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Instantaneous effect of dibromomethane on metamorphosis of larvae of the sea urchins Strongylocentrotus nudus and Strongylocentrotus intermedius
Aquaculture 251 (2006) 549 – 557
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Instantaneous effect of dibromomethane on metamorphosis of larvae of the sea urchins Strongylocentrotus nudus and Strongylocentrotus intermedius Yukio Agatsuma a,*, Tetsuo Seki b, Kazuya Kurata c, Kazuya Taniguchi a a b
Graduate School of Agricultural Science, Tohoku University, Tsutsumidori-amamiya, Aoba, Sendai, Miyagi 981-8555, Japan National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, Maruishi, Ohno, Saeki, Hiroshima 739-0452, Japan c Hakodate Technical College, Tokuracho, Hakodate, Hokkaido 042-0953, Japan Received 29 January 2005; received in revised form 30 May 2005; accepted 31 May 2005
Abstract A volatile chemical, dibromomethane (DBM), produced from red coralline algae is known as a chemical inducer of larval metamorphosis of the sea urchin Strongylocentrotus nudus. We performed experiments exposing DBM to the larvae of S. nudus and Strongylocentrotus intermedius through a hydrophobic membrane. Metamorphic rates resulting from different diluted DBM solutions and exposure times were ascertained. The highest metamorphic rate, more than 80% in both species, was found after 1 h exposure to 1/2 diluted DBM. With this dilution, more than 80% of S. nudus and S. intermedius larvae metamorphosed 1 h after start of the experiment after only 10 and 5 min exposure, respectively, which corresponded to the low concentrations of 52–61 ppm and 34–43 ppm DBM by GCMS analysis, respectively. These findings suggest that DBM has an instantaneous effect on high success of metamorphosis of larvae of S. nudus and S. intermedius. D 2005 Elsevier B.V. All rights reserved. Keywords: Sea urchin; Metamorphosis; Dibromomethane; Larvae; Strongylocentrotus nudus; Strongylocentrotus intermedius
1. Introduction Communities of crustose coralline red algae with no large erect macrophytes in subtidal rocky habitats
* Corresponding author. Tel.: +81 22 717 8899; fax: +81 22 717 8899. E-mail address: [email protected] (Y. Agatsuma). 0044-8486/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.05.043
are called bBarren groundQ (Pearse et al., 1970), bCoralline flatQ (Ayling, 1981), bIsoyake areaQ(Hagen, Hagen, 1983), bDeforested areaQ (Harrold and Pearse, 1987), bHeavy grazing bottomQ (Keats et al., 1990), or bUrchin barrenQ (Coyer et al., 1993). In these communities, a specific benthic animal community consisting of gastropods, limpets and sea urchins as herbivores is established (e.g. Andrew and Choat, 1982).
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It is considered that the high density of sea urchins in crustose coralline communities is attributed to the high success rate of recruitment involving metamorphosis, high survival rate of juveniles and/or migration after settlement. However, juvenile migration is generally discounted as a causative factor due to their limited movement and physical barriers such as sand (Rowley, 1989; Watanabe and Harrold, 1991). Metamorphosis of echinopluteus larvae is induced by chemicals produced from crustose corallines. The larvae of Strongylocentrotus purpuratus, S. franciscanus (Cameron and Schroeter, 1980; Rowley, 1989), Strongylocentrotus nudus (Sano et al., 1998) and Strongylocentrotus droebachiensis (Balch and Scheibling, 2000) metamorphose abundantly on crustose corallines. Taniguchi et al. (1994) found that two articulated coralline algae Serraticardia mazima and Calliarthron yessoense, the crustose coralline alga Lithophyllum yessoense (dominant species in coralline communities), and the green alga Ulvella lens all induce larval metamorphosis of S. nudus. They ascertained that dibromomethane (DBM), a volatile chemical produced by all these algae (Itoh and Shinya, 1994; Ohshiro et al., 1999), induced 100% of larvae to metamorphose within 2 h. In that experiment, larval metamorphic rate was examined in petri dishes with seawater in which DBM was dissolved to a relatively high concentration of approximately 700 ppm. Moreover, the effect of exposure time of the larvae to DBM was not examined. The fully developed 8-armed larvae contact the surface of the corallines just before metamorphosis (Taniguchi et al., 1994). Hence, metamorphosis appears to be induced by immediate reception of DBM, constantly released from the corallines (Itoh and Shinya, 1994). To simulate the relation between echinopluteus larvae and crustose corallines, we designed a new system that diffuses DBM through a hydrophobic membrane. In the present study, the optimum exposure time to DBM and the optimum concentration inducing larval metamorphosis were quantified.
2. Materials and methods 2.1. Metamorphosis by different DBM dilutions The experiments on inducing larval metamorphosis of S. nudus and Strongylocentrotus intermedius were
conducted at Fukushima Prefectural Fish Farming Center in October 2001 and 2002 and at Hokkaido Institute of Mariculture in September 2002, respectively. Larvae of both species were reared at a density of approximately 1 individual/ml in 0.5-m3 rectangular tank at the flow rate of 0.8 l/min and fed Chaetoceros gracilis with 50,000 cells/ml/day for ca. 1 week at water temperatures of 17–18 8C. Light and dark conditions were controlled every 12 h. They grew to 8-armed competent larvae with fully developed urchin rudiments. Filtered seawater to 5 Am (250 ml) was added to a light protected 500-ml erlenmeyer flask covered with black tapes that had a glass stopper. Twenty five grams of dibromomethane (DBM, CH2Br2) (Wako Pure Chemical Industries Ltd.) was added and dissolved by stirring for 24 h. As DBM is likely to chemically resolve under light condition for a long time, the light protected one was used. Seawater with insoluble DBM at the bottom was considered to be a saturated solution. To establish metamorphic rate at different concentrations of DBM, the saturated solution was diluted with filtered seawater to 1/8, 1/ 4, 1/3 and 1/2. The larvae of S. nudus were examined also at 1/16 diluted solution. The experimental vessel was a filter holder (SUS316, Shibata Scientific Technology Ltd.) cut into upper and lower halves and held upright in an acrylic stand (Fig. 1). Ten millilitres of each diluted DBM solution were added to the lower vessel and sealed with a silicon rubber plug. A filter (Shibata Support screen: 47 mm) was embedded immediately on the seal and a hydrophobic PTFE membrane (Advantec polymer: 47 mm diameter, 0.2-Am pore size, Toyo Roshi Kaisha, Ltd.) was placed on it. The upper vessel was clamped onto the lower one. Filtered seawater to 5 Am (28 ml), with ca. twenty 8armed larvae, was added to the upper vessel to initiate the experiment. Thus, DBM diffused from the lower vessel to larvae in the upper vessel through the hydrophobic membrane. After 1 h, the DBM solution in the lower vessel was discarded by removing the rubber plug. A plastic plate (35 40 mm) covered with the green alga U. lens was added to a petri dish (5.5 cm diameter, 3.0 cm deep) containing 28 ml of filtered seawater to 5 Am and ca. 20 larvae and served as a positive control. A petri dish with only filtered seawater to 5 Am was a negative control (blank). After 1, 2, 4, 8, 16 and 24 h from the start of the experiment,
Y. Agatsuma et al. / Aquaculture 251 (2006) 549–557
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75 mm 68 mm
30 mm
a) b)
g)
47 mm 120 mm
c)
55 mm
d) Larvae 13 mm
e) f)
20 mm
50 mm
100 mm
Fig. 1. Glass vessel used in this study for inducing larval metamorphosis of S. nudus and S. intermedius by dibromomethane (DBM). (a) Upper vessel, (b) hydrophobic membrane, (c) filter, (d) lower vessel, (e) silicon rubber plug, (f) acrylic stand, (g) clamp.
metamorphosed larvae were counted using a stereomicroscope under vertical illumination directly from above. The percent metamorphosis (number of metamorphosed individuals / total number of larvae added 100) was calculated. No observation on larvae of S. intermedius was made after 16 h. Metamorphosed individuals were defined as those with larval arms reabsorbed and having a globular test, tube feet and spines. 2.2. Metamorphosis by different exposure times to DBM To clarify induction of larval metamorphosis by different exposure times to DBM, the DBM solution diluted by half, that induced the highest percent metamorphosis in the first experiment, was added to the lower vessel. Simultaneously, 28 ml of filtered
seawater with ca. twenty 8-armed larvae were added to the upper vessel, which was separated from the lower one by the PTFE membrane. Six experimental treatments with different exposure times to DBM were designated. That is, the DBM solution was discarded after 1, 2.5, 5, 10, 20 and 40 min. Metamorphosed individuals were counted after 1, 2, 4, 8 and 24 h from the start of the experiment and percent metamorphosis was calculated. The metamorphosed larvae in positive and negative control petri dishes were also observed. In both experiments, each treatment consisted of 3 replicate assays. Abnormal and dead individuals were identified by the lack of larval swimming activity or lack of mobility of juvenile podia. Room temperature during all experiments was regulated at 20 8C. Statistical significance of percent larval metamorphosis among the treatments and times was analyzed by
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two-way repeated-measures ANOVA followed by multiple comparison test of Fisher’s LSD. All the data showed normal distribution and homogeneous variances by Shapiro-Wilk’s W-test and Cochran’s test, respectively. 2.3. Analysis of DBM concentration diffused To determine the DBM concentration diffused from the 1/2 diluted solution across the hydrophobic PTFE membrane, 10 ml of filtered seawater in the upper vessel was removed by pipet immediately after discarding the DBM solution diluted by half in the lower vessel 5, 10, 15, 20 and 30 min after diffusing, and added to vials for analysis in a headspace autosampler. The headspace DBM in the solutions was generated using a Hewlett Packard model HP 7694 headspace autosampler. The operating conditions were as follows: the oven temperature, 60 8C; the vial equilibration time, 30 min; the injection volume, 20 Al. Electron impact mass analysis of the headspace DBM was performed with a Hewlett Packard model HP 5972 MSD gas chromatograph-mass spectrometer,
100
a
consisting of a DB-624 column (30 m 0.32 mm 1.8 Am (cyanopropyl-phenyl) methylpolysiloxane, Aligent Technologies). The electron energy was 70 eV. The operating conditions were as follows: the injector temperature, 200 8C; the detector temperature, 260 8C; the oven temperature, from 40 to 180 8C at 8 8C/min and from 180 to 260 8C at 15 8C/min; the He carrier flow rate, 1.5 ml/min. The concentration of headspace DBM was determined by comparing with a standardized authentic. The analysis was performed twice. Room temperature was 20 8C.
3. Results 3.1. Metamorphosis of S. nudus larvae Changes in percent metamorphosis of S. nudus larvae induced by each diluted DBM solution (diffusing for 1 h) over 24 h and the statistical significance are shown in Fig. 2 and Table 1. Significant differences in percent metamorphosis were found among DBM dilutions and times ( p b 0.0001). Induction of
a
a
90 a*
b c
c
b
d d
d
c
4
8
1/2 dilution 1/3 dilution 1/4 dilution 1/8 dilution 1/16 dilution Ulvella lens Blank
b*
70 60 50 40
a*
a*
b* 80
Metamorphosis (%)
a
a
b
b
c
c
c
30 20 10 0 0
12
16
20
24
Time (hrs) Fig. 2. Percent metamorphosis of S. nudus after 1 h exposure to different dilutions of saturated dibromomethane (DBM) solution, a control with U. lens, and a negative blank (filtered seawater). Each treatment consists of 3 replicates with ca. 20 larvae per replicate. Each vertical bar indicates standard error. Different letters indicate significant differences among treatments at each time. Asterisks show significant differences of percent metamorphosis from that at 1 h.
Y. Agatsuma et al. / Aquaculture 251 (2006) 549–557 Table 1 Two-way repeated-measures ANOVAs of percent metamorphosis of S. nudus exposed to (A) different dilutions of dibromomethane and (B) different exposure times Source of variation
df
MS
F
p
A. Dilution Error Time Dilution time Error B. Exposure time Error Time Exposure time time Error
3 8 5 15 40 7 16 4 28 64
31,006.429 289.141 554.735 312.357 13.392 23,021.964 868.510 474.744 153.919 13.311
107.236
b0.0001
41.422 23.32
b0.0001 b0.0001
26.507
b0.0001
35.666 11.564
b0.0001 b0.0001
h in each DBM solution ( p N 0.05). Contrary, for the positive control with U. lens, percent metamorphosis rose significantly and reached 98% after 24 h ( p b 0.01). Changes in percent metamorphosis of S. nudus larvae with different exposure times using 1/2 dilution (that induced the highest percent metamorphosis) are shown in Fig. 3. Significant differences in the percent metamorphosis among exposure times and times were also found ( p b 0.0001) (Table 1). After 1 h, percent metamorphosis for larvae exposed for 40, 20 and 10 min was N80%. No statistical difference in the percent metamorphosis among those exposure times was found throughout the 24 h period ( p N 0.05). The percent metamorphosis for 5, 2.5 and 1 min exposure after 1 h were lower and fell with shortening exposure time. No larvae metamorphosed in the blank control. There was only a significant difference between the times of 1 and 2 h exposed for 20 min ( p b 0.01). The percent metamorphosis after 24 h for the positive control with U. lens reached 97% and rose significantly from 1, 2 h to 4, 8, 24 h ( p b 0.05). No abnormal or dead 8-armed larvae or metamorphosed juveniles were observed in any experimental or control groups.
metamorphosis was dependent on DBM concentration. After 24 h, percent metamorphosis was highest (96%) at 1/2 dilution. No larvae metamorphosed at 1/ 8 or 1/16 diluted solutions or in the blank control after 24 h. Percent metamorphosis at 1/2 and 1/3 dilutions rose rapidly during the first 2 h. In particular, percent metamorphosis reached 93% after 1 h at 1/2 dilution. After that, no significant difference in percent metamorphosis was found among times until 24 h after 2 a*
100
a a
90
a a
Metamorphosis (%)
80 a b
70
a*
a*
a*
a ab
a a
aab
bc
b
cd*
c bc*
b ab*
60
40 minutes 20 minutes 10 minutes 5 minutes 2.5 minutes 1 minute Ulvella lens Blank
bc
50 40 c c
30
cd
e
c
20 10
c cd
553
d
c
e
d
0 0
4
8
12
16
20
24
Time (hrs) Fig. 3. Percent metamorphosis of S. nudus after different exposure times to 1/2 diluted solution from saturated dibromomethane (DBM) solution, a control with U. lens, and a negative blank (filtered seawater). Each treatment consists of 3 replicates with ca. 20 larvae per replicate. Each vertical bar indicates standard error. Different letters indicate significant differences among treatments at each time. Asterisks show significant differences of percent metamorphosis from that at 1 h.
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3.2. Metamorphosis of S. intermedius larvae Changes in percent metamorphosis over 24 h of S. intermedius larvae induced with different diluted DBM solutions (1 h exposure) and the statistical significances are shown in Fig. 4 and Table 2. Significant difference in percent metamorphosis was found among DBM dilutions and times ( p b 0.0001). After 1 h, the percent metamorphosis at 1/2 and 1/3 dilution sharply rose to 82% and 73%, respectively, and did not differ significantly between them ( p N 0.05). The percent reached 100% after 2 h in both treatments. At 1/4 dilution, the percent rose to 36% after 2 h. Thus, larval metamorphosis of S. intermedius was induced by a less diluted solution than for S. nudus. However, no metamorphosis occurred at 1/8 dilution or in the blank control. Significant differences were found in the percent metamorphosis among 1/2, 1/3 and 1/4 dilutions after 1 h and also between the times of 1 and 2, 4, 8 and 24 h at each of those dilutions ( p b 0.05). In the control with U. lens, the percent rose significantly until 4 h ( p b 0.05) and reached 70% after 24 h. a*
a*
Table 2 Two-way repeated-measures ANOVAs of percent metamorphosis of S. intermedius exposed to (A) different dilutions of dibromomethane and (B) different exposure times Source of variation
df
MS
A. Dilution Error Time Dilution time Error B. Exposure time Error Time Exposure time time Error
3 8 4 12 32 7 16 4 28 64
18,338.529 297.822 2296.475 311.680 69.792 17,851.704 56.797 313.379 291.143 39.250
p
61.575
b0.0001
32.905 4.466
b0.0001 b0.001
314.308
b0.0001
7.984 7.418
b0.0001 b0.0001
Changes in percent metamorphosis of S. intermedius larvae with different DBM exposure times to 1/2 dilution are shown in Fig. 5. Significant differences in the percent metamorphosis among exposure times and times were also found ( p b 0.0001) (Table 2). All the larvae metamorphosed after 1 h for N 5 min exposure. After 1 h, percent metamorphosis of larvae exposed for 2.5 min was 75%, which did not
a*
a*
100 a
90
b*
80
Metamorphosis (%)
F
70 60
b*
b*
c*
c*
a
50 b*
c*
40 1/2 dilution 1/3 dilution 1/4 dilution 1/8 dilution Ulvella lens Blank
30 20 b
10
b b
0 0
4
8
12
16
20
24
Time (hrs) Fig. 4. Percent metamorphosis of S. intermedius after 1 h exposure to different dilutions of saturated dibromomethane (DBM) solution, a control with U. lens, and a negative blank (filtered seawater). Each treatment consists of 3 replicates with ca. 20 larvae per replicate. Each vertical bar indicates standard error. Different letters indicate significant differences among treatments at each time. Asterisks show significant differences of percent metamorphosis from that at 1 h.
Y. Agatsuma et al. / Aquaculture 251 (2006) 549–557
a a
a
100 90
b
Metamorphosis (%)
80 70
c
b
b
c
c
555
a
a
b
a* b
c
c
d*
d*
bc*
60
40 minutes 20 minutes 10 minutes 5 minutes 2.5 minutes 1 minute Ulvella lens Blank
50 40 30
d
20 10 0 0
4
8
12
16
20
24
Time (hrs) Fig. 5. Percent metamorphosis of S. intermedius after different exposure times to 1/2 diluted solution from saturated dibromomethane (DBM) solution, a control with U. lens, and a negative blank (filtered seawater). Each treatment consists of 3 replicates with ca. 20 larvae per replicate. Each vertical bar indicates standard error. Different letters indicate significant differences among treatments at each time. Asterisks show significant differences of percent metamorphosis from that at 1 h.
increase after this time. The percent metamorphosis of larvae exposed for 1 min sharply rose to 82% after 1 h and then gradually to 84.2% until 2 h when it ceased, significantly higher than that for larvae exposed for 2.5 min after 1, 2, 4, 8 and 24 h ( p b 0.05). No metamorphosis occurred in the blank control. In the control with U. lens, the percent metamorphosis rose to 97% after 24 h. No abnormal or dead 8-armed larvae or metamorphosed juveniles were observed in any experimental or control groups.
3.3. Concentration of DBM diffused Analysis by gas chromatograph-mass spectrometer showed that 10 and 5 min diffusing at 1/2 diluted DBM solution, which induced a high percent metamorphosis of more than 80% for S. nudus and S. intermedius at 1 h after start of the experiment, corresponded to the low concentrations of 52–61 ppm and 34–43 ppm, respectively (Table 3).
4. Discussion Table 3 Concentration of dibromomethane solution diluted to 1/2 with filtered seawater at different diffused times Dilution
Time diffused (min)
Concentration (ppm)
Control 1/2 1/2 1/2 1/2 1/2
5 10 15 20 30
0 42.5 60.9 61.3 55.9 64.8
0 34.3 51.7 59.0 58.6 65.8
In the present study, an exposure to 1/2 diluted DBM solution for 1 h induced a high percent larval metamorphosis (more than 80%) with 10 min exposure for S. nudus and 5 min exposure for S. intermedius. The concentrations of DBM diffusing over these time frames were 52–61 ppm and 34–43 ppm, respectively. This study showed that a high percent of sea urchin larvae settle and metamorphose following brief exposure to low concentrations of DBM. Significantly higher percent metamorphosis of S. interme-
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dius after 1 min exposure than after 2.5 min exposure is considered attributable to differences in probability whether larvae can respond to DBM during these short times. In the positive control with live U. lens, the percent metamorphosis rose sharply until 4 h and then more gradually after this time. Taniguchi et al. (1994) monitored larval percent metamorphosis of S. nudus induced by live L. yessoense and U. lens in petri dishes with filtered seawater after 2, 8, 16 and 24 h. Percent metamorphosis induced by L. yessoense was high at 2 h after the start of the experiment. Contrary, the percent induced by U. lens was lower than that by L. yessoense, while it rose continually until 8 h and then more gradually after this time. This suggests release of DBM from U. lens is less than from L. yessoense. Even though the amount of DBM released by the algae is low, continual release from the thalli probably leads to a gradual increase in percent metamorphosis. In this study the percent metamorphosis induced by U. lens differed among each experiment despite a similar gradual increase. These differences may be caused by different densities of U. lens used. Naidenko (1996) reported that glutamine or glutamine mimetics may be active components of natural inducers from calcareous algae. Kitamura et al. (1993) reported that larval metamorphosis of Pseudocentrotus depressus and Anthocidaris crassispina following larval contact with the articulated coralline alga Corallina pilulifera is due to eicosapentaenoic acid produced in the algal thalli. A chemical cue inducing high metamorphic rate of S. droebachiensis larvae after their contact with the coralline algae Lithothamnion glaciale, Phymatolithon laevigatum, P. rugulosum and Corallina officinalis is thought to be g-aminobutyric acid (GABA) (Pearce and Scheibling, 1990). The present study has shown instantaneous metamorphosis of a high percent of sea urchin larvae by DBM. Qualitative studies on the mechanism of release and how much DBM is being released from U. lens and crustose corallines are necessary. For S. nudus and S. intermedius larvae, responding to DBM instantly with a high percent of metamorphosis would ensure a high recruitment level in crustose coralline habitats even though high mortality might occur after settlement. The number of sea urchins that settle fluctuates temporally and spatially and is associated with abundance of larval supply caused by oceano-
graphic conditions, water temperature, salinity, predation, and starvation (reviewed by Ebert, 1983; Balch and Scheibling, 2001). However, it is probable that crustose corallines have an important role in determining sea urchin population structure and size in rocky subtidal bottoms.
Acknowledgements We sincerely thank Dr. John M. Lawrence, University of South Florida, for his insightful comments on this paper. We are also grateful to T. Ono (managing director) and T. Maruzoe of the Fukushima Prefectural Fish Farming Center and Dr. H. Uto (directorgeneral), S. Motoya and Y. Sakai of the Hokkaido Institute of Mariculture for their support and cooperation in this study.
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Instantaneous effect of dibromomethane on metamorphosis of larvae of the sea urchins Strongylocentrotus nudus and Strongylocentrotus intermedius Yukio Agatsuma a,*, Tetsuo Seki b, Kazuya Kurata c, Kazuya Taniguchi a a b
Graduate School of Agricultural Science, Tohoku University, Tsutsumidori-amamiya, Aoba, Sendai, Miyagi 981-8555, Japan National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, Maruishi, Ohno, Saeki, Hiroshima 739-0452, Japan c Hakodate Technical College, Tokuracho, Hakodate, Hokkaido 042-0953, Japan Received 29 January 2005; received in revised form 30 May 2005; accepted 31 May 2005
Abstract A volatile chemical, dibromomethane (DBM), produced from red coralline algae is known as a chemical inducer of larval metamorphosis of the sea urchin Strongylocentrotus nudus. We performed experiments exposing DBM to the larvae of S. nudus and Strongylocentrotus intermedius through a hydrophobic membrane. Metamorphic rates resulting from different diluted DBM solutions and exposure times were ascertained. The highest metamorphic rate, more than 80% in both species, was found after 1 h exposure to 1/2 diluted DBM. With this dilution, more than 80% of S. nudus and S. intermedius larvae metamorphosed 1 h after start of the experiment after only 10 and 5 min exposure, respectively, which corresponded to the low concentrations of 52–61 ppm and 34–43 ppm DBM by GCMS analysis, respectively. These findings suggest that DBM has an instantaneous effect on high success of metamorphosis of larvae of S. nudus and S. intermedius. D 2005 Elsevier B.V. All rights reserved. Keywords: Sea urchin; Metamorphosis; Dibromomethane; Larvae; Strongylocentrotus nudus; Strongylocentrotus intermedius
1. Introduction Communities of crustose coralline red algae with no large erect macrophytes in subtidal rocky habitats
* Corresponding author. Tel.: +81 22 717 8899; fax: +81 22 717 8899. E-mail address: [email protected] (Y. Agatsuma). 0044-8486/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.05.043
are called bBarren groundQ (Pearse et al., 1970), bCoralline flatQ (Ayling, 1981), bIsoyake areaQ(Hagen, Hagen, 1983), bDeforested areaQ (Harrold and Pearse, 1987), bHeavy grazing bottomQ (Keats et al., 1990), or bUrchin barrenQ (Coyer et al., 1993). In these communities, a specific benthic animal community consisting of gastropods, limpets and sea urchins as herbivores is established (e.g. Andrew and Choat, 1982).
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It is considered that the high density of sea urchins in crustose coralline communities is attributed to the high success rate of recruitment involving metamorphosis, high survival rate of juveniles and/or migration after settlement. However, juvenile migration is generally discounted as a causative factor due to their limited movement and physical barriers such as sand (Rowley, 1989; Watanabe and Harrold, 1991). Metamorphosis of echinopluteus larvae is induced by chemicals produced from crustose corallines. The larvae of Strongylocentrotus purpuratus, S. franciscanus (Cameron and Schroeter, 1980; Rowley, 1989), Strongylocentrotus nudus (Sano et al., 1998) and Strongylocentrotus droebachiensis (Balch and Scheibling, 2000) metamorphose abundantly on crustose corallines. Taniguchi et al. (1994) found that two articulated coralline algae Serraticardia mazima and Calliarthron yessoense, the crustose coralline alga Lithophyllum yessoense (dominant species in coralline communities), and the green alga Ulvella lens all induce larval metamorphosis of S. nudus. They ascertained that dibromomethane (DBM), a volatile chemical produced by all these algae (Itoh and Shinya, 1994; Ohshiro et al., 1999), induced 100% of larvae to metamorphose within 2 h. In that experiment, larval metamorphic rate was examined in petri dishes with seawater in which DBM was dissolved to a relatively high concentration of approximately 700 ppm. Moreover, the effect of exposure time of the larvae to DBM was not examined. The fully developed 8-armed larvae contact the surface of the corallines just before metamorphosis (Taniguchi et al., 1994). Hence, metamorphosis appears to be induced by immediate reception of DBM, constantly released from the corallines (Itoh and Shinya, 1994). To simulate the relation between echinopluteus larvae and crustose corallines, we designed a new system that diffuses DBM through a hydrophobic membrane. In the present study, the optimum exposure time to DBM and the optimum concentration inducing larval metamorphosis were quantified.
2. Materials and methods 2.1. Metamorphosis by different DBM dilutions The experiments on inducing larval metamorphosis of S. nudus and Strongylocentrotus intermedius were
conducted at Fukushima Prefectural Fish Farming Center in October 2001 and 2002 and at Hokkaido Institute of Mariculture in September 2002, respectively. Larvae of both species were reared at a density of approximately 1 individual/ml in 0.5-m3 rectangular tank at the flow rate of 0.8 l/min and fed Chaetoceros gracilis with 50,000 cells/ml/day for ca. 1 week at water temperatures of 17–18 8C. Light and dark conditions were controlled every 12 h. They grew to 8-armed competent larvae with fully developed urchin rudiments. Filtered seawater to 5 Am (250 ml) was added to a light protected 500-ml erlenmeyer flask covered with black tapes that had a glass stopper. Twenty five grams of dibromomethane (DBM, CH2Br2) (Wako Pure Chemical Industries Ltd.) was added and dissolved by stirring for 24 h. As DBM is likely to chemically resolve under light condition for a long time, the light protected one was used. Seawater with insoluble DBM at the bottom was considered to be a saturated solution. To establish metamorphic rate at different concentrations of DBM, the saturated solution was diluted with filtered seawater to 1/8, 1/ 4, 1/3 and 1/2. The larvae of S. nudus were examined also at 1/16 diluted solution. The experimental vessel was a filter holder (SUS316, Shibata Scientific Technology Ltd.) cut into upper and lower halves and held upright in an acrylic stand (Fig. 1). Ten millilitres of each diluted DBM solution were added to the lower vessel and sealed with a silicon rubber plug. A filter (Shibata Support screen: 47 mm) was embedded immediately on the seal and a hydrophobic PTFE membrane (Advantec polymer: 47 mm diameter, 0.2-Am pore size, Toyo Roshi Kaisha, Ltd.) was placed on it. The upper vessel was clamped onto the lower one. Filtered seawater to 5 Am (28 ml), with ca. twenty 8armed larvae, was added to the upper vessel to initiate the experiment. Thus, DBM diffused from the lower vessel to larvae in the upper vessel through the hydrophobic membrane. After 1 h, the DBM solution in the lower vessel was discarded by removing the rubber plug. A plastic plate (35 40 mm) covered with the green alga U. lens was added to a petri dish (5.5 cm diameter, 3.0 cm deep) containing 28 ml of filtered seawater to 5 Am and ca. 20 larvae and served as a positive control. A petri dish with only filtered seawater to 5 Am was a negative control (blank). After 1, 2, 4, 8, 16 and 24 h from the start of the experiment,
Y. Agatsuma et al. / Aquaculture 251 (2006) 549–557
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75 mm 68 mm
30 mm
a) b)
g)
47 mm 120 mm
c)
55 mm
d) Larvae 13 mm
e) f)
20 mm
50 mm
100 mm
Fig. 1. Glass vessel used in this study for inducing larval metamorphosis of S. nudus and S. intermedius by dibromomethane (DBM). (a) Upper vessel, (b) hydrophobic membrane, (c) filter, (d) lower vessel, (e) silicon rubber plug, (f) acrylic stand, (g) clamp.
metamorphosed larvae were counted using a stereomicroscope under vertical illumination directly from above. The percent metamorphosis (number of metamorphosed individuals / total number of larvae added 100) was calculated. No observation on larvae of S. intermedius was made after 16 h. Metamorphosed individuals were defined as those with larval arms reabsorbed and having a globular test, tube feet and spines. 2.2. Metamorphosis by different exposure times to DBM To clarify induction of larval metamorphosis by different exposure times to DBM, the DBM solution diluted by half, that induced the highest percent metamorphosis in the first experiment, was added to the lower vessel. Simultaneously, 28 ml of filtered
seawater with ca. twenty 8-armed larvae were added to the upper vessel, which was separated from the lower one by the PTFE membrane. Six experimental treatments with different exposure times to DBM were designated. That is, the DBM solution was discarded after 1, 2.5, 5, 10, 20 and 40 min. Metamorphosed individuals were counted after 1, 2, 4, 8 and 24 h from the start of the experiment and percent metamorphosis was calculated. The metamorphosed larvae in positive and negative control petri dishes were also observed. In both experiments, each treatment consisted of 3 replicate assays. Abnormal and dead individuals were identified by the lack of larval swimming activity or lack of mobility of juvenile podia. Room temperature during all experiments was regulated at 20 8C. Statistical significance of percent larval metamorphosis among the treatments and times was analyzed by
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two-way repeated-measures ANOVA followed by multiple comparison test of Fisher’s LSD. All the data showed normal distribution and homogeneous variances by Shapiro-Wilk’s W-test and Cochran’s test, respectively. 2.3. Analysis of DBM concentration diffused To determine the DBM concentration diffused from the 1/2 diluted solution across the hydrophobic PTFE membrane, 10 ml of filtered seawater in the upper vessel was removed by pipet immediately after discarding the DBM solution diluted by half in the lower vessel 5, 10, 15, 20 and 30 min after diffusing, and added to vials for analysis in a headspace autosampler. The headspace DBM in the solutions was generated using a Hewlett Packard model HP 7694 headspace autosampler. The operating conditions were as follows: the oven temperature, 60 8C; the vial equilibration time, 30 min; the injection volume, 20 Al. Electron impact mass analysis of the headspace DBM was performed with a Hewlett Packard model HP 5972 MSD gas chromatograph-mass spectrometer,
100
a
consisting of a DB-624 column (30 m 0.32 mm 1.8 Am (cyanopropyl-phenyl) methylpolysiloxane, Aligent Technologies). The electron energy was 70 eV. The operating conditions were as follows: the injector temperature, 200 8C; the detector temperature, 260 8C; the oven temperature, from 40 to 180 8C at 8 8C/min and from 180 to 260 8C at 15 8C/min; the He carrier flow rate, 1.5 ml/min. The concentration of headspace DBM was determined by comparing with a standardized authentic. The analysis was performed twice. Room temperature was 20 8C.
3. Results 3.1. Metamorphosis of S. nudus larvae Changes in percent metamorphosis of S. nudus larvae induced by each diluted DBM solution (diffusing for 1 h) over 24 h and the statistical significance are shown in Fig. 2 and Table 1. Significant differences in percent metamorphosis were found among DBM dilutions and times ( p b 0.0001). Induction of
a
a
90 a*
b c
c
b
d d
d
c
4
8
1/2 dilution 1/3 dilution 1/4 dilution 1/8 dilution 1/16 dilution Ulvella lens Blank
b*
70 60 50 40
a*
a*
b* 80
Metamorphosis (%)
a
a
b
b
c
c
c
30 20 10 0 0
12
16
20
24
Time (hrs) Fig. 2. Percent metamorphosis of S. nudus after 1 h exposure to different dilutions of saturated dibromomethane (DBM) solution, a control with U. lens, and a negative blank (filtered seawater). Each treatment consists of 3 replicates with ca. 20 larvae per replicate. Each vertical bar indicates standard error. Different letters indicate significant differences among treatments at each time. Asterisks show significant differences of percent metamorphosis from that at 1 h.
Y. Agatsuma et al. / Aquaculture 251 (2006) 549–557 Table 1 Two-way repeated-measures ANOVAs of percent metamorphosis of S. nudus exposed to (A) different dilutions of dibromomethane and (B) different exposure times Source of variation
df
MS
F
p
A. Dilution Error Time Dilution time Error B. Exposure time Error Time Exposure time time Error
3 8 5 15 40 7 16 4 28 64
31,006.429 289.141 554.735 312.357 13.392 23,021.964 868.510 474.744 153.919 13.311
107.236
b0.0001
41.422 23.32
b0.0001 b0.0001
26.507
b0.0001
35.666 11.564
b0.0001 b0.0001
h in each DBM solution ( p N 0.05). Contrary, for the positive control with U. lens, percent metamorphosis rose significantly and reached 98% after 24 h ( p b 0.01). Changes in percent metamorphosis of S. nudus larvae with different exposure times using 1/2 dilution (that induced the highest percent metamorphosis) are shown in Fig. 3. Significant differences in the percent metamorphosis among exposure times and times were also found ( p b 0.0001) (Table 1). After 1 h, percent metamorphosis for larvae exposed for 40, 20 and 10 min was N80%. No statistical difference in the percent metamorphosis among those exposure times was found throughout the 24 h period ( p N 0.05). The percent metamorphosis for 5, 2.5 and 1 min exposure after 1 h were lower and fell with shortening exposure time. No larvae metamorphosed in the blank control. There was only a significant difference between the times of 1 and 2 h exposed for 20 min ( p b 0.01). The percent metamorphosis after 24 h for the positive control with U. lens reached 97% and rose significantly from 1, 2 h to 4, 8, 24 h ( p b 0.05). No abnormal or dead 8-armed larvae or metamorphosed juveniles were observed in any experimental or control groups.
metamorphosis was dependent on DBM concentration. After 24 h, percent metamorphosis was highest (96%) at 1/2 dilution. No larvae metamorphosed at 1/ 8 or 1/16 diluted solutions or in the blank control after 24 h. Percent metamorphosis at 1/2 and 1/3 dilutions rose rapidly during the first 2 h. In particular, percent metamorphosis reached 93% after 1 h at 1/2 dilution. After that, no significant difference in percent metamorphosis was found among times until 24 h after 2 a*
100
a a
90
a a
Metamorphosis (%)
80 a b
70
a*
a*
a*
a ab
a a
aab
bc
b
cd*
c bc*
b ab*
60
40 minutes 20 minutes 10 minutes 5 minutes 2.5 minutes 1 minute Ulvella lens Blank
bc
50 40 c c
30
cd
e
c
20 10
c cd
553
d
c
e
d
0 0
4
8
12
16
20
24
Time (hrs) Fig. 3. Percent metamorphosis of S. nudus after different exposure times to 1/2 diluted solution from saturated dibromomethane (DBM) solution, a control with U. lens, and a negative blank (filtered seawater). Each treatment consists of 3 replicates with ca. 20 larvae per replicate. Each vertical bar indicates standard error. Different letters indicate significant differences among treatments at each time. Asterisks show significant differences of percent metamorphosis from that at 1 h.
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3.2. Metamorphosis of S. intermedius larvae Changes in percent metamorphosis over 24 h of S. intermedius larvae induced with different diluted DBM solutions (1 h exposure) and the statistical significances are shown in Fig. 4 and Table 2. Significant difference in percent metamorphosis was found among DBM dilutions and times ( p b 0.0001). After 1 h, the percent metamorphosis at 1/2 and 1/3 dilution sharply rose to 82% and 73%, respectively, and did not differ significantly between them ( p N 0.05). The percent reached 100% after 2 h in both treatments. At 1/4 dilution, the percent rose to 36% after 2 h. Thus, larval metamorphosis of S. intermedius was induced by a less diluted solution than for S. nudus. However, no metamorphosis occurred at 1/8 dilution or in the blank control. Significant differences were found in the percent metamorphosis among 1/2, 1/3 and 1/4 dilutions after 1 h and also between the times of 1 and 2, 4, 8 and 24 h at each of those dilutions ( p b 0.05). In the control with U. lens, the percent rose significantly until 4 h ( p b 0.05) and reached 70% after 24 h. a*
a*
Table 2 Two-way repeated-measures ANOVAs of percent metamorphosis of S. intermedius exposed to (A) different dilutions of dibromomethane and (B) different exposure times Source of variation
df
MS
A. Dilution Error Time Dilution time Error B. Exposure time Error Time Exposure time time Error
3 8 4 12 32 7 16 4 28 64
18,338.529 297.822 2296.475 311.680 69.792 17,851.704 56.797 313.379 291.143 39.250
p
61.575
b0.0001
32.905 4.466
b0.0001 b0.001
314.308
b0.0001
7.984 7.418
b0.0001 b0.0001
Changes in percent metamorphosis of S. intermedius larvae with different DBM exposure times to 1/2 dilution are shown in Fig. 5. Significant differences in the percent metamorphosis among exposure times and times were also found ( p b 0.0001) (Table 2). All the larvae metamorphosed after 1 h for N 5 min exposure. After 1 h, percent metamorphosis of larvae exposed for 2.5 min was 75%, which did not
a*
a*
100 a
90
b*
80
Metamorphosis (%)
F
70 60
b*
b*
c*
c*
a
50 b*
c*
40 1/2 dilution 1/3 dilution 1/4 dilution 1/8 dilution Ulvella lens Blank
30 20 b
10
b b
0 0
4
8
12
16
20
24
Time (hrs) Fig. 4. Percent metamorphosis of S. intermedius after 1 h exposure to different dilutions of saturated dibromomethane (DBM) solution, a control with U. lens, and a negative blank (filtered seawater). Each treatment consists of 3 replicates with ca. 20 larvae per replicate. Each vertical bar indicates standard error. Different letters indicate significant differences among treatments at each time. Asterisks show significant differences of percent metamorphosis from that at 1 h.
Y. Agatsuma et al. / Aquaculture 251 (2006) 549–557
a a
a
100 90
b
Metamorphosis (%)
80 70
c
b
b
c
c
555
a
a
b
a* b
c
c
d*
d*
bc*
60
40 minutes 20 minutes 10 minutes 5 minutes 2.5 minutes 1 minute Ulvella lens Blank
50 40 30
d
20 10 0 0
4
8
12
16
20
24
Time (hrs) Fig. 5. Percent metamorphosis of S. intermedius after different exposure times to 1/2 diluted solution from saturated dibromomethane (DBM) solution, a control with U. lens, and a negative blank (filtered seawater). Each treatment consists of 3 replicates with ca. 20 larvae per replicate. Each vertical bar indicates standard error. Different letters indicate significant differences among treatments at each time. Asterisks show significant differences of percent metamorphosis from that at 1 h.
increase after this time. The percent metamorphosis of larvae exposed for 1 min sharply rose to 82% after 1 h and then gradually to 84.2% until 2 h when it ceased, significantly higher than that for larvae exposed for 2.5 min after 1, 2, 4, 8 and 24 h ( p b 0.05). No metamorphosis occurred in the blank control. In the control with U. lens, the percent metamorphosis rose to 97% after 24 h. No abnormal or dead 8-armed larvae or metamorphosed juveniles were observed in any experimental or control groups.
3.3. Concentration of DBM diffused Analysis by gas chromatograph-mass spectrometer showed that 10 and 5 min diffusing at 1/2 diluted DBM solution, which induced a high percent metamorphosis of more than 80% for S. nudus and S. intermedius at 1 h after start of the experiment, corresponded to the low concentrations of 52–61 ppm and 34–43 ppm, respectively (Table 3).
4. Discussion Table 3 Concentration of dibromomethane solution diluted to 1/2 with filtered seawater at different diffused times Dilution
Time diffused (min)
Concentration (ppm)
Control 1/2 1/2 1/2 1/2 1/2
5 10 15 20 30
0 42.5 60.9 61.3 55.9 64.8
0 34.3 51.7 59.0 58.6 65.8
In the present study, an exposure to 1/2 diluted DBM solution for 1 h induced a high percent larval metamorphosis (more than 80%) with 10 min exposure for S. nudus and 5 min exposure for S. intermedius. The concentrations of DBM diffusing over these time frames were 52–61 ppm and 34–43 ppm, respectively. This study showed that a high percent of sea urchin larvae settle and metamorphose following brief exposure to low concentrations of DBM. Significantly higher percent metamorphosis of S. interme-
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dius after 1 min exposure than after 2.5 min exposure is considered attributable to differences in probability whether larvae can respond to DBM during these short times. In the positive control with live U. lens, the percent metamorphosis rose sharply until 4 h and then more gradually after this time. Taniguchi et al. (1994) monitored larval percent metamorphosis of S. nudus induced by live L. yessoense and U. lens in petri dishes with filtered seawater after 2, 8, 16 and 24 h. Percent metamorphosis induced by L. yessoense was high at 2 h after the start of the experiment. Contrary, the percent induced by U. lens was lower than that by L. yessoense, while it rose continually until 8 h and then more gradually after this time. This suggests release of DBM from U. lens is less than from L. yessoense. Even though the amount of DBM released by the algae is low, continual release from the thalli probably leads to a gradual increase in percent metamorphosis. In this study the percent metamorphosis induced by U. lens differed among each experiment despite a similar gradual increase. These differences may be caused by different densities of U. lens used. Naidenko (1996) reported that glutamine or glutamine mimetics may be active components of natural inducers from calcareous algae. Kitamura et al. (1993) reported that larval metamorphosis of Pseudocentrotus depressus and Anthocidaris crassispina following larval contact with the articulated coralline alga Corallina pilulifera is due to eicosapentaenoic acid produced in the algal thalli. A chemical cue inducing high metamorphic rate of S. droebachiensis larvae after their contact with the coralline algae Lithothamnion glaciale, Phymatolithon laevigatum, P. rugulosum and Corallina officinalis is thought to be g-aminobutyric acid (GABA) (Pearce and Scheibling, 1990). The present study has shown instantaneous metamorphosis of a high percent of sea urchin larvae by DBM. Qualitative studies on the mechanism of release and how much DBM is being released from U. lens and crustose corallines are necessary. For S. nudus and S. intermedius larvae, responding to DBM instantly with a high percent of metamorphosis would ensure a high recruitment level in crustose coralline habitats even though high mortality might occur after settlement. The number of sea urchins that settle fluctuates temporally and spatially and is associated with abundance of larval supply caused by oceano-
graphic conditions, water temperature, salinity, predation, and starvation (reviewed by Ebert, 1983; Balch and Scheibling, 2001). However, it is probable that crustose corallines have an important role in determining sea urchin population structure and size in rocky subtidal bottoms.
Acknowledgements We sincerely thank Dr. John M. Lawrence, University of South Florida, for his insightful comments on this paper. We are also grateful to T. Ono (managing director) and T. Maruzoe of the Fukushima Prefectural Fish Farming Center and Dr. H. Uto (directorgeneral), S. Motoya and Y. Sakai of the Hokkaido Institute of Mariculture for their support and cooperation in this study.
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