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.G. Gallardo et al. J. Exp. Mar. Biol. Ecol. 240 1999 179 –191
1. Introduction
Environmental stress such as food limitation and high free ammonia concentration is common in rotifer mass cultures when population density increases, resulting in
decreased reproduction and survival. To mitigate the stress of high population densities, hormonal treatment may be effective. In an earlier paper Gallardo et al., 1997, we
reported that in batch cultures of the rotifer Brachionus plicatilis, population growth of
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rotifers treated with growth hormone GH, 0.0025 and 0.025 I.U. ml or g-amino-
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butyric acid GABA, 50 mg ml was 1.7 and 2 times higher than that of controls,
respectively. It was surprising to find that a vertebrate hormone is effective on rotifers, but vertebrate growth hormone has also been found to be effective on other invertebrates
such as oysters Paynter and Chen, 1991 and abalone Taylor et al., 1996. Also, GABA has been found to be effective for inducing metamorphosis in the red abalone
Haliotis rufescens Searcy-Bernal et al., 1992.
To confirm our batch culture results Gallardo et al., 1997 and examine the effect on subsequent generations of hormone-treated rotifers, we conducted individual culture
experiments on isolated females carried through the third generation Parent, F , F . It
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is possible that significant effects would be observed in subsequent generations even if these were not treated with hormones.
Results of our individual culture experiment aimed at confirming the batch culture result showed the absence of a positive effect of GABA in contrast to our batch culture
results. This led us to hypothesize that GABA may be effective in enhancing rotifer reproduction when culture conditions are not optimum. Therefore, in our succeeding
experiments, the rotifers were exposed to low food and high free ammonia levels to determine whether GABA and possibly GH also could mitigate the effects of these
stressors. In the batch cultures where GH and GABA were effective, food became limited and water quality deteriorated as a result of increasing population density. This
paper reports the effect of GH and GABA on rotifer reproduction in optimal and limited environments or in the presence of toxic levels of free ammonia.
2. Materials and methods
2.1. Effective hormone concentration GH porcine and GABA were purchased from Sigma. Individual culture experiments
were carried out in 24-well microplates Iwaki, Japan. Concentrations tested were 0.0025, 0.025, 0.25 and 2.5 I.U. of GH and 0.05, 0.5, 5, 50 and 500 mg of GABA per ml
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of Nannochloropsis oculata suspension at 7 3 10 cells ml . For each concentration
eight replicate wells were prepared. In order to use rotifers B. plicatilis NH3L strain, Balompapueng et al., 1997 of
similar age, amictic eggs were collected by shaking egg-bearing females in a screw- capped bottle. These eggs were placed in a petri dish with food suspension for hatching.
When the hatchlings became amictic females bearing only one egg, they were pipetted individually into the well plates with 1 ml of medium at the designated hormone
W .G. Gallardo et al. J. Exp. Mar. Biol. Ecol. 240 1999 179 –191
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concentration. These isolated rotifers were exposed for 48 h which was the same time as our batch culture experiments Gallardo et al., 1997. After 24 h the first offspring were
removed so as not to confuse it with the mother during transfer to the new well on the 48th hour. Thereafter, the mother was transferred daily to new culture medium without
hormone. The number of offspring were counted daily until the mother died. When the offspring reached sexual maturity egg-bearing, they were classified as to amictic or
mictic based on the type of egg they carried Hagiwara et al., 1988. The experimental condition was 258C, 22 ppt salinity, and darkness.
The intrinsic rate of natural increase r was computed based on Birch 1960. From the raw data, fecundity or net reproduction rate Ro, lifespan and reproductive period
were calculated.
2.2. Effect of hormone at different food levels
4 5
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Rotifers were cultured in three food levels 7 3 10 , 7 3 10 , and 7 3 10 N . oculata
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cells ml with or without hormone 0.0025 I.U. ml
GH or 50 mg ml GABA. The
same procedure was followed as in the first experiment. From the survival and fecundity schedules, the r values, Ro, lifespan and reproductive period were calculated.
A separate experiment was conducted to determine the effect of GH and GABA on
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parent rotifers and on the generation time and size of their F and F offspring at 7 3 10
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and 7 3 10 cells ml food levels. As in the previous experiment, egg-bearing amictic
females P of similar age were used. For each treatment, 12 females each bearing one egg were pipetted individually into culture wells and incubated at 258C in darkness.
After 16 h, these rotifers were observed every hour at 100 3 magnification, and as soon as neonates F appeared, they were transferred to a new food suspension without
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hormone. The time of neonate appearance was recorded and the mother was removed. After 20 h, observations were made every hour and the time was recorded at the
appearance of eggs and neonates F . To determine if generation time is related to body
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size, the lorica length and width of the mother were measured using a compound microscope at 100 3 magnification with an ocular micrometer. For easy measurement
without significant shrinkage, rotifers were fixed with 6.5 HCl Fu et al., 1991. Body size of maternal rotifer at first offspring was measured to determine if hormone affects
both reproductive and somatic growth because normally, high reproduction causes smaller size because more energy is allocated for reproduction over somatic growth
King and Anderson, 1971; Duncan, 1989.
2.3. Effect of hormone at different free ammonia levels
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Free ammonia concentrations of 2.4 and 3.1 mg ml were tested. In each ammonia
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concentration, rotifers were cultured with or without hormone 0.0025 I.U. ml GH or
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50 mg ml GABA. Experiments using GH and GABA were done separately. We
followed the protocol of Yu and Hirayama 1986 and the procedure in our first individual culture experiment. From the survival and fecundity schedules, the r values,
Ro, lifespan and reproductive period were calculated.
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.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
