182 W
.G. Gallardo et al. J. Exp. Mar. Biol. Ecol. 240 1999 179 –191
2.4. Statistical analyses Two-way ANOVA was performed to determine any interaction between hormone
concentration and rotifer generation. One-way ANOVA was conducted to identify significant differences among treatment means within each generation. Multiple com-
parison by Tukey’s method was conducted to determine which treatments were significantly different. We used
SYSTAT
version 5.0 Systat for all statistical analyses.
3. Results
3.1. Effective hormone concentration GH effect on Ro, lifespan and reproductive period differed among generations
two-way ANOVA, P , 0.05 but effect on r did not differ. One-way ANOVA showed significant differences between treatment means in each generation. Parental rotifers P
21
treated with GH at 0.25 I.U. ml had significantly lower net Ro and a shorter
reproductive period than the control, but the first generation offspring F of rotifers
1 21
treated with GH at 0.0025, 0.025 and 0.25 I.U. ml had significantly higher Ro Table
1. In F rotifers, there was no significant difference in any parameter between
2
treatments. No significant difference was found in the intrinsic rate of natural increase r and the lifespan of rotifers between treatments and control in all generations.
GABA effect on r, Ro, lifespan and reproductive period did not differ significantly among generations two-way ANOVA, P . 0.05 but within each generation, significant
differences in r and Ro were observed one-way ANOVA, P , 0.01. Parental rotifers
21
treated with 50 and 500 mg ml GABA had significantly lower Ro compared to the
21
control. Furthermore, the r and Ro of F and F of rotifers exposed to 500 mg ml
1 2
GABA was significantly lower than the controls Table 2. There was no significant difference in lifespan and length of reproductive period between treatments in each
generation. Mictic female production of rotifers did not differ between rotifers with or without hormone treatment.
3.2. Effect of hormone at different food levels
21
GH 0.0025 I.U. ml resulted in higher r, Ro and lifespan of F rotifers when food
1 6
21 5
level is at 7 3 10 N . oculata cells ml
, but not at lower food levels of 7 3 10 and
4 21
6 21
7 3 10 cells ml Table 3. On the other hand, at 7 3 10 cells ml
control, there was no significant difference in Ro between rotifers with or without GABA treatment
21 5
21
50 mg ml , but at a lower food density of 7 3 10 cells ml
, the Ro of the F of
1
GABA-treated rotifers was significantly higher than those without GABA treatment. The
6 21
r values of P and F of GABA-treated rotifers at 7 3 10 cells ml were significantly
1 6
21
higher, but the lifespan and reproductive period of F at 7 3 10
cells ml were
1
W .G. Gallardo et al. J. Exp. Mar. Biol. Ecol. 240 1999 179 –191
183 Table 1
Intrinsic rate of increase r, net reproduction rate Ro, lifespan, and reproductive period of GH-treated B.
a
plicatilis cultured through the third generation Parent, F , F
1 2
GH r
Ro Lifespan
Reproductive
21
I.U. ml days
period days Parent
P 5 0.012 P 5 0.001
P 5 0.012 P 5 0.000
0.92 0.02 b 23.6 0.5 b
9.5 0.3 b 7.4 0.3 a
0.00025 0.92 0.01 b
22.7 0.7 b 12.4 1.8 ab
6.9 0.3 acd 0.0025
0.90 0.02 bc 24.6 0.4 ab
12.0 1.0 ab 8.7 0.4 ab
0.025 0.96 0.01 ab
22.2 0.6 b 9.2 0.4 b
6.6 0.3 ad 0.25
0.95 0.01 b 20.6 0.7 c
8.0 0.6 bc 5.7 0.4 de
F P 5 0.334
P 5 0.000 P 5 0.161
P 5 0.000
1
0.99 0.02 20.0 0.5 c
6.6 0.7 4.6 0.2 c
0.00025 1.00 0.02
21.5 0.5 bc 8.7 1.1
5.2 0.2 a 0.0025
1.01 0.02 22.4 0.7 b
8.6 0.9 5.2 0.2 a
0.025 1.01 0.02
23.2 0.4 ab 8.6 0.8
5.1 0.2 b 0.25
0.96 0.02 24.4 0.6 a
9.5 0.4 6.0 0.2 a
F P 5 0.106
P 5 0.538 P 5 0.054
P 5 0.396
2
0.83 0.02 25.2 0.8
9.0 0.6 6.6 0.2
0.00025 0.82 0.02
25.9 0.7 10.7 0.8
7.1 0.3 0.0025
0.87 0.03 24.9 1.1
8.2 0.6 6.8 0.4
0.025 0.89 0.02
25.6 0.7 8.4 0.6
6.4 0.2 0.25
0.82 0.02 23.7 1.3
9.1 0.4 6.6 0.2
Two-way ANOVA Source of variation
F P
F P
F P
F P
Concentration 2.95
0.023 8.47
0.000 3.62
0.008 4.88
0.001 Generation
90.09 0.000
20.70 0.000
6.00 0.003
61.56 0.000
Conc 3 Gen 1.66
0.116 2.15
0.037 2.49
0.016 7.77
0.000
a
Values are mean and standard error in parenthesis of 6–8 replicates per treatment. One-way ANOVA P values are indicated for each generation. In a column, means followed by a common letter are not significantly
different at the 5 level. Means lacking letters not significantly different. Two-way ANOVA results for each variable are shown at the bottom of the table.
5
significantly shorter than the untreated rotifers. At a lower food density of 7 3 10 cells
21
ml the r values of F and F of GABA-treated rotifers were significantly higher than
1 2
untreated rotifers, whereas the lifespan and reproductive period did not differ sig- nificantly between treated and untreated rotifers.
6 21
At 7 3 10 cells ml food level, the time to first egg and first offspring of GH-treated
5
rotifers was significantly shorter than the untreated rotifers. However, at 7 3 10 cells
21
ml food level, there was no significant difference between treatments Table 4. On
the other hand, with GABA treatment, the time to first egg and first offspring of
5 21
GABA-treated rotifers at 7 3 10 cells ml food level was significantly shorter than the
untreated rotifers. There was no significant difference in time to first egg and first
6 21
offspring between rotifers with or without GABA treatment at 7 3 10 cells ml food
level. Size at first offspring of maternal rotifers with or without hormone treatment did not differ significantly.
184 W
.G. Gallardo et al. J. Exp. Mar. Biol. Ecol. 240 1999 179 –191 Table 2
Intrinsic rate of increase r, net reproduction rate Ro, lifespan, and reproductive period of GABA-treated B.
a
plicatilis cultured through the third generation Parent, F , F
1 2
GABA r
Ro Lifespan
Reproductive
21
mg ml days
period days Parent
P 5 0.0069 P , 0.001
P 5 0.081 P 5 0.024
0.68 0.01 a 24.0 1.5 a
11.3 0.3 9.0 0.5 a
0.05 0.70 0.01 ab
23.6 0.5 a 12.5 0.5
9.5 0.5 a 0.5
0.70 0.01 ab 22.3 0.4 a
12.0 0.5 8.1 1.0 a
5 0.70 0.01 ab
24.1 0.6 a 12.9 0.3
10.3 0.4 ab 50
0.65 0.02 ac 17.4 2.2 b
11.2 0.6 8.0 0.6 a
500 0.64 0.01 ac
14.8 1.4 b 10.5 0.7
7.2 0.8 ac F
P 5 0.0023 P 5 0.110
P 5 0.381 P 5 0.696
1
0.82 0.02 a 22.1 1.5 a
12.6 0.8 8.4 0.7
0.05 0.80 0.03 a
21.2 1.2 a 11.5 0.8
8.0 0.4 0.5
0.82 0.04 a 22.4 1.0 a
12.9 0.5 9.1 0.6
5 0.85 0.05 a
24.0 0.3 ab 11.2 0.8
8.6 0.6 50
0.78 0.04 a 18.4 2.3 ac
10.4 0.9 7.5 1.0
500 0.60 0.03 b
17.2 2.8 ac 11.4 1.6
8.2 1.0 F
P 5 0.0005 P , 0.001
P 5 0.911 P 5 0.375
2
0.84 0.03 a 25.0 0.5 a
12.6 0.7 8.5 0.4
0.05 0.76 0.05 a
25.8 1.0 a 12.7 0.4
9.7 0.6 0.5
0.86 0.03 a 26.6 0.7 a
12.5 0.5 8.9 0.4
5 0.79 0.04 a
24.6 1.3 a 12.2 1.3
9.2 0.8 50
0.83 0.04 a 25.8 0.8 a
12.8 0.7 8.8 0.7
500 0.61 0.04 b
18.0 2.2 b 11.7 1.2
7.7 1.0 Two-way ANOVA
Source of variation F
P F
P F
P F
P Concentration
10.26 0.000
10.64 0.000
1.13 0.351
2.28 0.052
Generation 23.02
0.000 11.80
0.000 1.62
0.203 1.14
0.323 Conc 3 Gen
1.92 0.051
1.33 0.225
0.90 0.536
0.82 0.613
a
Values are mean and standard error in parenthesis of 6–8 replicates per treatment. One-way ANOVA p values are indicated for each generation. In a column, means followed by a common letter are not significantly
different at the 5 level. Means lacking letters are not significantly different. Two-way ANOVA results for each variable are shown at the bottom of the table.
3.3. Effect of hormone at different free ammonia levels In controls lacking hormone, r, Ro, lifespan and reproductive period decreased with
increasing free ammonia concentration particularly among parent rotifers, but with the addition of GABA, the r of parent rotifers at 3.1 ppm free ammonia was significantly
higher than the untreated rotifers Table 5. Higher lifespan and reproductive period
21
were observed in the F of GABA-treated rotifers at 2.4 mg ml and in the F at 3.1 mg
1 2
21 21
ml . On the other hand, at 2.4 mg ml
free ammonia, the F of GH-treated rotifers had
1
an r value significantly higher than those without GH treatment. There was no significant difference in Ro, lifespan and reproductive period of rotifers with or without
GH treatment at any free ammonia level.
W .G
. Gallardo
et al
. J
. Exp
. Mar
. Biol
. Ecol
. 240
1999 179
– 191
185 Table 3
21 21
Intrinsic rate of increase r, net reproduction rate Ro, lifespan, and reproductive period of B. plicatilis treated with GH 0.0025 I.U. ml or GABA 50 mg ml
a
at different food levels, compared with the controls. N 5 8 for each treatment
Food density r
Ro Lifespan days
Reproductive period days
21
cells ml Control
GH GABA
Control GH
GABA Control
GH GABA
Control GH
GABA Parent
6
7 3 10 0.56
0.60 0.70
23.1 19.8
21.8 13.9
13.0 11.8
9.1 9.1
8.1
5
7 3 10 0.52
0.55 0.58
14.6 15.6
15.4 13.4
15.9 14.1
11.0 12.4
12.6
4
7 3 10 0.34
0.38 0.31
5.1 5.4
4.1 16.1
18.0 14.0
10.5 11.4
10.4 F
1 6
7 3 10 0.66
0.73 0.88
24.4 28.3
26.1 12.1
14.3 9.4
10.4 11.1
7.8
5
7 3 10 0.53
0.52 0.65
11.1 11.8
15.9 12.7
14.1 13.4
10.6 12.6
11.3
4
7 3 10 –
– –
– –
– –
– –
– –
– F
2 6
7 3 10 0.84
0.83 0.82
23.0 25.1
26.0 11.1
12.3 12.6
9.9 11.0
10.5
5
7 3 10 0.52
0.57 0.64
9.0 10.4
12.2 12.2
12.3 11.8
11.2 11.0
10.0
4
7 3 10 –
– –
– –
– –
– –
– –
–
a 4
Level of significant difference with the control: P , 0.05, P , 0.01. No survivors at 7 3 10 in F and F .
1 2
186 W
.G. Gallardo et al. J. Exp. Mar. Biol. Ecol. 240 1999 179 –191 Table 4
21
Generation time and lorica size at first offspring of B. plicatilis treated with GH 0.0025 I.U. ml or GABA
21 a
50 mg ml at different food levels
Food density Time to first egg
Time to first offspring Size at first offspring
h h
Length Width
mm
6 21
7 3 10 cells ml Control
26.9 0.5 48.5 0.8
301.0 2.8 199.0 1.8
GH 24.7 0.5
46.2 0.4 305.8 4.2
200.8 2.9 GABA
27.1 0.6 50.2 1.0
292.2 4.5 196.2 1.8
5 21
7 3 10 cells ml Control
33.7 0.7 56.3 0.9
287.3 3.5 190.0 2.0
GH 33.3 0.7
55.1 0.9 288.3 2.2
186.7 2.0 GABA
30.3 1.4 52.0 1.1
284.7 4.2 185.0 3.3
a
Values are mean and standard error in parenthesis of 6-8 replicates females per treatment. Level of significant difference with the control: P , 0.05, P , 0.01.
4. Discussion
