Abstract

We experimentally examined the influence of suspended kaolinite particles on the hatching rate of eggs and on the development and growth of larvae of the Manila clam, Ruditapes philippinarum. 1) The relative hatching rate of fertilized eggs decreased linearly with increasing concentrations of suspended particles in seawater, from 100% in filtered seawater with no suspended particles to about 17% at suspended particle concentrations of
200 mg L⁻¹. 2) The proportion of trochophore larvae that developed normally, decreased with increasing concentration of suspended particles to about 80% at 150 mg L⁻¹. 3) The shell length of veliger larvae increased with elapsed time in filtered seawater and in suspended particle concentrations of 10 mg L⁻¹, reaching 196–203 µm at 12 days (17 days after fertilization). However, in particle concentrations of 30 and 50 mg L⁻¹, the larvae did not grow and the shell length did not increase. The experiments indicated that the development and growth of Manila clam larvae are inhibited when suspended particle concentrations reach 30 mg L⁻¹ or more. No significant correlations were found between the abundance of Manila clam larvae and the concentrations of suspended solids (SS) or particulate inorganic matter (PIM) found in Tokyo Bay. However, our experimental results strongly suggest that high levels of suspended particles in seawater, are likely to have an impact on the early life history of this species.

Keywords Manila clam, Ruditapes philippinarum, suspended matter, hatching rate, larva, fertilized egg, trochophore, shell length, veliger.

Author and Article Information

Author info
1 Tokyo University of Marine Science and Technology, Tokyo, Japan.
2 Tokyo Bay Fisheries Laboratory, Chiba Prefectural Fisheries Research Center, Chiba, Japan.
3 Fukui Prefectural University, Obama, Fukui, Japan.

RecievedApr 12 2014　　AcceptedAug 15 2014　　PublishedAug 27 2014

CitationArakawa H, Akatsuka T, Nemoto M, Matsumoto A, Kobayashi Y, Seto M (2014) The influence of suspended particles on larval development in the Manila clam Ruditapes philippinarum. Science Postprint 1(1): e00028. doi: 10.14340/spp.2014.08A0002

Copyright©2014 The Authors. Science Postprint is published by General Healthcare Inc. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 2.1 Japan (CC BY-NC-ND 2.1 JP) License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

FundingThis study was funded by MEXT Revitalization Project for the creation of Fisheries Research and Education Center in Sanriku.

Competing interestNo relevant competing interests were disclosed.

Donation messageYour kind support would be highly appreciated to further advance this research.

Corresponding authorHisayuki Arakawa
AddressTokyo University of Marine Science and Technology, Konan-4, Minato, Tokyo, Japan.
E-mailarakawa@kaiyodai.ac.jp

Introduction

The Manila clam, Ruditapes philippinarum (Veneridae) is an important fishery resource of tidal areas. This bivalve forms shoals in bays and functions in the purification of seawater on tidal flats 1. At the peak of this fishery in 1983, approximately 140,000 t of Manila clams were harvested in Japan, but the catch decreased to less than 50,000 t in 1994 2. The quantity of Manila clams harvested does not necessarily reflect the size of the resource but, according to Kakino 3, the condition of the shallow sea fishery indicates that it is unlikely that the decrease in catch was caused by lack of fishing effort. He concluded that the resource is actually decreasing.

Suggested causes for the decrease in the Manila clam population include: over-fishing, disappearance of fishing grounds, deterioration of the quality of the water and of the seabed environment, increased chemical pollution of seawater, and interactions with other organisms 2-6. However, none of these have been definitively identified as causative.

Previous investigations have reported that the survival and growth of several species of shellfish larvae (including Venus Mercenaria, Mercenaria mercenaria, Haliotis diversicolor, Haliotis discus and Mytilus edulis) were impaired by turbidity 7-14 and it is possible that this is also a factor affecting the planktonic larval stages of the Manila clam.

To investigate the effects of suspended particles on early development of the Manila clam, we experimentally examined the effects of different particle concentrations in seawater, on the hatching rate of fertilized eggs, the growth of trochophore larvae, and the growth of veliger larvae. In addition, we compared the abundance of planktonic larvae with suspended particle concentrations in seawater in Manila clam harvesting grounds in the northern part of Chiba Prefecture, central Japan.

Materials and Methods

Fertilized eggs and planktonic larvae

Full-grown adult Manila clams, R. philippinarum (shell length, 30–40 mm) were collected in Futtsu, Chiba Prefecture. The Manila clams were pre-cultured in filtered seawater for a few days and fed with phytoplankton (Pavlova lutheri (Haptophyta)). The clams were induced to spawn by repeated thermal stimulation and fertilized eggs were obtained. The fertility rate of the eggs (percentage that commenced cleavage 2 h after fertilization) was determined and samples with >90% fertilization were selected for the experiment. Fertilized eggs were left for 12 h after fertilization to obtain trochophore larvae. Trochophores were cultured in a 10-L tank (water temperature 20 ± 2°C) for 5 days to obtain veliger larvae. They were not fed until 3 days after fertilization and subsequently they were fed daily with P. lutheri, adjusting the concentration according to the growth of the larvae 15.

Inorganic particles

Kaolinite (produced in California) was used for the inorganic particles. Seawater used for this experiment was drawn from the fishing port of Onuki, Chiba Prefecture, filtered through a vacuum filtering apparatus and sterilized through an ultraviolet irradiation device (Rei-sea Co. Ltd, Tokyo, Japan).

The turbidity of the water (concentration of suspended particles) sample was adjusted by adding different quantities of kaolinite at each experiment. In this paper, the term turbidity refers to the concentration of kaolinite in milligrams per liter. The mean diameter of the suspended particles was 2.6 µm, ranging from 2.0 to 62.4 µm. Particle size was measured with a Coulter Multisizer (Beckman Coulter, Inc., CA, USA) using a 100 µm orifice.

Effect of turbidity on hatching of fertilized eggs

Centrifuge tubes (12 cm long, 15 mL) were used as breeding vessels and were attached to a tube rotator (2 rpm, AS ONE Corporation, Osaka, Japan) to ensure uniform turbidity.

The experiment was performed in a room with a constant temperature (20 ± 1°C). One thousand eggs just after fertilization that had commenced cleavage were added to a series of breeding vessels containing either filtered seawater or turbid seawater at concentrations of 10, 20, 30, 50, 75, 100, 125, 150, or 200 mg L⁻¹ particles. The rotation was stopped after 7 h, the breeding vessels were removed from the equipment and left for 1 h. Hatched larvae floating on the surface were collected and immediately fixed in 1% formalin solution. They were then removed with a plankton net (30 µm mesh) and counted under a microscope. The actual hatching rate was obtained from the following formula:

where Hr is the actual hatching rate, and Nh and Nm are the numbers of hatched larvae and fertilized eggs, respectively. The experiment was performed six times at each turbidity level. The relative hatching rate at each turbidity level was calculated by assuming the actual hatching rate in filtered seawater was 100%.

Effect of turbidity on the growth of trochophore larvae

Breeding vessels containing water samples of different turbidities (filtered seawater and 10, 30, 50, 100, and 150 mg L⁻¹ particles) were each stocked with 200 trochophore larvae, 12 h after hatching. Ten trials were performed at each turbidity level and the rotator was stopped 24 h after the start of the experiment (36 h post-fertilization). The larvae were fixed by formalin added to the vessels and the fixed larvae were separated from the turbid water using a plankton net. They were then transferred to watch glasses and the larvae were observed under a microscope.

Effect of turbidity on the growth of veliger larvae

Breeding vessels were filled with water samples of different turbidities (filtered seawater or turbid seawater at 10, 30, and 50 mg L⁻¹) and each stocked with approximately 40 trochophore larvae, obtained 5 days after fertilization. In one series of 1) filtered seawater and turbid seawater at 2) 10 mg L⁻¹, 3) 30 mg L⁻¹, and 4) 50 mg L⁻¹, food was supplied and, in another 5) filtered seawater series, no food was provided. The no food condition experiment was conducted to clarify the cause of low growth in high concentration of turbid seawater. The water was changed daily. When the water samples were changed, P. lutheri was given as food at a concentration of 8×10³–20×10³ cells L^-1 depending on shell length 15. During the experiment, one tube was removed from each treatment group every 2 days, the larvae were fixed with formalin, and shell length was measured under a microscope.

Field observations

To examine the effects of turbidity on larvae in a natural environment, larvae were collected and measurements of seawater turbidity were made in the northern part of Chiba Prefecture (Funabashi Offshore; 35° 38' 3.169"N, 139° 58' 1.205"E), one of the main clam fishing grounds in Tokyo Bay, central Japan. Observations were made once a month from June 2006 to January 2007. Water samples were collected from a depth of 6 m (bottom layer) and from the surface water layer using a magnetic pump. Larvae were collected with a hand net of 50 µm mesh by drawing approximately 150 L of seawater from each layer. The collected samples of larvae were identified by an immunological method 16, using monoclonal antibodies. Manila clam larvae were identified and counted under a fluorescent microscope and their shell lengths were measured.

At the same time, a water sample was taken from the surface and bottom layers (6 m depth) and the concentration of suspended solids (SS, mg L⁻¹) was measured. The filter used for the SS measurement was then placed in a ceramic crucible and calcined for an hour in a muffle furnace at 600°C. The weight of the residue was used to calculate the concentration of particulate inorganic matter (PIM, mg L⁻¹).

Results

Hatching rate of fertilized eggs

The mean actual hatching rate of fertilized eggs in filtered seawater was 92.4% in the experiments. The relative hatching rates in the experimental turbidities are shown in Figure 1. Relative hatching rates decreased markedly with increasing turbidity (94.6 ± 4.2% at
10 mg L⁻¹, 41.0 ± 7.3% at 100 mg L⁻¹, and 16.8 ± 4.3% at 200 mg L⁻¹). There was a significant negative correlation between the relative hatching rate of Manila clams and the concentration of suspended particles (Spearman’s rank correlation, r_s = −0.94, P <0.01, n = 60).

Figure 1Relationship between experimental concentrations of suspended particles (kaolinite) and the hatching rate of Manila clam eggs

Vertical bars indicate standard deviations, n = 60.

Growth of trochophore larvae

Among the larvae that were maintained in filtered seawater after hatching, 94.4% grew normally into D-shape larvae (Figure 2a). Larvae raised in turbid water had many adherent particles and exhibited a behavior of repeatedly opening and closing their shells. In addition, a number of abnormalities were observed in individual trochophore larvae, including inability to withdraw the body into the shell (Figure 2b), deformed shell and/or reduced shell (Figure 2c), and deformed velum (Figure 2d).

The proportion of larvae that developed normally decreased with increasing levels of turbidity, 95.9 ± 3.0% at 10 mg L⁻¹, 92.7 ± 3.3% at 50 mg L⁻¹, and 68.6 ± 14.3% at 150 mg L⁻¹, relative to the proportion of normal larvae in filtered seawater (Figure 3). There was a significant difference between the rates of the normal development in larvae reared in conditions where turbidity was below 10 mg L⁻¹, compared with treatments where turbidity was at a concentration of 100 mg L⁻¹ or more (Steel-Dwass multiple comparison test, P <0.05).

Figure 2Plates of normal and abnormal trochophore larvae of Ruditapes philippinarum at 36 h after fertilization

(a) Normal D-shape larvae, (b) larvae displaying an inability to withdraw the body into the shell, (c) larvae with deformed shells and reduced shells, (d) deformed velum and body.

Figure 3Relationship between experimental concentrations of suspended particles and the proportion of normally developed Manila clam trochophore larvae

Vertical bars indicate standard deviations, n = 10.

Growth of veliger larvae

The mean shell length of veligers maintained in an indoor tank (10 L) with food was 105.7 ± 3.5 μm (n = 50) on the fifth day after fertilization. In the larvae maintained in filtered seawater and provided with food, the shells grew to an average length of 202.3 ± 16.9 µm (n = 39) at 12 days (17 days after fertilization).

The changes in shell length of veligers grown for 12 days at different levels of turbidity are shown in Figure 4. The average shell lengths at 12 days were 196.4 ± 15.0 µm (n = 46) at 10 mg L⁻¹, 120.1 ± 2.8 µm (n = 27) at 30 mg L⁻¹, and 105.9 ± 3.5 µm (n = 21) at 50 mg L⁻¹, and the shell length of larvae bred without food was 110.4 ± 4.3 µm (n = 43). After 12 days, there was no significant difference between the larvae grown in filtered seawater and those in 10 mg L⁻¹ turbidity. However, there were significant differences between the larvae in filtered seawater and those in turbidities of 30 and 50 mg L⁻¹, and those without food (Scheffe’s F multiple comparison test, P <0.05). In short, at higher turbidities growth rates were lower. There was no significant difference in mean shell length between the larvae bred in a turbidity of 50 mg L⁻¹ and those bred without food.

Figure 4Comparison of growth of Manila clam veliger larvae, measured as shell length, at different particle concentrations

The open circles joined by a solid line and the dotted line refer to filtered seawater with food (n = 39) and without food (n = 43), respectively. Filled squares, triangles, and circles refer to filtered seawater with particle concentrations of 10 (n = 46), 30 (n = 27), and 50 mg L⁻¹ (n = 21), respectively. Different letters at the right indicate significant differences (P <0.05) among treatments after 12 days. There were six replicates for each SS concentration.

Larval abundance and concentrations of suspended particles in the fishing ground

Monthly variations of larval abundance and the concentrations of SS and PIM are shown in Table 1. More than 100 individuals/100 L were recorded in surface waters in June, July, September, and November. The greatest abundance of larvae was observed in the surface layer in July (401.4 individuals/100 L). About 40 individuals/100 L were present in the bottom layer (at a depth of 6 m) in June but less than 20 individuals/100 L were recorded in other months, and none at all in September, December, and January. Spawning of the Manila clam in Tokyo Bay takes place twice a year: in spring and autumn 17. Correspondingly, we observed high concentrations of larvae during June–July and September–November.

The highest value of suspended sediments (SS) was 21.8 mg L⁻¹, observed in the bottom layer (at a depth of six meters) in July, and the lowest value was 2.1 mg L⁻¹ in the bottom layer in June. The mean value of SS in the bottom layer over the entire period was 5.7 ± 4.2 mg L⁻¹. The SS concentrations in the surface layer were higher than in the bottom layer, except during July and January (Table 1). The highest value of particulate inorganic matter (PIM) was 14.2 mg L⁻¹, observed in the bottom layer in July, the lowest value was
0.6 mg L⁻¹, in the bottom layer in June, and the overall mean value was 2.3 ± 1.9 mg L⁻¹ (Table 1). The ratio of PIM to SS was on average 57.7% in waters from a depth of six meters, and 33.7% in surface waters, indicating that there were a high number of inorganic particles in seawater collected from a depth of six meters. There were no significant correlations between the density of Manila clam larvae and the concentrations of particles in seawater samples from Chiba Prefecture (Spearman’s rank correlation, P <0.05).

Table 1Monthly variation in the abundance of R. philippinarum larvae and in the concentrations of suspended solids (SS) and particulate inorganic matter (PIM) at a clam fishing ground in Tokyo Bay from June 2006 to July 2007.

r_s indicates correlation coefficients between the larvae abundance and SS and PIM.

Discussion

Effects of suspended particles on eggs and larvae of the Manila clam

It has been reported that Manila clam resources along the coast of Japan are decreasing 2. Potential causes for the decline include: a reduction in suitable habitat (e.g., tidal flats), an increase in the number of predators and environmental degradation 2-6. An increase in the number of suspended particles in seawater has contributed to environmental degradation. The effects of suspended particles in seawater on bivalves have been reported for a number of species, such as Manila clams, surf clams, mussels and oysters 7-14, 18-23.

Wilber and Clarke 24 reviewed research on the influence of seawater turbidity on bivalves. According to this review, effects of turbidity on the eggs and larvae of bivalves have only been reported for three species, the eastern oyster 8, the northern quahog 8, 13 and the Pacific oyster 25. Our research showed that an increase in the concentration of suspended particles in seawater was strongly associated with a decline in hatching rate and in the survival and growth of the larvae of the Manila clam.

We observed that higher concentrations of suspended inorganic particles led to lower hatching rates of Manila clam eggs. The absence of floating larvae immediately after hatching is consistent with microscopic observations that particles of kaolinite covered the bodies of the larvae. Presumably, this impaired floating and swimming of the larvae.

With respect to the development of trochophore larvae, we observed an inverse relationship between the proportions of normal larvae and the concentrations of suspended particles. Large quantities of coagulated particles were observed adhering to the abnormal larvae. Likewise, in an experimental study of the trochophores of Scapharca kagoshimensis, Terashima and Takagi 26 reported adherence of coagulated particles to floating larvae. The survival rate of S. kagoshimensis decreased to one third at a concentration of about 30 mg L⁻¹. Thus, the effects of turbidity on the Manila clam were a little lower than those of S. kagoshimensi. However, it remains unclear why adherence of particles to larvae was associated with abnormalities in the growth of larvae and in the formation of their shells.

The hatching rate decreased to below 50% at 100 mg L⁻¹ kaolinite. However, the proportion of normal trochophores was greater than 80% at the same turbidity. It appears that Manila clams are more susceptible to suspended particles during hatching than at the trochophore stage. Larvae immediately after hatching are possibly less efficient swimmers and were greatly impaired by the particles. The growth rates of veliger larvae decreased markedly at suspended kaolinite particle concentrations greater than 30 mg L⁻¹. Observations of veliger larvae maintained in turbid water revealed large quantities of coagulated kaolinite particles adhering to the velum, particularly in the higher turbidities. The action of opening and closing of shells was often observed, presumably a response to suspended particles touching and adhering to the soft parts of the body. It was also observed that veligers stopped swimming when the concentration of particles was high. In summary, the swimming behavior of veligers was impaired, apparently resulting from the adherence of coagulated suspended particles to their juvenile shells and to the velum.

Veligers in 50 mg L⁻¹ kaolinite showed very low growth rates that were similar to those deprived of food (Figure 4). However, we observed one individual with stomach contents, indicating that it had ingested food. This suggests that the extremely fine suspended inorganic particles may also affect digestive functions of the larvae.

In contrast to our findings, there was no effect on the larvae of eastern oysters at a turbidity concentration of 300 mg L⁻¹ 8. Abnormal shell development occurred in Pacific oyster larvae at a concentration of 1,200 mg L⁻¹ or more 25. It was found out that the Manila clam larvae are more sensitive to the effects of turbidity than the larvae of oysters.

The effects of PIM in the field

We examined the distribution of the larvae of Manila clams in relation to the particle concentrations of seawater near Funabashi, one of the main Manila clam fishing grounds in Tokyo Bay. The maximum values of SS and PIM concentrations of 21.8 and 14.2 mg L⁻¹, respectively, were observed in the bottom layer (depth of six meters) of seawater in July. The high PIM concentration in seawater collected from the bottom layer in July, suggests that the hatching rate of Manila clams may have been reduced by 10–20% at this time (Figure 1). The eggs of Manila clam have demersal characteristics. It is considered that this reduction in hatching rate may have occurred in natural sea area.

No significant difference in growth was observed between the veliger larvae maintained in filtered seawater and those bred in a turbidity of 10 mg L⁻¹ kaolinite in the laboratory. The growth of larvae was much reduced in a turbidity of 30 mg L⁻¹. The concentration of PIM (14.2 mg L⁻¹) in summer at Tokyo Bay exceeded the lower threshold value of 10 mg L⁻¹ that appears the effects of the growth of Manila clam larvae. Our field observations were conducted on sunny days. In a natural environment, high turbidity is considered to be caused by the flow of particles from rivers and the resuspension of the particles from the sea bottom during rough weather. When rough weather continues for a long time, it is likely that turbidity increases. This may inhibit larvae from feeding and swimming, which might affect their growth. To quantitatively assess these influences, we must conduct long-term monitoring of turbidity at the Manila clam harvesting grounds.

Iwamoto and Hamada 27 reported turbidities exceeding 100 ppm in an estuarine clam ground in Yamaguchi Prefecture in western Japan at the time of a large flood. If Manila clam spawned in an environment of such high turbidity, egg-hatching and growth of trochophore larvae would be inhibited. Therefore, it is suggested that in the approach to the Manila clam regeneration project, including the development, improvement, and management of clam harvesting grounds, consideration should be given to the mechanisms of movement and accumulation of suspended matter in the sea.

Kakino 4 summarized eight physiochemical factors affecting the survival rate of adult Manila clams and identified turbidity as one. Kurashige and Matsumoto 20, Chiba and Oshima 18, and Tabata et al. 28 reported on the effects of suspended inorganic matter on adult Manila clams. They observed a decline in the filtering rate, increased discharge of pseudo-feces, and decreased particle retention.

Wilber and Clark 24 reviewed the influence of turbidity on adult bivalves. They indicated that a concentration of 10,000 mg L⁻¹ of suspended particles was lethal to adult bivalves. Based on the turbidity levels measured in our field observation, the turbidity in natural conditions may not greatly affect the survival of adult Manila clams in Tokyo Bay. However, the presence of suspended sediments may contribute to worsen the health of the Manila clam 29-31. In some previous reports, seawater turbidity is not regarded as having a serious effect on Manila clam resources. However, Manila clam larvae spend about 2 weeks in the plankton before recruitment. Our results suggest that seawater turbidity may have a strong detrimental effect on the early life stages of the Manila clam and may have long term effects on the population.

Turbidity caused by organic particles is also a serious problem in enriched areas of the sea such as Tokyo Bay e.g., 32. The effect of organic particles on Manila clams should be investigated in the future.

Conclusions

We examined the influence of suspended particles on the hatching of fertilized eggs and on the development and growth of larvae of the Manila clam, R. philippinarum. In our laboratory experiment the hatching rate of fertilized eggs declined with increasing concentrations of suspended particles in seawater. There was little effect of suspended particles on the development and growth of larvae at concentration of 10 mg L⁻¹. However, at suspended particle concentrations of 30 mg L⁻¹ and greater, there was an increasingly higher proportion of larvae that failed to develop normally and grow. No significant correlations were found between the abundance of Manila clam larvae and the concentrations of suspended solids (SS) or particulate inorganic matter (PIM) found in Tokyo Bay. However our experimental results strongly suggest that high levels of suspended particles in seawater, are likely to have an impact on the early life history of this species.

Acknowledgements

We sincerely appreciate the cooperation extended to our research team by Dr. Mitsuharu Toba, the president of the Tokyo Bay Fisheries Laboratory, Chiba Prefectural Fisheries Research Center, and all of their staff.

Author Contributions

Arakawa H: Wrote the article and designed the discussion.
Akatsuka T, Kobayashi Y, Matsumoto A: Treated the samples, and analyzed the data.
Seto M, Nemoto M: Edited and approved the article.

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