in 2007.
1. Extracts by crushing treatment: Whole plants of M. crassissima were cut into pieces with scissors. Algal fragments were soaked in a 10% povidone iodine solution for 10 min and rinsed in FSW. The algal pieces were crushed and homogenized in 50 mM Tris-HCl pH 7.5 (0.33 g wet wt. algae/mL) and stirred for 20 min on ice. After filtration with gauze, homogenates were centrifuged at 3,000 rpm for 10 min. The supernatant created by this procedure (crude extract) was kept on ice before use. Piece of filter paper (2 cm x 2 cm) were soaked in the crude extracts and used as settlement substrata for top shell larvae.
2. Extracts by soaking treatment: ACA fragments, which were prepared in the same way as the algal fragments for the positive controls in each experiment, were soaked in FSW (0.025 g wet wt. algae/mL) for 48 h after sterilization with a 10% povidone iodine solution as in the Extracts by crushing treatment. The ratio of algal weight to FSW volume was almost equivalent to the positive control below. The ACA-soaked seawater was filtered through a filter paper (Whatman, No. 2) and added to wells instead of FSW.
3. Control treatment for the medium: Pieces of filter paper (2 cm x 2 cm) were soaked in Tris-HCl buffer that had been kept on ice. These filter papers containing the extraction medium were then presented to larvae.
4. Positive control: Fragments of the unprocessed ACA.
5. Negative control: Wells without substrata.
The percentage of individuals that settled in each treatment was measured at 24 and 48 h after the addition of larvae.
All statistical analyses were performed using the R-2.6.2 (R Development Core Team 2008) computer package. The percentages of settled individuals were transformed using the arcsine transformation to achieve homogeneity, after which statistical tests were performed. We used one-way ANOVA with the Tukey-Kramer HSD test to examine the differences in the settlement percentages among the experimental treatments when the data in the experiments were homogeneous in variance (Levene test, P > 0.05). The Steel-Dwass test (Dwass 1960), a nonparametric method for multiple comparison, was used to evaluate the differences in the percentages of settlement among the substrata, when homogeneity of variances was not confirmed.
The densities of bacteria and diatoms on ACA surface were compared between before, and after the experiments; and significant differences were tested by the Mann-Whitney U-test. Significance level for all tests was P < 0.05.
At the start of all experiments, top shell larvae showed vertical swimming behaviors and repeatedly contacted the surface of the experimental substrata or the bottoms of the wells. This was believed to be the exploratory behavior for settlement, similar to that reported for abalone larvae (Seki & Kan-no 1981). Settled individuals were observed crawling on algal surfaces or on well bottoms. Whereas larvae in the negative control wells without the algal fragments, continued swimming for at least 48 h, larvae in the positive controls settled on the ACA fragments at high percentages (>80%) in all experiments. These results indicate that larvae and settled juveniles were healthy and competent. Almost no larvae settled without an inducer during the experimental period.
Settlement in Response to ACA Morphology
The mean percentage of larvae that settled on dead ACA fragments (10.5 %) was significantly lower (P < 0.05) than on live ACA fragments (94.0%), but significantly higher (P < 0.05) than on the artificial algae or in the negative control (0.3% and 0.0% respectively; Fig. 1). Few larvae settled on the artificial plastic algae, and no significant difference was detected in the mean settlement percentage from the negative control (P > 0.05).
Settlement in Response to Diatoms Growing on ACA Surfaces
Germanium dioxide significantly reduced the diatom density (3,000 [+ or -] 2,000 cells / [cm.sup.2]) on the ACA fragments compared with densities before the soaking process (17,000 [+ or -] 2,000 cells/[cm.sup.2]; P < 0.05). In contrast, soaking in Jorgensen medium significantly increased the diatom density (59,000 [+ or -] 27,000 cells/[cm.sup.2]; P < 0.05). The density of diatoms on the ACA fragments did not change significantly in the control treatment for soaking (15,000 [+ or -] 6,000 cells/[cm.sup.2]; P > 0.05).
[FIGURE 1 OMITTED]
The percentages of larval settlement on ACA fragments in the germanium dioxide solution treatment, the Jorgensen medium treatment, the FSW treatment and in the positive control were all high (78.6% to 91.0%), and no significant differences were observed in larval settlement percentages among treatments, except for the negative control both after 24 h and 48 h (P > 0.05; Fig. 2), despite large differences in diatom densities among the treatments.
Settlement in Response to Bacteria Growing on A CA Surfaces
The antibiotic treatment significantly decreased the density of bacteria on the algal surface ([0.5 [+ or -] 0.3] x [10.sup.7] cells/[cm.sup.2]) compared with that before soaking ([2.2 [+ or -] 0.5] x [10.sup.7] cells/[cm.sup.2]; P < 0.05).
Despite the large reduction in bacterial density, the mean larval settlement percentage in the antibiotic treatment was nearly equal to the positive control (94.7% and 93.1%, respectively; Fig. 3). Larvae settled on the algal fragments at high percentages (>90%) in all treatments, with the exception of the negative control. No significant differences were detected among treatments (P > 0.05).
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
Settlement in Response to Algal Extracts
A significantly higher percentage (32.0%) of larvae settled in response to the crude extract with Tris-HCl buffer (extracts by crushing), compared with the negative control (0.4%), after 48 h (P < 0.05), although no significant difference was found between the two treatments after only 24 h (P > 0.05; Fig. 4). The settlement percentages in the extracts by crushing treatment were significantly lower than in the positive control, after 24 h and 48 h. Most larvae neither attached nor metamorphosed in the extracts by soaking treatment, in the control treatment for the medium, or in the negative control. Settlement percentages were not significantly different among those three treatments (P > 0.05).
Settlement of top shell larvae was strongly induced by live fragments of the ACA M. crassissima in all experiments in this study. Whereas the experiments included three different batches of larvae, and the ACA were collected in multiple years, the high percentages of settlement (>80%) on the intact ACA (positive controls) were stable throughout the experiments. In our previous study (Hayakawa et al. 2007), settlement of top shell larvae that were derived from two different batches was also induced at high percentages (>60%) by the fragments of M. crassissima, in August and September 2005. These results indicate that the settlement induction by M. crassissima is consistently very strong, regardless of differences in both larval batches and the season in which ACA fragments are collected. The spawning season of the top shell is long (late June to early October), and several cohorts occur in each year (Yamada 1998). Stability in the strength of settlement induction by ACA species during the long spawning season is important for the settlement of top shell larvae on coralline algal turfs.
[FIGURE 4 OMITTED]
The strong induction of larval settlement did not change even when the density of diatoms on the ACA surface was artificially modified in the experiments. In previous studies, benthic diatom films induced the settlement of diverse marine invertebrates including a limpet (Chiu et al. 2007) and abalone species (Kawamura & Kikuchi 1992, Gordon et al. 2004, Roberts et al. 2007). For abalone larvae, however, the strength of the settlement induction by diatom films varied considerably with the species, density, age and culture condition of the diatoms (Roberts 2001, Roberts et al. 2007). Kawamura and Kikuchi (1992) found a correlation between the larval settlement percentage of the abalone Haliotis discus hannai and diatom density. However, the density of benthic diatoms on the ACA was not related to the settlement proportion of top shell larvae in our study, although the species composition of benthic diatoms was not considered in the experiments. From observations of algal surfaces under the SEM, the dominant diatom species on the ACA fragments used in these experiments were Cocconeis spp. and Amphora spp., which were listed as relatively strong inducers for abalone larval settlement (Kawamura & Kikuchi 1992, Roberts et al. 2007). Larger colonial species also appeared on the ACA fragments in the accelerating diatom growth treatment. Diatoms on the ACA M. crassissima seem to have small effect on larval settlement of the top shell T. cornutus.
Bacteria are ubiquitous in diatom films and on algal surfaces, but the roles they play in settlement induction for marine invertebrates have not been thoroughly studied. Bacteria located on algal surfaces induced larval settlement of a polychaete (Kirchman et al. 1982), a starfish (Johnson & Sutton 1994), a sea urchin (Huggett et al. 2006), and a mussel (Bao et al. 2007). In the induction of larval settlement of a limpet, the presence of both bacteria and diatoms in biofilms was important (Chiu et al. 2007). For some abalone species, bacteria induced larval settlement, but settlement proceeded slowly (Roberts 2001). The results of our study indicate that the effects of bacteria on the larval settlement of top shell onto ACA are negligible, even if bacterial induction of larval settlement requires longer periods. The effects of both ACA surface-associated bacteria and diatoms do not seem important in the induction of larval settlement of the top shell T. cornutus. The principle cues for settlement were found to be derived from the ACA itself. Huggett et al. (2005) also reported that the densities of bacteria and diatoms on ACA surfaces did not affect larval settlement of the abalone Haliotis rubra on the ACA Corallina officinalis.
Roberts et al. (2004) reported that larvae of the abalone Haliotis iris selectively settled on CCA species with thin encrusting morphologies but not on those with thick encrusting or warty growth forms. The settlement preferences of planktonic larvae onto substrata with specific morphologies were believed to influence the spatial distribution of juveniles in some marine invertebrates such as a scallop (Harvey et al. 1993) and a barnacle (Herbert & Hawkins 2006). For the top shell, the passive accumulation of larvae by small-scale swirling currents created by the complex morphology of ACA species is assumed to determine the specific distribution of the juveniles (Uchiba et al. 1982, Yamazaki & Ishiwata 1987). In the present study, however, top shell larvae settled on dead ACA fragments and the plastic artificial algae at significantly lower percentages than on live ACA fragments with similar morphologies. These results indicate that the growth form of the ACA itself is not a major factor inducing larval settlement of the top shell. The selective settlement of top shell larvae thus does not result only from the trapping of larvae by the complex external morphology of ACA species.
Chemical substances have been identified as settlement cues for marine invertebrate larvae, such as abalone (Morse & Morse 1984), a barnacle (Larman et al. 1982) and a sea urchin (Swanson et al. 2006). Chemical compounds produced by ACA species have also been assumed to induce the settlement of the top shell larvae (Uchiba et al. 1982, Hayakawa et al. 2007), but no evidence has yet been reported. In our experiments, top shell larvae were induced to settle by crude extracts from the ACA species, although the strength of induction was weaker than the ACA fragments. In contrast, the filtered seawater in which the algal fragments were immersed induced neither larval attachment nor metamorphosis. These results indicate that chemical compounds could be the main settlement cue of the ACA M. crassissima for top shell larvae, and that the chemicals are not water-soluble and are therefore unlikely to be widely distributed in the water. Thus, larval contact with ACA surfaces may be essential to accept chemicals inducing settlement, which is possibly available only on and/or near the algal surface.
ACA species have calcified thalli and strongly adhere to rocks with their basal crusts; thus they are highly resistant to various physical and biological disturbances, such as strong water currents, waves, and grazing pressures (Littler & Kauker 1984, Stewart 1989). Therefore, ACA turfs usually dominate in areas with strong physical and biological disturbances where other algal species cannot form dense communities. If top shell larvae are induced to settle by specific water-soluble chemical substances from ACA, which would easily diffuse in the water column from the algal surfaces, larvae could not identify the direction in which ACA are located under strong water currents. Also, the weak swimming abilities of larvae can make it difficult for them to approach suitable settlement substrata by themselves in flowing water. We suggest a hypothesis that the capture of top shell larvae from water columns around ACA turfs, which are composed of ACA species with complex morphologies, increases the opportunities for larvae to come in contact with the algal surfaces where they can detect the settlement cues.
If the main settlement cues are chemical compounds contained in the ACA, the large reduction in the settlement proportion on dead ACA fragments in the algal growth-form experiment could be explained by the loss and/or deterioration of specific chemicals by soaking in 90% ethanol and heating. Larvae settled on filter paper containing algal extracts at a significantly higher percentage than in the control and Tris-HCl buffer treatment, but at a much lower proportion than on the ACA fragments. Whereas most larvae settled on the algal fragments within 24 h, the larval settlement percentage on the algal extracts increased between 24 and 48 h. Some potential reasons for this weaker and slower induction by the algal extracts are a shortage in the quantity of the specific chemicals in the ACA extract, the loss and/or deactivation of specific chemicals by the extraction process, and any negative effects of the filter paper.
In conclusion, the settlement induction of top shell larvae by the ACA M. crassissima is primarily related to the alga itself. The main settlement cue seems to be chemical compounds contained in the algal body. Coralline algal turfs composed of ACA species have been considered favorable nurseries for juveniles of top shell species in previous studies (Yamazaki & Ishiwata 1987, Worthington & Fairweather 1989). The present study strongly supports the close relationships between ACA species and top shells in their early life stages. Further studies, especially ones that identify the chemical materials inducing larval settlement, ate needed to clarify the mechanism of the strong settlement response to ACA species by top shell larvae. The ecological implications of the selective larval settlement on ACA surfaces should also be considered to understand the early life ecology of the top shell Turbo cornutus.
The authors thank the staff of the Nagasaki Prefectural Institute of Fisheries for their cooperation in rearing the top shell larvae.
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JUN HAYAKAWA, (1), * TOMOHIKO KAWAMURA, (1) SATOSHI OHASHI, (2) TOYOMITU HORII (3) AND YOSHIRO WATANABE (1)
(1) Ocean Research Institute, The University of Tokyo, 1-15-1 Minamidai, Nakano, Tokyo 164-8639, Japan; (2) Nagasaki Prefectural Institute of Fisheries, Taira, Nagasaki, Nagasaki 1551-4, Japan; (3) National Fisheries Research Institute, Fisheries Research Agency, Nagai, Yokosuka, Kanagawa 238-0316, Japan
* Corresponding author. E-mail: email@example.com
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|Author:||Hayakawa, Jun; Kawamura, Tomohiko; Ohashi, Satoshi; Horii, Toyomitu; Watanabe, Yoshiro|
|Publication:||Journal of Shellfish Research|
|Date:||Aug 1, 2009|
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