S .K. Meidel et al. J. Exp. Mar. Biol. Ecol. 240 1999 161 –178
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from a single locality and rear them to reproductive maturity under tightly controlled conditions. Because food quality and quantity varies widely between adult populations in
kelp beds and barrens Meidel and Scheibling, 1998, any effect of parental nutrition on larval survival and metamorphosis may influence the overall reproductive success of this
species. Understanding the potential importance of parental condition in determining larval quality may shed light on the population dynamics of sea urchins in shallow water
communities.
2. Materials and methods
2.1. Experimental design To investigate the simple and interactive effects of parental conditioning and larval
nutrition on larval development and metamorphosis of Strongylocentrotus droebach- iensis, we conducted a 2 3 2 factorial experiment in the laboratory. To attain different
levels of parental conditioning, we collected juvenile sea urchins horizontal test diameter: 13–17 mm from a barren ground at 6–8 m depth off Little Duck Island in
Mahone Bay, Nova Scotia, Canada, and reared them to reproductive maturity as part of a broader 22-month May 1995 to March 1997 feeding experiment Meidel and
Scheibling, 1999. Feeding treatments used in the present study are 1 KM, a high
21
ration of kelp blades of Laminaria longicruris and L . digitata for 6 days week
21
augmented with mussel flesh Mytilus edulis and M . trossulus for 1 day week
, and
21
2 KL, a low ration of kelp 1 day week . Kelp and mussel flesh were provided ad
libitum for the prescribed periods and unconsumed portions removed as necessary see Meidel and Scheibling, 1999, for details of this experiment. At the peak of their second
reproductive season March 1997, sea urchins of both sexes in the KM treatment had a significantly higher mean gonad index gonad wet weight expressed as a percentage of
total body wet weight than those in the KL treatment Meidel and Scheibling, 1999; Table 1. Sea urchins in the KM treatment also were significantly larger and heavier than
those in the KL treatment. Mean egg size as cross-sectional area, however, did not differ significantly between the two treatments Table 1.
On March 20, 1997, we induced spawning in ten randomly selected sea urchins from
Table 1 Summary of somatic and reproductive parameters in adult sea urchins fed kelp plus mussel flesh KM or a
a
low ration of kelp KL Parameter
KM KL
Test diameter mm 49.060.6 31
25.260.6 22 Total body weight g
54.761.7 31 7.860.5 22
Gonad index Female
38.361.1 7 11.261.0 7
Male 28.961.0 8
8.060.6 7
4 2
Egg cross-sectional area 310 mm 1.2660.05 16
1.1260.03 22
a
Data are mean6S.E.; sample size is in parenthesis.
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.K. Meidel et al. J. Exp. Mar. Biol. Ecol. 240 1999 161 –178
each of the two parental treatments by coelomic injection of 0.5–1.5 ml of 0.53 M KCl. Females five and six in the KM and the KL treatment, respectively spawned into glass
beakers containing | 120 ml of chilled, 0.45-mm Millipore -filtered seawater hereafter referred to as filtered seawater and males five and four spawned into dry, chilled trays.
After ensuring that female spawn contained only ripe eggs identified by the absence of a large nucleus and nucleolus suggestive of breakdown of the germinal vesicle, we
washed it three times with filtered seawater. We checked dry sperm from three males for motility and mixed it in a chilled tray. We collected sperm by inserting the tip of a clean
spatula into the mixture and then added it to the eggs of individual females. After stirring the egg–sperm mixtures gently for 10 min, we rinsed the eggs three times with
filtered seawater to remove excess sperm. Fertilization rates of individual females were calculated as the percentage of 100 eggs showing an elevated perivitelline membrane.
For each parental treatment, the fertilized eggs of the three females with the highest fertilization rates mean6S.E.: KM, 94.0 60.9; KL, 98.7 60.6 were mixed and
distributed among six glass finger bowls containing 135 ml of filtered seawater, to form a monolayer in each bowl.
We reared embryos for 48 h in finger bowls at 128C before transferring them to 12 six each for the KM and KL treatments 4-l glass jars containing 2 l of filtered seawater.
For the remainder of the experiment, larvae were kept at 98C under a 12:12-h light dark
22 21
photoperiod at a light intensity of 39.4 62.9 mEin m s
mean6S.D., n 5 3 hereafter referred to as standard culture conditions. Cultures were stirred with T-
paddles rotating at 10 rpm. At the start of the experiment, larval density in culture jars
21
was | 1 larva ml . From each parental treatment KM, KL, we randomly assigned
21
three replicate jars to a high ration 5000 cells ml and three to a low ration 500 cells
21
ml of the green alga Dunaliella tertiolecta
, grown in f 2 nutrient medium under constant fluorescent illumination at 238C. Every 2 days, we replaced 60–70 of the
culture water with freshly filtered seawater and, starting at 4 days after fertilization when larvae were in the late prism stage, added exponentially growing algae. Cultures of the
high ration treatment were terminated at 39 days when most larvae had settled. Cultures of the low ration treatment were terminated at 55 days when development had
effectively ceased and only few larvae had reached competency. We chose larval food rations and the temperature regime for consistency with Hart and Scheibling 1988a.
2.2. Larval development and morphology At 6, 12, 19, 22, 27, and 33 days after fertilization for all treatment combinations, and
at 55 days for the low ration treatment only, we removed 8–29 larvae from each jar and videotaped them on glass microscope slides using a Hi8 camera Panasonic WV-3170A
and tapedeck Sony EV-S900 NTSC connected to a binocular microscope Leitz Labovert. We analysed video records using an image analysis system NIH Image,
Version 1.60; National Institutes of Health, Bethesda, Maryland, USA. We classified larvae as being in the 4, 6, or 8-arm developmental stage. When first analysed at 6
days, all larvae were clearly in the 4-arm stage with arm lengths . 165 and . 80 mm for the 1st and 2nd arm pair, respectively. We considered larvae as having attained the
6- or 8-arm stage when the 3rd or 4th arm pair, respectively, exceeded a length of 25
S .K. Meidel et al. J. Exp. Mar. Biol. Ecol. 240 1999 161 –178
165
mm. For each replicate jar, we calculated the frequency of each developmental stage as a percentage of the total number of larvae sampled.
For measurements of growth and morphology, we analysed subsamples of six to ten larvae per jar at each sampling date. We measured the length of the postoral,
anterolateral, posterodorsal and preoral arms from the base to the arm tip; the body length from the posterior tip to the transverse ciliary band between the postoral arms;
and the body width at the base of the postoral arms or between the posterior epaulettes once they had developed.
To record the decline in larval number over time due to mortality or metamorphosis, we counted larvae in three to four replicate subsamples of 5 ml from each jar at 2 upon
transfer to the culture jars, 22, 39, and 55 days.
2.3. Metamorphosis and size of settlers At 33 and 36 days after fertilization high ration treatment or 55 days low ration
treatment, we induced metamorphosis of larvae in the 8-arm stage with a well- developed rudiment. We pipetted 30 high ration or 15 low ration larvae from each
culture jar into each of three finger bowls containing 100 ml of filtered seawater. Each bowl contained one pebble | 2 cm in diameter collected from a nearby subtidal site
and encrusted with coralline algae Phymatolithon laevigatum, Lithothamnion glaciale, which are known to induce metamorphosis in Strongylocentrotus droebachiensis Pearce
and Scheibling, 1990, 1991. Before use in the induction trials, we scrubbed pebbles to remove any organisms adhering to the algae. We calculated the rate of metamorphosis
after 24 h as the number of settlers expressed as a percentage of the total number of individuals larvae and settlers retrieved on average, . 98 were retrieved, and
videotaped settlers on glass slides for measurement of test diameter. All settlers from replicate bowls from each culture jar were then pooled in a clean glass bowl and kept for
1 week without added food under standard culture conditions before being videotaped again.
2.4. Statistical analysis To determine the effects of parental conditioning and larval food ration on larval
morphology, we used principal components analysis PCA based on measurements of arm length mean of two measurements for each of four arm pairs, body length and
body width. All measures were log transformed, and a small constant 1 was added to allow inclusion of zero lengths for preoral arms in larvae fed the low ration. For PCA,
we used the correlation rather than covariance matrix because some measurements preoral arm lengths differed by two orders of magnitude between larvae from the high
and low rations Reyment et al., 1984. We conducted three separate analyses to compare fully developed larvae at 33 days for the high ration or at 55 days for the low
ration between parental conditioning treatments: within the high PCA 1, n 5 60 and the low PCA 2, n 5 59 larval ration, and between parental and larval treatment
combinations PCA 3, n 5 119. For each larva included in a PCA, we then calculated
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the mean score of the six characters measured for the first PC 1 and second PC 2 principal components.
We used two or three-way analysis of variance ANOVA to compare mean principal component scores among larvae in the different parental conditioning and or larval
ration treatments. Parental Conditioning KM, KL and Larval Ration high, low were fixed factors, and Jar three levels was a random factor nested within Parental
Conditioning two-way ANOVA or within the interaction of Parental Conditioning and Larval Ration three-way ANOVA. In each analysis, there were ten replicate mean
scores nine in one case per jar. Despite log-transformation, variances remained heterogeneous as shown by Cochran’s C-test at a 5 0.05 in PCA 2 and 3. We
maintained the factor designation, factor levels, and nesting terms used here in all other analyses.
We compared the percentage of larvae remaining at 39 days relative to the number transferred to culture jars at 2 days between treatments using two-way ANOVA, with
the factors Parental Conditioning and Larval Ration, and three replicate measures means of three to four counts per jar per treatment combination. We compared the percentage
of larvae remaining at 55 days between parental treatments in the low ration with a t-test.
We analysed differences in the rate of metamorphosis between treatments using counts of larvae and settlers transformed using the following equation Zar, 1984:
]]]] X 1 0.375
]]] ]]]
S D
p9 5 n 1 0.5 arcsin
œ
œ
n 1 0.75 where p9 is the transformed rate, n is the number of retrieved larvae and settlers, and X
is the number of retrieved settlers. This transformation is appropriate when data are at the extreme ends of the range of possible values Zar, 1984. We measured the rate of
metamorphosis twice in the high ration and once in the low ration. For the high ration and for each parental treatment, two-way ANOVA with the random factors Trial two
levels and Jar, and three replicate bowls per jar, showed that there was no significant difference p .0.05 in the rate of metamorphosis between the first and second trials. We
therefore pooled trials for the high ration to compare rates of metamorphosis among parental and larval treatment combinations using three-way ANOVA with the factors
Parental Conditioning, Larval Ration, and Jar. There were six replicates per jar for the high ration pooled from two trials and three for the low ration.
We measured horizontal test diameters of settlers 24 h and 1 week after induction twice in the high ration and once in the low ration. Using the same analysis as for rate of
metamorphosis in the high ration, we found no significant difference p .0.05 in the test diameter of settlers after 24 h or 1 week between trials and thus pooled them. To
compare test diameter among parental and larval treatment combinations after 24 h and 1 week, we used two-way ANOVA with the factors Parental Conditioning and Larval
Ration but were unable to test for the factor Jar because of insufficient replication in the low ration zero to three settlers per jar.
In analyses where replication levels differed among groups, we applied ANOVA techniques for unbalanced data using type III sums of squares. Because the sums of
squares in an unbalanced model are not necessarily independent, the denominator mean
S .K. Meidel et al. J. Exp. Mar. Biol. Ecol. 240 1999 161 –178
167
square of the F-ratio generally is constructed from a linear combination of mean squares, based on the variance components. The degrees of freedom for the divisor are estimated
using the Satterthwaite approximation. For further discussion of these techniques, see Minor and Scheibling 1997. We used t-tests to carry out post-hoc comparisons between
means in analyses where interaction terms were significant. To determine the relative importance of each factor in analyses with significant results, we calculated the
magnitude of the experimental effect using a fixed model Howell, 1987 which excluded the factor Jar not significant in these analyses.
3. Results
