W .G. Gallardo et al. J. Exp. Mar. Biol. Ecol. 252 2000 97 –107
99
present study will provide insights into the regulation of the rotifer life cycle by hormonal signals and provide means for manipulating mictic reproduction of B
. plicatilis cultures.
2. Materials and methods
2.1. Effective hormone concentration JH and 5-HT were purchased from Sigma Chemical Company product numbers
J2000 and H7752, respectively. Individual culture experiments were done in 24-well microplates Iwaki Co., Japan in volumes of 1 ml of Nannochloropsis oculata
6 21
suspensions of 7 3 10 cells ml . Concentrations of JH and 5-HT tested were 0.05, 0.5,
21
5, and 50 mg ml same concentrations as in Gallardo et al., 1997. For each hormone
concentration and the control no hormone eight replicate wells were prepared, each with one rotifer.
Amictic eggs of the rotifer B . plicatilis NH3L strain, Balompapueng et al., 1997
were collected by shaking egg-bearing females vigorously in a screw-capped bottle. Detached eggs were placed in a Petri dish with algal suspension until hatching. When the
newly hatched amictic females extruded their first egg, they were pipetted individually
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into the well plates with 1 ml of culture medium 7 3 10 N . oculata cells ml
at the
designated hormone concentration. The experimental conditions were 258C, 22‰ salinity, pH 8 and darkness. These isolated rotifers eight per treatment were exposed to
JH or 5-HT for 48 h, the same time with our batch culture experiments Gallardo et al., 1997. After 24 h, offspring were removed to avoid confusing them with the mother
during subsequent observations. After 48 h, the mother was transferred daily to a new well containing food suspension without hormone. The first offspring which were
exposed to the hormone together with the mother in the first well was not used for F
1
culture. The offspring produced in the second well after 48 h were not exposed to the hormone. They were transferred to a new set of multi-well plates for F culture. For F ,
1 2
the first offspring of F were transferred and cultured in another set of multi-well plates.
1
For each generation, the number of offspring was counted daily and reared until the mother died. When the offspring reached maturity egg bearing, they were classified as
amictic or mictic based on the type of egg they carried Hagiwara et al., 1988. The intrinsic rate of natural increase r was computed based on Birch 1948. From the raw
data, fecundity or net reproduction rate Ro, lifespan and reproductive period were calculated.
2.2. Effect of hormone at low food or high free ammonia
4 5
6
Rotifers were cultured in three food levels 7 3 10 , 7 3 10 , and 7 3 10 N . oculata
21 21
21
cells ml with or without hormone 0.5 mg ml
JH or 5 mg ml 5-HT. Addition of
21
free ammonia at 2.4 and 3.1 mg ml pH 8.5 and 8.3, respectively were also tested. In
each free ammonia concentration, rotifers were cultured with or without hormone 0.5
100 W
.G. Gallardo et al. J. Exp. Mar. Biol. Ecol. 252 2000 97 –107
21 21
mg ml JH or 5 mg ml
5-HT. The procedure in the first experiment effective hormone concentration was followed in this experiment.
2.3. Statistical analysis Paired t-tests Systat, 1997 were performed to identify significant differences
between treatments and controls of the r, Ro, reproductive period, lifespan, and the arcsine-transformed mictic female percentage data Zar, 1999.
3. Results
