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3. Results
3.1. Metamorphosis and settlement During the late doliolaria and early pentactula stages, the larvae repetitively touched
the substrate for hours each day, crawling and exploring the bottom with their five buccal podia. Given the relatively short monitoring period of 1 h, no behavioural
difference was noted between night and day. Overall, late doliolaria larvae spent 8463 of the time swimming and attached to the substrate only for short periods of time,
21
around 60635 s. Doliolaria larvae were estimated to move . 3 m h under static
laboratory conditions. Early pentactula larvae lost their ciliated band and their capacity to swim. They moved by small successive jumps, spending 9362 of their time
2
attached to the substrate, restricted roughly within 1 cm and exhibiting only minimal movements. Once settled using their ambulacral podia, the larvae moved on the substrate
and fed using their feeding podia. Most of the time, four buccal podia were attached to the substrate and one was transferred to the mouth. At this stage, the larvae were
negatively phototaxic and migrated to the shaded side of the substrate. Feeding of older pentactula larvae was continuous for periods of 2.160.5 h, separated by intervals of
immobility lasting 30 min to 1 h.
The doliolaria and pentactula larvae responded differently to a variety of settling conditions Fig. 1. Metamorphosis and settlement were delayed for nearly 96 h in the
absence of an adequate substrate. Ultimately, the vast majority of larvae did not reach the late auricularia and pentactula stages in control containers and died before settlement
Fig. 1. Metamorphosis into pentactulae occurred after ca. 48 h in the presence of crushed coral, 3 ml of sand or complete sand cover, whether conditioned or not Figs. 1
and 2, but settlement rates remained comparable to controls one-way ANOVA, F 5 19.0, df 5 10,22, P , 0.0001; SNK test, P . 0.05. Rates of settlement were greater
on all other substrates and substrate combinations than for the controls SNK test, P , 0.05. Overall, significantly higher proportions of settled pentactulae ca. 4.8–
10.5 were observed whenever T
. hemprichii leaves were present SNK test, P , 0.05. Cleaned and uncleaned seagrass leaves yielded equally high settlement rates when
present in a combination SNK test, P . 0.05. 3.2. Settlement preferences between different substrates and seagrass leaves
Settlement was consistently greater on T . hemprichii leaves than on all other
substrates Fig. 2. When clean and uncleaned leaves were offered in separate trials, a greater number of pentactulae were recovered from the former one-way ANOVA,
F 5 21.4, df 5 6,14, P , 0.0001; SNK test, P , 0.05. However, when clean and unclean leaves were offered to larvae simultaneously, there was no significant difference in
settlement paired t-test, P 5 0.640. The number of settled pentactulae on sand and crushed coral was consistently low , 20 larvae, regardless of whether they were
offered as a single substrate or in combination with another Fig. 2. There was no significant difference in abundance of pentactula larvae on adult-conditioned sand and
ordinary sand SNK test, P . 0.05. However, the presence of 3 ml of sand resulted in
A .
Mercier et
al .
J .
Exp .
Mar .
Biol .
Ecol .
249 2000
89 –
110
97 Fig. 1. Holothuria scabra. Abundance of larval stages mean6S.D. on different substrates during settlement. The final settlement rate is indicated as a mean for
the three replicates of each treatment.
98 A
. Mercier et al. J. Exp. Mar. Biol. Ecol. 249 2000 89 –110
Fig. 2. Holothuria scabra. General substrate preferences trial. Number of late pentactulae mean6S.D. settled on each substrate in different treatments.
significantly greater settlement than complete coverage with ordinary or adult-con- ditioned sand SNK test, P , 0.05. Crushed coral supported significantly greater
settlement than sand, both in paired comparisons paired t-test, P 5 0.033 and when essayed alone SNK test, P , 0.05.
3.3. Settlement preferences between seagrass and other natural or artificial plants In the trial comparing settlement on different species of plants from soft-bottom
habitats, pentactula larvae settled preferentially on seagrass leaves Fig. 3. Among single substrate treatments, there was significantly greater settlement on E
. acoroides compared to C
. racemosa and H. gracilis one-way ANOVA, F 5 27.8, df 5 2,6, P 5 0.0009; SNK test, P , 0.05. Pairwise comparisons of combination assays showed
A . Mercier et al. J. Exp. Mar. Biol. Ecol. 249 2000 89 –110
99
Fig. 3. Holothuria scabra. Soft bottom plants trial. Number of late pentactulae mean6S.D. settled on each substrate in different treatments.
that T . hemprichii leaves were preferred to C. racemosa and H. gracilis paired t-tests,
P , 0.05, whereas there was similar settlement on T . hemprichii and E. acoroides
paired t-test, P 5 0.585; Fig. 3. Very few late pentactulae settled on plants from hard substrates compared to the
numbers settling on T . hemprichii leaves Fig. 4. In single treatments, B. coacta
received more larvae than S . cristaefolium one-way ANOVA, F 5 8.42, df 5 2,6,
P 5 0.0181; SNK test, P , 0.05, but similar settlement to E . breviarticulatus SNK test,
P . 0.05. Conditioned artificial seagrass was an effective substrate for settlement of H
. scabra larvae but clean artificial leaves were not Fig. 5. Significantly more larvae settled on T
. hemprichii than conditioned artificial seagrass in paired comparisons paired t-tests,
P , 0.05. However, there was no significant difference in settlement of H . scabra
larvae on conditioned artificial seagrass and the alga C . racemosa paired t-test,
P , 0.5.
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Fig. 4. Holothuria scabra. Hard bottom plants trial. Number of late pentactulae mean6S.D. settled on each substrate in different treatments.
3.4. Effect of plant extracts Settlement varied significantly among containers given different plant extracts one-
way ANOVA, F 5 248.3, df 5 3,8, P , 0.0001; Fig. 6. H . gracilis extracts did not
induce high rates of settlement; the larvae remained pelagic until they died, responding similarly to the larvae that were kept under control conditions SNK test, P . 0.05.
However, extracts of E . acoroides and T. hemprichii induced a similar response SNK
test, P . 0.05, both yielding significantly higher settlement rates than controls or H .
gracilis after 24 h SNK test, P , 0.05. Nonetheless, the fastest response was obtained when the larvae were exposed to extracts from T
. hemprichii Fig. 6: the larvae began to settle ca. 6 h after the first addition of extract and 100 of individuals were found on
the bottom after 21 h. In comparison, few larvae exposed to extracts from E. acoroides were still floating after 24 h Fig. 6.
A . Mercier et al. J. Exp. Mar. Biol. Ecol. 249 2000 89 –110
101
Fig. 5. Holothuria scabra. Artificial seagrass trial. Number of late pentactulae mean6S.D. settled on each substrate in different treatments.
3.5. Effect of substrate on growth Pentactulae that settled on cleaned and uncleaned T
. hemprichii leaves were of the same average size at the end of the experiment Student t-test, P . 0.05; Table 1.
Pentactulae that settled on sand and crushed coral were also the same average size Mann–Whitney U-test, P . 0.05. However, pentactulae from T
. hemprichii leaves were about 10–35 larger than those from all other substrates, including sand and coral.
This difference in size was significant Mann–Whitney U-test, P . 0.05 Table 1.
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Fig. 6. Holothuria scabra. Abundance of different larval stages mean6S.D. over time after addition of various plant extracts.
3.6. Post-settling migration After 16–17 days of pelagic stages, the larvae of H
. scabra settled on the leaves of the seagrass T
. hemprichii and remained there for about 12 days until they reached a size of 3.960.4 mm long Fig. 7. The next ca. 6 days where spent moving between the leaves
and the sand until day 35 after fertilisation. A negative phototaxic response was not observed during this period. The juveniles of 6.160.3 mm long were associated only
with the sandy habitat, although the characteristic burrowing behaviour of the adults and
A . Mercier et al. J. Exp. Mar. Biol. Ecol. 249 2000 89 –110
103 Table 1
a
Holothuria scabra: size and survival rate of late pentactulae settled under different conditions Conditions tested
Length mm n
Survival rate in bowl
Clean T. hemprichii 592.8691.1
83 10.561.9
vs T. hemprichii 574.1686.7
83 Clean T. hemprichii
594.3689.1 88
5.261.7 vs sand
500.0650.0 3
T. hemprichii 578.5688.6
100 6.062.3
vs sand 430.0683.7
5 Clean T. hemprichii
577.96103.5 122
8.360.7 vs crushed coral
521.96103.2 16
T. hemprichii 575.0684.0
78 4.861.1
vs crushed coral 470.0657.0
5 Sand
– 1.160.5
vs crushed coral 415.8655.4
19 T. hemprichii
625.9693.2 111
6.761.5 Crushed coral
419.6649.4 23
1.460.6 Adult-conditioned sand
421.8660.1 4
1.260.7 Sand
450.06100.0 3
0.260.8 Three-ml sand
375.06143.6 7
0.860.4 Water only
416.7660.6 6
0.560.5
a
The number n of pentactulae measured on each substrate is shown and survival rate was calculated for three replicate containers. All data as mean6S.D.
Fig. 7. Holothuria scabra. Growth mean6S.D., n 5 38–183 and behaviour of settling larvae and newly settled juveniles over time.
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larger juveniles Mercier et al., 1999 was recorded only when individuals reached ca. 11 mm, approximately 45 days after fertilisation Fig. 7.
4. Discussion
