Settlement of mussels is commonly associated with macroalgae. The effects of 19 macroalgal species on the settlement and metamorphosis of pediveliger ...
Mar Biol (2007) 152:1121–1132 DOI 10.1007/s00227-007-0759-0
R ES EA R C H A R TI CLE
Larval settlement and metamorphosis of the mussel Mytilus galloprovincialis on diVerent macroalgae Jin-Long Yang · Cyril Glenn Satuito · Wei-Yang Bao · Hitoshi Kitamura
Received: 21 April 2007 / Accepted: 21 June 2007 / Published online: 17 July 2007 © Springer-Verlag 2007
Abstract Settlement of mussels is commonly associated with macroalgae. The eVects of 19 macroalgal species on the settlement and metamorphosis of pediveliger larvae of the mussel Mytilus galloprovincialis were investigated in the laboratory. Settlement and metamorphosis inducing activities of macroalgae Chlorodesmis fastigiata and Ceramium tenerrimum collected each month during the period between January 2005 and April 2006 were also investigated. Furthermore, C. fastigiata and C. tenerrimum were subjected to various treatments to investigate the roles of bacteria and diatoms on the algal surface in the induction of larval settlement and metamorphosis of M. galloprovincialis and the characteristics of the cues in these two macroalgae. Pediveliger larvae of M. galloprovincialis settled and metamorphosed in high percentages on Cladophora sp., Chlorodesmis fastigiata, Centroceras clavulatum, and Ceramium tenerrimum, all of which were Wlamentous in morphology. Macroalgae that were cylindrical, phylloid, Xabellate, palmate and pinnate all showed low (288 and >309 m, respectively (Satuito et al. 2005). Straight-hinge veliger larvae were also stored inside a refrigerator for a maximum period of 3 months and were cultured inside an incubator to the pediveliger stage when needed in assays. This ensured the supply of pediveligers almost all year round. Refrigeration had no adverse eVects on the survival, growth, settlement behavior and metamorphosis of larvae (Satuito et al. 2005). Conditions for storing larvae in the refrigerator were the same as that reported by Satuito et al. (2005). Conditions for culturing the refrigerated larvae were the same as described above. Larval settlement and metamorphosis bioassays Twenty pediveliger larvae were released to each glass Petri dish (Ø 60 mm £ 20 mm height) containing 20 ml of 0.22 m Millipore Wltered seawater (0.22 m FSW) and a piece of the test macroalga (fresh alga, treated alga, etc.). Settlement inducing activities of these macroalgae were evaluated by the percentage of metamorphosis to post-larvae obtained after 48 h from the start of assays. Post-larvae were veriWed under the microscope as individuals with post-larval shell growth. Petri dishes, each containing 20 larvae and 20 ml of 0.22 m FSW were also set as controls during assays. All assays were conducted at 17 § 1°C in a dark environment. In assays to screen the settlement inducing activities of the diVerent algal species, wet weights of macroalgae used were from 0.025 to 0.2 g per Petri dish. In the other assays to test the eVect of treatments (formalin, ethanol, heat, and antibiotics) and culture conditions (culturing with GeO2 added into the culture medium and culturing the apical segments of
1123
the alga) on C. fastigiata and C. tenerrimum activities, the wet weight of the treated or cultured alga used per Petri dish was 0.1 g. For each algal species, treatment, and culture condition, at least six replicate assays were conducted using larvae from at least two separate culture batches. In assays to investigate the eVects of the conditioned water and crude extracts of C. fastigiata and C. tenerrimum at least six replicate assays were also conducted using larvae from at least two separate culture batches. During assays with treated C. fastigiata and C. tenerrimum and with their respective conditioned water and crude extracts, algae collected on the same day of the assays were also set for comparison. Collection of macroalgae Nineteen diVerent macroalgal species including six green algae (Enteromorpha compressa, Ulva pertusa, Cladophora sp., Chlorodesmis fastigiata, Codium fragile, and Bryopsis plumosa), Wve brown algae (Dilophus okamurae, Padina arborescens, Myelophycus simplex, Hizikia fusiformis, and Sargassum thunbergii) and eight red algae (Amphiroa zonata, Corallina pilulifera, Gelidium elegans, Grateloupia Wlicina, Grateloupia turuturn, Centroceras clavulatum, Ceramium tenerrimum, and Heterosiphonia pulchra) were collected from a rocky shore in Koe-machi, Nagasaki (129°50⬘E; 32°45⬘N), and a wharf of the Nagasaki Prefectural Institute of Fisheries, Taira-machi, Nagasaki (129°51⬘E; 32°43⬘N), Japan, and evaluated for their ability to induce larval metamorphosis in M. galloprovincialis. Thalli that appeared healthy and clean were selected for the assays. These were brought back to the laboratory on the day of the assay and were thoroughly washed with FSW prior to use in assays. Young bivalves (SL: 75% metamorphosis to post-larvae. The other 15 species all showed low settlement and metamorphosis inducing activities. The other four Wlamentous macroalgae (B. plumose, M. simplex, G. elegans, and H. pulchra) induced 0.05, ANOVA, Fig. 1), implying that activities of these two algae were not inXuenced by seasonal variations. EVects of treatments on activities of C. fastigiata and C. tenerrimum Percentages of post-larvae and corresponding cell survivals of C. fastigiata and C. tenerrimum treated with formalin, ethanol and heat are as shown in Fig. 2. The average percentage of post-larval metamorphosis on UT C. fastigiata was 78% (Fig. 2a). However, percentages of post-larvae on FA, 10E, 20E, 50E, 100E, 35H, 40H, and 100H C. fastigiata signiWcantly decreased (P < 0.001, ANOVA), suggesting that the cue in C. fastigiata may have been signiWcantly or completely destroyed by the above treatment conditions. Algal cell survival in UT C. fastigiata was 94%. Algal cell survival in treated (FA, 10E, 20E, 50E, 100E, 35H, 40H, and 100H) C. fastigiata where percentages of post-larvae signiWcantly decreased, also decreased and were between 0 and 37% (P < 0.001, ANOVA, Fig. 2a). A similar tendency was observed in the case of C. tenerrimum where the UT alga, which induced 77% post-larval metamorphosis, had 96% algal cell survival (Fig. 2b). Moreover, treated (FA, 10E, 20E, 50E, 100E, 35H, 40H, and 100H) C. tenerrimum that had signiWcantly lower percentages of post-larvae (P < 0.001, ANOVA) also had signiWcantly lower percentages of algal cell survivals (0–56%) than the UT alga (P < 0.05, ANOVA followed by Tukey– Kramer HSD test: UT alga versus each treated alga, Fig. 2b). These results imply that settlement inducing activities
123
1126 Table 1 Taxonomic classiWcation of the 19 species of macroalgae and their settlement and metamorphosis inducing activities expressed in percentages of post-larvae
Mar Biol (2007) 152:1121–1132
Phyla
Morphology
Weight (g)
Enteromorpha compressa
c
0.1
6
8
Ulva pertusa
ph
0.1
8
4
8
Cladophora sp.
W
0.1
9
81
3
0.2
6
89
0.025
6
12
0.05
9
53
0.1
21
83
0.2
6
90
Algal species
n
Post-larvae (%)
Kruskal–Wallis (rank sums)a
CHLOROPHYTA
Chlorodesmis fastigiata
W
6
2
Codium fragile
c
0.1
10
7
7
Bryopsis plumosa
W
0.1
14
13
5
0.2
6
13
PHAEOPHYTA Dilophus okamurae
X
0.1
7
4
9
Padina arborescens
X
0.1
7
1
15
Myelophycus simplex
W
0.1
7
2
14
Hizikia fusiformis
c
0.1
7
2
11
Sargassum thunbergii
c
0.1
7
6
10
Amphiroa zonata
c
0.1
10
3
17
Corallina pilulifera
pa
0.1
7
1
13
Gelidium elegans
W
0.1
11
1
18
0.2
6
2 2
16
RHODOPHYTA
Grateloupia Wlicina
pi
0.1
9
Grateloupia turuturu
ph
0.1
9
1
19
Centroceras clavulatum
W
0.1
6
48
4
0.2
6
76
0.025
6
26
0.05
6
55
0.1
30
78
0.2
6
85
0.1
9
3
0.2
6
6
Ceramium tenerrimum The letters of c, ph, W, X, pa, pi indicate cylindrical, phylloid, Wlamentous, Xabellate, palmate, pinnate, respectively a Score sums were ranked from 1 to 19
Heterosiphonia pulchra
W
W
1 12
of these two algae were signiWcantly aVected by their cell survival and that these algae induced mussel larval settlement and metamorphosis only when alive. Alternatively, results also suggest that treatments possibly altered the surface chemistry of these algae either by cross-linking (formalin treatment), denaturing (heat treatment) or extracting lipid components (ethanol treatment) of extracellular products on the surface of algae.
at all concentrations (10, 100, and 1,000%) tested (Table 2). Percentages of post-larval metamorphosis on the conditioned water and crude extracts of C. tenerrimum were between 0 and 2% at all concentrations (10, 100, and 1,000%) tested (Table 2).
Activities of conditioned water and crude extracts of C. fastigiata and C. tenerrimum
Percentages of post-larvae on C. fastigiata and C. tenerrimum and their corresponding numbers of bacteria after treatment with antibiotic solution are as shown in Fig. 3. In C. fastigiata, the percentage of post-larvae on the UT alga was 79% and the corresponding number of bacteria was
Percentages of post-larval metamorphosis on the conditioned water and crude extracts of C. fastigiata were all 0%
123
EVects of bacteria and diatoms on the settlement inducing activities of C. fastigiata and C. tenerrimum
Temperature (°c )
Mar Biol (2007) 152:1121–1132
1127 100
30 20
100
(a)
80
80
60
60
100
40
40
80
20
20
0
0
10
60 40 20
(b)
100
100
80
80
60
60
40
40
20
20
0
0
Algal cell survival (% )
Post-larvae (% )
Post-larvae ( % )
0
Ja n Fe b M ar A pr M ay Ju n Ju l A ug Se p O ct N ov D ec Ja n Fe b M ar A pr
0 2005
2006
Fig. 1 Percentages of post-larvae (Mytilus galloprovincialis) on Chlorodesmis fastigiata and Ceramium tenerrimum collected each month from January 2005 to April 2006 and the corresponding seawater temperature. Open and shaded boxes indicate C. fastigiata and C. tenerrimum, respectively. Shaded circles indicate temperature of seawater measured at the time of the sampling. Data are means (+SD) of six replicates
7.7 £ 106 cells per 0.1 g alga. Treatment of C. fastigiata with diVerent concentrations of the antibiotic mixture signiWcantly reduced the number of bacteria (P < 0.001, ANOVA). The number of bacteria in 10 AB C. fastigiata was 1.3 £ 106 cells per 0.1 g alga; »80% reduction from that of the UT alga (P < 0.05). However, antibiotic treatment did not aVect the inducing activity of the alga and percentages of post-larvae in 1 AB and 10 AB algae were at the same level as that of the UT algae (P > 0.05). Similarly, treatment of C. tenerrimum with antibiotic solution signiWcantly reduced the numbers of bacteria in treated algae (P < 0.001, ANOVA), and was 1.3 £ 106 cells per 0.1 g alga in the 10 AB alga; 91% reduction in number as compared to the control (UT alga). However, percentages of post-larvae on antibiotic treated algae remained constantly high (P > 0.05, ANOVA); 62 and 65% post-larvae for 1 AB and 10 AB algae, respectively. Percentages of post-larvae on C. fastigiata and C. tenerrimum cultured in SW media with and without GeO2, and their corresponding numbers of diatoms are as shown in Fig. 4. Freshly collected alga induced 80% post-larval metamorphosis and had 3.2 £ 104 diatom cells per 0.1 g of the alga. When cultured in the laboratory for 3 weeks, the number of diatoms per 0.1 g of the cultured alga increased nearly seven-fold in SW media without GeO2 but decreased signiWcantly to 2.0 £ 103 cells per 0.1 g alga in the SW medium containing GeO2 (P < 0.05); a nearly 94% decrease as compared to that of the freshly collected alga. In contrast, no signiWcant diVerence in percentages of post-larvae was observed between algae cultured in media
UT
FA
10
E
20
E
50
E
10
0E 30H 35H 40H 00H 1
Treatments Fig. 2 Percentages of post-larvae (Mytilus galloprovincialis) and algal cell survivals in treated Chlorodesmis fastigiata (a) and Ceramium tenerrimum (b). Shaded squares indicate algal cell survival. UT indicates algae that were not treated. FA indicates 5% formalin treated algae. 10E, 20E, 50E, and 100E indicate algae treated with ethanol at concentrations of 10, 20, 50, and 100%, respectively. 30H, 35H, 40H, and 100H indicate algae heated at 30, 35, 40, and 100°C, respectively. Data are means (+SD) of 6–15 replicates Table 2 Settlement and metamorphosis inducing activities, expressed in percentages of post-larvae, of conditioned water and crude extracts prepared from Chlorodesmis fastigiata and Ceramium tenerrimum Algal species
Concentration (%)
n
Post-larvae (%)
21
83 § 7
Chlorodesmis fastigiata UT Conditioned water
10, 100, 1,000
6–12
0§0
Crude extracts
10, 100, 1,000
6
0§0
36
75 § 13
Conditioned water
10, 100, 1,000
6–29
0§0
Crude extracts
10
14
1§3
100
25
2§4
1,000
16
1§3
Ceramium tenerrimum UT
Hundred percentage of concentration means 0.1 g alga per 20 ml FSW
with and without GeO2 even after 3 weeks of culture (1 week: algae cultured with GeO2 versus without GeO2, P > 0.05; 3 weeks: algae cultured with GeO2 versus without
123
1128
Mar Biol (2007) 152:1121–1132
P o s t - l ar v a e ( % )
80 60 10 7 40
Bacteria (cells . 0.1g)
10 8
100
20 10 6
0
UT
1 AB
10 AB
Antibiotic treatments Fig. 3 Percentages of post-larvae (Mytilus galloprovincialis) and the corresponding numbers of bacteria in untreated algae (UT) and antibiotic solution treated (AB) algae. Open and shaded boxes indicate Chlorodesmis fastigiata and Ceramium tenerrimum, respectively. Closed triangles indicate numbers of bacterial cells. 1 AB and 10 AB indicate algae treated with antibiotics solution. 1 AB = streptomycin sulphate 20 mg l¡1, penicillin G 10 mg l¡1, neomycin 2 mg l¡1, and kanamycin 10 mg l¡1, 10 AB = 1 AB concentration increased tenfold. Data are means (+SD) of six replicates
100
(a)
10 6
80 10 5
40
Post-larvae (% )
10 4 20 10 3
0 100
(b)
10 6
Diatoms (cells . 0.1g)
60
80 10 5 60 40
10 4
20 10 3 0
0
1
2
3
Culture Periods (weeks) Fig. 4 Percentages of post-larvae (Mytilus galloprovincialis) and the corresponding numbers of diatoms in Chlorodesmis fastigiata (a) and Ceramium tenerrimum (b) cultured in SW media with and without GeO2. Shaded diamond shaped marks indicate numbers of diatom cells. Open and shaded boxes indicate fresh C. fastigiata and C. tenerrimum, respectively. Striped boxes indicate C. fastigiata and C. tenerrimum cultured in SW without GeO2; grid boxes, C. fastigiata and C. tenerrimum cultured in SW with GeO2. Data are means (+SD) of 6–12 replicates
123
GeO2, P > 0.05). In addition, the percentage of post-larvae on C. fastigiata cultured for 3 weeks in SW medium with GeO2 remained constantly high and was at the same level as that of the fresh alga (P < 0.05, Fig. 4a). A similar tendency was observed when C. tenerrimum thalli were cultured in SW media with and without GeO2. After 3 weeks of culture, the number of diatoms per 0.1 g C. tenerrimum cultured in SW medium with GeO2 decreased by 92% as compared to that of the freshly collected alga (P < 0.05). However, percentages of post-larvae on all cultured C. tenerrimum remained constantly high and were at the same level as that of the fresh alga (P > 0.05, Fig. 4b). In addition, no signiWcant diVerence in percentages of post-larvae was observed between algae cultured in media with and without GeO2 for 3 weeks (P > 0.05). These results suggest that bacteria and diatoms may not play a crucial role in the production of the potential cue of C. fastigiata and C. tenerrimum. The activity of C. tenerrimum cultured from apical segments Percentages of post-larvae on C. tenerrimum cultured from naturally grown thalli and those from apical segments, and their corresponding numbers of bacteria are as shown in Fig. 5. The percentage of post-larvae on fresh C. tenerrimum was 77%. When naturally grown thalli of C. tenerrimum were brought to the laboratory and cultured in SW medium, the settlement inducing activity of this cultured alga remained constantly high until after 4 weeks of culture (P > 0.05). However, the percentage of post-larvae on this cultured alga gradually decreased to 63% after 5 weeks (P < 0.05). In contrast, C. tenerrimum cultured from apical segments for 3 weeks showed no inducing activity and the percentage of post-larvae remained 0% even after 5 weeks of culture. Addition of bacteria, obtained from freshly collected naturally grown alga, into the SW medium containing C. tenerrimum cultured for 4 weeks and then culturing it for another week had no eVect and post-larval metamorphosis on this cultured alga remained 0% (Fig. 5a). The number of bacteria on fresh C. tenerrimum was 1.3 £ 107 cells per 0.1 g alga. This number gradually increased with the culture period and reached 6.6 £ 107 cells after 5 weeks. After 5 weeks, the numbers of bacteria on the two types of algae cultured from apical segments were 2.5 to 4.4 £ 107 cells per 0.1 g alga. These numbers were signiWcantly higher than that of the fresh alga (P < 0.001, ANOVA, Fig. 5b).
Discussion and conclusions Macroalgae had been reported to aVect larval settlement and metamorphosis of many invertebrates, e.g., two bryozoan
Mar Biol (2007) 152:1121–1132
(a)
100
Post-larvae (% )
1129
80 60 40 20
Bacteria (cells . 0.1g)
0 10 8
(b)
10 7
0
10
20 30 Culture Periods (days)
40
Fig. 5 Percentages of post-larvae (a) (Mytilus galloprovincialis) and the corresponding numbers of bacteria (b) in Ceramium tenerrimum cultured in the laboratory from naturally growth thalli (open triangle) and apical segments (open circle, open square). Open triangles, circles, and squares indicate naturally grown alga, cultured apical segments and cultured apical segments with the addition of bacteria from fresh alga on the 4th week of culture, respectively. Shaded triangles, circles, and squares indicate corresponding bacterial numbers of fresh alga, cultured apical segments and cultured apical segments with the addition of bacteria from fresh alga on the 4th week of culture, respectively. Data are means (+SD) of six replicates
species (Walters et al. 1996; Schmitt et al. 1998), a polychaete species (Walters et al. 1996; Harder et al. 2004), mollusks (Morse and Morse 1984; Yvin et al. 1985; Dobretsov 1999; Krug and Manzi 1999; Daume et al. 2000; Huggett et al. 2005) and echinoderms (Pearce and Scheibling 1991; Kitamura et al. 1993; Takahashi et al. 2002; Swanson et al. 2004; Li et al. 2004a, b). Mussel larval settlement has also been associated with macroalgae, and a large volume of literature is available on this subject (e.g., Bayne 1964, 1965; Eyster and Pechenik 1987; CáceresMartínez et al. 1994; Davis and Moreno 1995; Dobretsov 1999; Alfaro et al. 2006; Bulleri et al. 2006). In the present investigation, the authors have demonstrated through laboratory experiments that larvae of the mussel M. galloprovincialis responded diVerently to diVerent macroalgae and settled and metamorphosed in high percentages on the algae C. fastigiata, Cladophora sp., C. clavulatum and C. tenerrimum, all of which were Wlamentous in morphology. In addition, the settlement and metamorphosis inducing activities of C. fastigiata and C. tenerrimum remained constantly high throughout the investigation, regardless of the season. The Wnding that M. galloprovincialis settled and metamorphosed on Wlamentous algae is consistent with
reports on M. edulis larvae (Bayne 1964; Petersen 1984; Eyster and Pechenik 1987; Dobretsov 1999). CáceresMartínez et al. (1994) concluded from Weld observations that M. galloprovincialis used Wlamentous, thallus and membranous algae among other substrates for direct settlement. In the present investigation, algae that were not Wlamentous all showed low (
