Thresholds for mictic female production in the rotifer Brachionus plicatilis (Muller)

73

J. Enp. Mar. Biol. Ecof., 1988, Vol. 124, pp. 73-85

Elsevier

JEM 01176

Thresholds for mictic female production in the rotifer Brachionus plicatilis (Muller) Terry W. Snell and Emily M. Boyer Divifion of Science,Univers@ of Tampa, Tampa, Florida, U.S.A.

(Received 16 February 1988; revision received 2 August 1988; accepted 23 August 1988) Abstract: Food concentration, free ammonia, and population density thresholds for mictic female production were characterized for the rotifer Bra&onus plica&s (Muller). Mictic female production ceased at 15.3 x IO3 Dunaliella cells . ml- ’ (5.7 pg dry weight 1ml- ‘) while amictic female production continued at 0.1 this food level. Free ammonia also repressed mictic and amictic female production differentially. A free :ammonia threshold of 24.4 was recorded for mictic female production. Increasing population density over the range of 140-7400 females. l- ’ promoted mictic female production. The percentage of mictic daughters produced at 260 females. l- ’ was 20 times less than that at 7400 females - l- ‘, The threshold population density for mictic female production was 147 females .l- ‘, The rate of amictic female production strongly correlated with mictic female production. Most mictic female production occurred when the rate of amictic female production exceeded 5.2 females . day- ‘. A hypothesis is proposed that high rates of amictic female production trigger mixis in B. plicatih Key words: Food level; Mixis; Population density threshold; Reproduction;

Rotifer; Sex

INTRODUCTION

In the cyclically parthenogenetic life cycle of monogonont rotifers, asexual reproduction in the absence of males (amictic phase) is intermixed with sexual reproduction (mictic phase). Amictic females are diploid and produce eggs mitotically which develop into females (Birky & Gilbert, 197 1; Ruttner-Kolisko, 1974; Gilbert, 1983). Most of the rotifer life cycle is spent in the an&tic phase but, in certain environments, sexual reproduction occurs concomit~tly. Upon receiving the mictic stimulus, amictic females begin producing both mictic and amictic daughters. The proportion of mictic daughters and the duration of their production depends on the strength of the mictic stimulus. Mictic female production is the initial step in mictic reproduction and is followed by male production, fertilization, and, finally, cyst formation. Mictic females produce haploid eggs that, if unfertilized, develop into haploid males. Fertilized mictic eggs become diploid and develop into resting eggs (cysts) which are dormant resistant stages (Gilbert, 1974). After a period of dormancy that varies among species, cysts respond Correspondence 1J.S.A.

address: T.W. Snell, Division of Science, University of Tampa, Tampa, FL 33606,

0022-0981/88/$03.50 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)

14

T.W.SNELLANDE.M.BOYER

to species-specific hatching cues and hatch into amictic females (Pout-riot & Snell, 1983), entering again into the asexual phase of the life cycle. The stimulus for initiating sexual reproduction is still poorly understood for most rotifer species, the tocopherol response of Aspfunchna (Gilbert, 1980) and the photoperiod response of Nozommatu (Pourriot & Clement, 1981) being exceptions. In the genus Bruchionus, population density has been most frequently cited as a stimulus for mictic female production (Buchner, 1941; Gilbert, 1963, 1977; Hino & Hirano, 1976; Pourriot & Clement, 1981; Pourriot & Snell, 1983). In addition to environmental factors, genetic factors also play a major role in determining the sensitivity of particular strains to mictic stimuli (Buchner, 1977, 1987; Hino & Hirano, 1977; Lubzens et al., 1985; Snell & Hoff, 1985; Hagiwara et al., 1988a). The ecological significance of mictic reproduction in rotifers is based upon its effect on population dynamics and adaptation to adverse environments. As products of mictic reproduction, cysts enable rotifers to escape adverse environments through dormancy. The significance of dormancy as an adaptive strategy has been discussed by several authors (e.g., Cohen, 1968; Templeton & Levin, 1979; Elgmork, 1980). A second consideration is the sexual origin of cysts. Sexual reproduction and its accompanying genetic recombination contribute to phenotypic variability in a population and promote its adaptation. A third ecological consequence of mictic reproduction is its suppression of population growth. The 2-90-day innate minimum period of dormancy of rotifer cysts (Pourriot & Snell, 1983) removes females from the population. Dormancy in effect lengthens generation time, greatly delaying a female’s reproductive contribution to population growth. As a result, rotifer populations reproducing exclusively amictically have higher population growth rates than populations also reproducing mictically (Snell, 1987). A key to understanding the timing of sexual reproduction in rotifer life cycles is knowledge of the environmental requirements of amictic and mictic reproduction. Snell (1986) showed that amictic and mictic female B. plicutilis differ in their reproductive responses to environmental extremes. The reproductive rates of mictic females were more strongly repressed by temperature and salinity extremes or by low food levels than amictic females. In this paper, we continue the examination of the ecological differences between amictic and mictic reproduction by investigating how the rate of mictic female production is affected by starvation, free ammonia, and population density. We describe threshold food levels for both amictic and mictic reproduction and free ammonia thresholds for repression of reproduction. Population density thresholds for mictic reproduction are also described and a hypothesis is proposed which identities the rate of amictic female production as a stimulus for mixis in B. plicutilis. METHODS

Bruchionusplicatilis (Muller), Russian strain (Snell & Carillo, 1984), was used for all experiments. This strain has been in laboratory culture since 1980, either as live animals

THRESHOLDS

FOR MICllC

REPRODUCIYION

15

or cysts. Standard culture conditions were artikial seawater made from Ocean 50 Seamix (Jtmgle Laboratories, Cibolo, Texas) dissolved in deionized water, errriched with F medium nutrients (Guihard, 1983). Experimental temperature was 25 “C, salinity 15x,, and light 4000 lux on a 16 : 8 L : D cycle. Rotifers were fed the green alga Dunalieilu tertiolectu (Utex 999) which was cultured in the same conditions as the rotifers. Experiments were begun by hatching cysts and collecting the neonate females within 1 h of their births. In the food threshold experiment, neonates were randomly assigned to six food concentrations ranging from 5 x lo3 to lo6 cells * ml- ’ (lo6 Du~ulielZucells weigh 370 pg dry weight). Each food treatment consisted of eight isolated females, each in 0.1 ml on a 96-well tissue culture plate (Falcon 3911). Experimental females were transferred daily to fresh medium with the appropriate food concentration. Offspring were collected and transferred to a 24-well plate (Falcon 3047) where they were cultured individu~ly until they reproduced. These F, daughters were classified as amictic if they produced female and mictic if they produced male progeny. Regression analysis was performed on log-transformed cell densities and progeny numbers. One was added to progeny numbers (Y + 1) before log transformation to avoid negative values. A second experiment to examine the effects of starvation on mixis also was begun with neonates hatched from cysts. Females were randomly assigned to six treatments: 0 (control), 1, 2, 3, and 4 days of starvation, and a no-food treatment. Females were placed singly into 0.1 ml of an appropriate food concentration on a 96-well plate. Starvation conditions were lo3 Dunaliella cells * ml- l. After starvation, females were transferred to 3 x lo6 cells *ml-‘, a concentration that avoided food limitation. Females were transferred daily to new wells with fresh medium and algae. F, daughters were collected and cultured singly to determine whether they were mictic or amictic. Free ammonia effects on mixis were examined by assigning eight neonates hatched from cysts to each of five free ammonia concentrations: 0, 5, 10, 15, and 20 mg * l- ’ Females were isolated in 0.1 ml on a 96-well plate and transferred daily to fresh medium, algae, and the appropriate free ammonia concentration. Food concentration was lo6 Du~fflje~lacells . ml - ‘. F, daughters were collected, isolated, and scored as mictic or amictic on the basis of their offspring. Regression analysis was performed on log,transformed progeny numbers. One was added to progeny numbers (Y + 1) before log transformation to avoid negative values. The rate of amictic and mictic female production was calculated using the controls from the three experiments above. The number of amictic and mictic daughters produced * day-’ was averaged for eight females on each day of their reproductive period. These values were treated as x,y pairs, combined from all three experiments and used to calculate a linear regression (n = 26). Population density experiments were initiated by placing neonates hatched from cysts into 50 ml of medium containing lo6 Dunalieflu cells * ml - ’ . The experiment was conducted in small plastic vials with the following population densities: 1,2, 5, 10, and 50 females *50 ml- ’ (20, 40, 100, 200, and 1000 females * I- ‘). There were 10 replicates for the lowest density, five replicates for the 2 females 450 ml- ’ density, and two

T. W. SNELL AND E.M. BOYER

76

replicates each for the remaining densities. Each day, all experimental females were transferred to a new vial with fresh medium and algae. Maternal females were distinguished from F, daughters by size and the fact that maternal females bore multiple eggs while F, daughters were non-ovigerous or bore at most one egg. The actual densities experienced by the maternal females were their density plus the offspring they produced that day. Actual densities were calculated as the total number of rotifers in the vial before transfer of the maternal females and ranged from 140 to 7400 females * l- ‘. A linear regression was calculated on the log-transformed population densities and the percentage of mictic daughters produced.

RESULTS

The threshold food concentrations for amictic and mictic female production are compared in Fig. 1. When plotted on a log-log scale, amictic female production increases linearly with increasing food concentrations from 5 to 1000 x lo3 Dunaliella cells * ml- ’ (1.9-370 pg dry weight. ml- ‘). The regression statistics are: (Y + 1) = 0.83 X“.44,R2 = 0.92, df = 6, F = 59.1, P < 0.001. This regression line intersects the X axis at 1.5 x lo3 cells * ml- ‘, indicating the threshold food level for amictic female production. Below this food concentration, amictic female production ceases. Mictic female production also increases linearly with increasing food concentration but has a different threshold. The regression statistics for mictic reproduction are: 100

1

100 Dunaliella

cells.

ml-’

x IO’

Fig. 1. Threshold food concentration for amictic and mictic female production. Open symbols arc amictic females and solid symbols are mictic females. Each point is mean number of amictic and mictic daughters produced by eight females over their lifetimes. Heavy line indicates the predicted log-log least squares linear regression calculated from equation (Y + 1) = 0.83x0-” for amictic females and (Y + 1) = 1.58.!?~” for mictic females. Light lines on either side of regression line indicate 95% confidence limits for predicted values. Food concentration threshold for arnictic reproduction is 1.5 x lo3 cells ‘ml- ’ and for mictic females 15.3 x 10’ cells. ml- ‘.

THRESHOLDS

FOR MICTIC REPRODUCTION

II

(Y + 1) = 1.58J?“, R2 = 0.84, df = 4, F = 15.8, P = 0.03. The threshold food concentration for mictic female production is 15.3 x lo3 cells . ml - ’ which is w 10 times higher than the threshold for amictic female production. A second experiment was performed to examine further the effects of food limitation on amictic and mictic female production. Fig. 2 illustrates the effect of starvation for

30~ EI Amictic I Mictic

0

1

2 Days

3

4

No food

starvation

Fig. 2. Effects of starvation on amictic and mictic female production. Columns are mean number of amictic daughters (diagonal stripes) and mictic daughters (solid black) produced by eight females over their lifetimes.

0, 1,2,3, or 4 days prior to feeding at optimal levels. Control females (O-day starvation) were fed a food concentration of 3 x lo6 cells * ml - ’ their entire lives and produced an average of 24.5 amictic and 3 mictic daughters * female- *. l-day-starved females were fed lo3 cells * ml- ’ for the 1st day oftheir lives and 3 x IO6cells * ml- ’ thereafter. These females averaged 20.6 amictic and 1.7 mictic daughters. As the duration of starvation became longer, there was a decline in both amictic and mictic female production but the response was not identical Starvation of maternal females for 0, 1, or 2 days produced a relatively small reduction in amictic and, especially, mictic female production. If starvation persisted for 3 days, however, amictic female production was drastically reduced and mictic female production ceased (Table I). These data reinforce the results of the first experiment, indicating that mictic female production is more sensitive to starvation than amictic female production. The free ammonia threshold for amictic and mictic female production is illustrated in Fig. 3. Amictic female production decreased linearly over the O-20 mg * l-- ’ range, with (Y + 1) = 30.6 e- oo6w (R2 = 0.88, df = 4, F = 22.3, P = 0.02). The free ammonia

78

T. W. SNELL AND E. M. BOYER TABLE I

One-way ANOVA for amictic and mictic female production among groups starved for O-4 days and Sheffe’s test (Sokal& Rohlf, 1981, p. 253) for pairwise comparison among means. Underlined means in Sheffe test are not significantly different at 0.05 level. Amictic female production Source df

Sum squares

Mean square

F

P

Between groups Within groups Total

5 43 48

4741 1590 6331

948 36.9

25.6

< 0.001

Mictic female production Source

df

Sum squares

Mean square

F

P

Between groups Within groups Total

5 42 47

66.4 59.6 126

13.3 1.42

9.35


1 20.6

2 3 4 No food 0 11.5 1.6 0.6

Sheffe’s test Days of starvation 0 Amictic means 24.5

Days of starvation Mictic means

0 3

2 2.1

1 3 4 1.6 0.1 0

Free

ammonia

(pg.ml-‘)

Fig. 3. Free ammonia thresholds for amictic and mictic female production. Open symbols are amictic females and solid symbols are mictic females. Each point is mean number of amictic and mictic daughters produced by eight females over their lifetimes. Heavy line indicates the predicted log least squares linear regression calculated from equation (Y + 1) = 30.6 e- ooa8xfor amictic females and (Y + 1) = 2.32 e-“.03hx for mictic females. Light lines on either side of regression line indicate 95% confidence limits for predicted values. Free ammonia threshold for mictic female production is 24.4 mg .I_ ‘.

THRESHOLDS

19

FOR MICTIC REPRODUCTION

threshold for amictic female production cannot be accurately estimated from these data since the threshold dies beyond the concentrations investigated. However, it appears to be considerably > 20 mg * l- ‘. Iv&tic female production also decreased linearly with increasing free ammonia concentration: (Y + 1) = 2.32 e-o.o36x, R2 = 0.77, df = 4, F = 10.1, P = 0.05. The free ammonia threshold for mictic female production is 24.4 mg - l- r. Females, therefore, appear to be better able to produce amictic daughters in the presence of these levels of free ammonia than mictic daughters.

*

.Z

.+ 0.1 .u_ i 2 oO*-+*-Y-+--+* Amictic

l * I

4

6

8

IO

(daughters-female-‘.

12

day-l)

Fig. 4. Comparison of amictic and mictic female production rates. Rates are given as daughters. female _ ’ . day - ‘. X value of each point is mean number of amictic daughters produced. female - ’ . day - ’ and Y value is mean number of mictic daughters produced. female - ’ . day- r, Each point is mean production from eight females on each day of their reproductive lives.

Population

density

(females

’ I“)

Fig. 5. Population density threshold for production of mictie daughters. Heavy line indicates predicted log least squares linear regression calculated from equation Y = 12.610#-27.2. Light lines on either side of regression line indicate 95 y0 confidence limits for predicted values. Population density threshold for mictic female production is 147 females . I - ‘.

80

T. W. SNELL AND E.M. BOYER

In Fig. 4, the relationship between the rate of amictic and mictic female production is explored. Most mictic female production occurred when amictic female production exceeded 4 offspring. female - ’ . day- i. If a linear regression is calculated for the points above the 4 offspring * female - ’ * day - 1 reproductive rate, the X intercept is 5.2 (Y = 0.16X-0.83, R2 = 0.66, df = 11, F = 19.1, P = 0.001). Reproductive rates ~5.2 offspring +female- ’ * day- ’ resulted in nearly all daughters becoming amictic females. Only when reproductive rates exceeded 5.2 were mictic females produced in abundance. These data clearly link mictic female production to high rates of amictic female production. The relationship between population density and mictic female production can be seen in Fig. 5. Mictic female production increased linearly with population density over the range of 140-7400 females al- ‘, giving the regression: Y = 12.6 logX-27.2 (R2 = 0.95, df = 4, F = 57.2, P = 0.005). The percentage of mictic daughters produced at a density of 260 females * l- ’ was 20 times less than that at 7400 females. 1~ ‘. The threshold population density for mictic female production is 147 females * 1- I. In other words, when population density is -C147 females .I - ‘, all daughters are amictic and no males are produced.

DISCUSSION

Attention was focused on the importance of threshold food concentrations in zooplankton by Lampert & Schober (1980). They argued that food thresholds are important structuring forces in zooplankton communities. This theme was amplified and extended by Stemberger & Gilbert (1985,1987) who showed that the threshold food concentrations of 17 rotifer species was lowest for smaller species and used these results to explain rotifer community structure in nature. Our work applies threshold food concentration analysis to the population level and describes how different food thresholds for amictic and mictic female production affect rotifer population structure. We have used threshold food level along with free ammonia and population density thresholds to define the environmental conditions where mictic reproduction occurs. These observations illustrate the physiological constraints on the timing of mictic reproduction in rotifer life cycles. The differential response of B. plicatilis amictic and mictic females to environmental stress was reported by Snell (1986) for the McK strain. This strain is subtropical in origin and probably an S type (small morphotype), as classified by the Japanese, whereas the Russian strain we used in the present study is the L type (large morphotype). The systematic relationship between the S and L morphotypes ofB. plicatilis is currently being investigated (Sudzuki, 1987). When Snell compared populations of amictic and mictic females in the McK strain, he found that starvation reduced the reproductive rate of mictic females to a greater extent than amictic females. Rather than examining the reproductive rate of mictic females, like Snell(1986), in this study we investigated the

THRESHOLDS

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81

rate at which mictic females were produced. Females required IO times higher food concentrations to produce mictic daughters than amictic daughters. The reasons for this greater food requirement for mictic daughter production are unknown. One plausible hypothesis is based on the fact that fertilized mictic females produce cysts which are energetically more demanding than amictic eggs (Gilbert, 1980). Mictic females are committed to producing cysts or dying without contributing to future generations. The proportion of a female’s daughters that are mictic is therefore an important aspect of fitness and is expected to be strongly regulated by selection. In order to insure mictic females will have the resources to form cysts, mictic daughters should be produced only when food levels are relatively high. When food concentrations are low, the production of amictic daughters will be more effective for exploiting limited resources through parthenogenesis. The effects of free ammonia on rotifer reproduction are becoming better appreciated. Hirata & Nagata (1982) found that z 50 % of nitrogenous wastes in B. piicatilis are excreted as ammonium. Depending on temperature and pH, ammonium can be converted to free ammonia which is very toxic to aquatic invertebrates (Russo, 1985). Yu & Hirayama (1986) recorded free ammonia EC,, values for I (the intrinsic rate of increase) and R, (net reproductive rate) of B. plicatilis of 13.2 and 7.8 mg . l- ‘, respectively. These authors suggested that high free ammonia concentrations may be one of the most important factors restricting rotifer population growth in mass cultures. Snell et al. (1988) recorded and EC,, value for sw~ming activity of 2.3 mg - I- 1 free ammonia in B. ~licati~is.S~rn~g activity is an important parameter in rotifer reproduction because it plays a role in determining male-female encounter probabilities (Snell & Garman, 1986). In this study, we found that free ammonia differentially repressed mictic female production. It has now been demonstrated that extremes of temperature and salinity (Lubzens etal., 1985; Snell, 1986) as well as starvation and free ammonia depress mictic reproduction relatively more than amictic reproduction. Some authors, however, have not found a differential response to temperature and salinity by mictic and amictic B. plicatilisfemales (Hagiwara et al., 1988b). Nevertheless, our results suggest that repression of mixis is a general response to environmental stress of many types. Such a response is probably adaptive considering the higher energetic demands of cyst production mentioned above. Unlike starvation and free ammonia, population density had a positive effect on mictic female production over the range of 100-7500 females +1- i. Population density has long been regarded as an inducer of mictic female production in 3. plicatilis (Ito & Iwai, 1956; Ito, 1960; Hino & Hirano, 1976, 1977; Pozuelo, 1977; Scott, 1977) but the consensus is not unanimous. Pourriot & Rougier (1979) could not demonstrate a population density effect in their B. plicatilis strain. They presented data showing the effect detected by previous authors was really a grouping effect. Once this grouping effect was removed, population density no longer affected mixis. The grouping effect may yet be more complicated because it was detected on a ~~naZiellQdiet but disappeared when the rotifers were fed ~ann~c~~o~~.

82

T. W. SNELL AND E.M. BOYER

An analysis of the probabi~ty of male-female

encounters in B. pl~cat~l~(Snell &

Garman, 1986) suggested that, if density effects exist, they probably operate at population densities of w l-25 females * I- *. Our findings indicate a population density

threshold of 147 females * I- 1 for mictic female production in B. plicatilis. This value is somewhat higher than that calculated by Snell & Garman (1986) and higher than the density threshold for mictic reproduction of 2.3 females * 1- 1 recorded by King & Snell (1980) in a natural population of the freshwater rotifer Asplanchna girodi. It is not surprising that species as different as B. plicatilis and A. girodi differ markedly in their density threshold for mictic female production. While population density appears to be an important ingredient in triggering mictic female production in 3. p~ica~l~s,its positive effects can be repressed by other factors. As rotifer populations grow to high densities and deplete food supplies, starvation and/or free ammonia accumulate and begin to repress mixis. The mode of action of population density and the mechanism of repression are currently unknown. We have conducted several experiments to investigate whether conditioned medium has the same inducing properties as live females but have obtained only negative results. Crude rotifer homogenates likewise lack any mixis inducing capability. If the population density effect in rotifers is chemically based, the molecules involved are probably unstable and difficult to work with experimentally. The observation that n&tic female production is associated with high rates of amictic reproduction is a novel and intriguing result. The hypothesis that high rates of amictic reproduction trigger mixis clearly requires further testing. However, iftrue, it represents an eflective reproductive tactic for timing n&tic reproduction to periods of high resource availability and good water quality. Gilbert (1980) has suggested that rotifer populations which inhabit unpredictable environments will use some aspect of their own population dynamics as a trigger for mixis. We suggest here that amictic reproductive rate is just such a mictic cue for B. plicatilis. The association of mictic reproduction with a general population growth response in B. plicatilis is also similar to what Gilbert has described for the tocopherol response in Asplanchna. It should be noted, however, that no relationship between amictic reproductive rate and mixis was detected by Rougier et al. f 1977) or Pourriot et al. (1987) in B. rubens. They used a different experimental design and may not have been able to detect a relationship by averaging many females to calculate their R, statistic. Consequently, further expe~mentation is necessary before the generality of this hypothesis can be determined. Recently, Lubzens & Minkoff (1988) speculated that the trigger for mixis in B. plicatilis is related to dietary factors associated with algal nutritional quality. They argued that algal nutritional quality changes as algae cultures grow through log, stationary, and senescent phases. They hypothesized that as yet unspecified dietary factors, like a-tocopherol (Gilbert, 1980), or the general nutritive quality of the algae act as a trigger for mixis. They further suggested that a population density effect does not exist in their system because renewal of the medium at 8-10-h intervals did not reduce mixis. Our data suggest that Lubzens & Minkoff actually were detecting a continuous

THRESHOLDS

FOR MICIIC REPRODU~ION

83

density effect since their experimental density was 1 female - ml- ‘, far higher than the threshold density of 147 females - i- ’ that we observed. Their failure to record a reduction in mixis with medium renewal was probably due to the long interval between changes. Instead of every 8-10 h, they probably needed to renew every 2 h as did Hino & Hirano (1976) who successfully suppressed mixis with this renewal schedule. In summary, the view of mictic reproduction in B. plicatilis proposed here is one of a complex reproductive response regulated by several environmental factors, some repressing some promoting mixis. Mixis is integrated into a general reproductive response to exploit temporarily abundant resources. Because mictic reproduction is more physiolo~c~ly difIicult, it is more ecolo~c~ly restricted than amictic reproduction. Mixis does not occur during times of en~onmental stress as has been suggested by many authors. Mictic reproduction occurs during periods of high food availability which are the most favorable environments for cyst formation.

ACKNOWLEDGEMENTS

Comments by two reviewers substantially improved this work. This material is based on work supported by the National Science Foundation under Grant OCE-8600305. REFERENCES BIRKY,

C.W., JR. & J.J. GILBERT, 1971. Parthenogenesis in rotifers: the control of sexual and asexual reproduction. Am. Zool., Vol. 11, pp. 245-266. BUCHNER,H.,, 1941. Experimentelle Untersuchungen iiber den Generationswechsel .der Rgdertiere. II. Zool. Jahrb. Abt. Allg. Zool. Physiol., Vol. 60, pp. 219-344. BUCHNER,H., 1977. Physiological basis of reproduction of heterogenous Rotatoria. Arch. Hydrobiol. Beih., Vol. 8, pp. 167-168. BUCHNER,H., 1987. Untersuchungen tiber die Bedingungen der heterogenen Fortplanzungsarten bei den Radertieren. III. Uber den Verlust der miktischen Potenz bei Brachionus urceolati. Arch. Hydrobiol., Vol. 109, pp. 333-354. COHEN, D., 1968. A general model of optimal reproduction in a randomly varying environment. J. Ecol., Vol. 56, pp. 219-228. ELGMORK,E,, 1980. evolutional aspects of diapause in freshwater copepods. In, Evolution and ecology of ~oopian~on ~omm~ities, edited by W.C. Kerfoot, University Press of New England, Hanover, New Hampshire, pp. 411-417. GILBERT,J.J., 1963. Mictic female production in the rotifer Brachionw ca~c~o~s. J. Exp. Zool., Vol. 153, pp. 113-124. GILBERT,J.J., 1974. Dormancy in rotifers. Trans. Am. Micros. Sot., Vol. 93, pp. 490-513. GILBERT, J.J., 1977. Mictic-female production in monogonont rotifers. Arch. Hydrobiol. Beih., Vol. 8, pp. 142-155. GILBERT,J. J., 1980. Female polymorphism and sexual reproduction in the rotifer Asplanchna. Evolution of their relationship and control by dietary tocopherol. Am. Nat., Vol. 116, pp. 409-431. GILBERT,J. J., 1983. Rotifera. In, Reproductive biology of invertebrates. I. Oogenesis, ovopositionand oosorption, edited by K.G. Adiyodi & R.G. Adiyodi, John Wiley & Sons, New York, pp. 181-209. GUILLARD,R. R. L., 1983. Culture of phytoplankton for feeding marine invertebrates. In, Culture of marine invertebrates, edited by C. J. Berg, Jr., Hutchinson-Ross, Stroudsberg, Pennsylvania, pp. 108-132. HAGIWARA,A., A. HINO & R. HIRANO, 1988a. Comparison of resting egg formation among five Japanese stocks of the rotifer Brachion~ plicatiI~. Bull. Jpn. Sot. Sci. Fish., Vol. 54, pp. 577-580.

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