AM. ZOOLOCIST, 11:245-266 (1971).
Parthenogenesis in Rotifers: The Control of Sexual
and Asexual Reproduction
C. WILLIAM BIRKY, JR.
Faculty of Genetics, The Ohio Slate University, Columbus, Ohio 43210
AND
JOHN J.
GILBERT
Department of Biological Sciences, Dartmouth College,
Hanover, New Hampshire 03755
SYNOPSIS. The class Rotifera includes species which reproduce solely by apomictic
female parthenogenesis and species which alternate this "asexual" reproduction with
ordinary sexual reproduction. The transition between asexual and sexual reproduction
is controlled by the environment. Laboratory studies with the genus Asplanchna have shown that it is possible to identify specific molecules as inducers, which act
on embryos in utero to modify their development and determine whether they will
mature as sexually or asexually reproducing females. Moreover, an evolutionary
rationale can be provided for the response to these particular environmental controlling agents, which are such that sexual reproduction will occur only when it will result
in successful fertilization.
Rotifers are opportunistic or colonising organisms, which implies selection for rapid
reproduction. We suggest that this may account, at least in part, for the origin of both
apomicitc parthenogenesis and certain features of the pattern of macromolecular
syntheses during development. To account for the success of those rotifers which have
lost sexual reproduction entirely, we note that accumulation of mutations during
periods of exponential apomictic parthenogenetic reproduction, together with "mitotic"
crossing-over, could theoretically produce sufficient genotypic diversity to provide
evolutionary flexibility. This would eliminate a major advantage of sexual reproduction.
ROTIFER LIFE CYCLES
rotifers were hermaphroditic. This was the
result of a general failure to find, or to
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The discovery of parthenogenesis
in ro.
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recognize, male rotifers,
and led to some
tilers, and the elucidation of the hie cycles
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amusing attempts to identify male reproof the maior rotifer groups, has been re, . °
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ductive organs in females. The confusion
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viewed in detail by Lange (1913), Remane
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as eliminated by Bnghtwell s (]848a,b)
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(1929), and others and we give only the
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highlights here. The first clear descriptions ^
described
A°planchna
brightwellL
and drawings of rotilers were those or „ . , . , ,
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cies, the male testis, and the female reprotemales.
Later
naturalists,
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ductive organs; further, he observed and
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described oogenesis and copulation.
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tunately,
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observations
and
experiments
The authors' own research in this area has been w ere not extensive enough to demonstrate
greatly facilitated by discussions with numerous the existence of parthenogenesis, and he
friends, colleagues, and students at Dartmouth Col- concluded with a half-truth: "At least one
le ge Th e O h i
° S t a t e Y"!VerSi,ty> a " d t h e V" i v e r s i , t y
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of California at Berkeley, by grants from the
NSF and the USPHS, and by a USPHS Special
of the more highly organized species is
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dioecious.
Research Fellowship to C. W. Birky, Jr.
On the basis of Studies of natural popu245
246
C. WILLIAM BIRKY, JR. AND JOHN J. GILBERT
lations of Brachionus urceolaris, Cohn
(1856) worked out the correct rotifer life
cycle, including the existence of two kinds
of females (Fig. 1), but two years later
discarded his earlier views. It was left to
Maupas (1890^6, 1891) to carry out the
required laboratory experiments with Hydatina (Epiphanes) senta and to clearly
demonstrate the existence of amictic females, which produce daughters by parthenogenesis, and mictic females, which
produce males by parthenogenesis or, if
impregnated by a male, resting eggs Which
hatch into amictic females. He also showed
that amictic females can produce either
amictic or mictic daughters, and that the
proportion of mictic daughters is markedly
greater when the animals are reared at
higher temperatures. We have here the
first demonstration that an extrinsic (environmental) signal or stimulus can modify
embryonic development and the mode of
reproduction of female rotifers.
Subsequent work has led to the generalization that there are, in fact, three different types of life cycles in rotifers, corresponding to the three major subdivisions
of the Class Rotifera. Alternating amictic
and mictic reproduction as described by
Maupas is found in the Order Monogononta; it should be noted that no males
have been found for a number of species
of monogononts, so that these species may
consist of amictic females only and be
strictly parthenogcnetic. No males have
been found for any species in the Order
Bdelloidea; reproduction is strictly by female parthenogenesis, although the cytogenetic mechanism may differ from that
found in amictic monogononts (see below). Finally, the small, parasitic marine
rotifers which comprise the Order Seisonidea appear to be strictly dioecious; one
female can produce both male and female
offspring, no resting eggs are known, and
the males are not "degenerate" as are those
of the monogononts.
Mictic and amictic females may differ
markedly in reproductive parameters (e.g.
Miller, 1931; Burhner, Kiechle, and
Hamm, 1965), but are generally difficult
(de Beauchamp, 1935) or impossible to
distinguish morphologically until they contain nearly mature embryos whose sex can
be determined. Successfully impregnated
mictic females are recognizable by the appearance of numerous particles of granular
material in the vitellarium, identified as
shell precursors by Bentfeld (1968). Mictic
females can only be impregnated with
sperm during the first few hours after
birth, when the female is still growing and
her first oocyte is undergoing maturation
(See BuchtleT, MllUJcllkii, and Kindile,
1967, for a thorough study and references). This is probably attributable to
hardening of the female cuticle following
the growth period, as suggested by Whitney (1913; see also Brodie, 1970). Impregnated mictic females generally produce
only fertilized (resting) eggs, but in at
least some species some females can give
birth to males as well. We find such females to be rare in Asplanchna and have
been unable to hatch any of their resting
eggs, but in Buchner's clone of this genus,
40% of all impregnated mictic females produce both males and resting eggs, and at
least some of the latter are viable (Buchner e t a l , 1967).
ROTIFER CYTOGENETICS AND GENETICS
It is generally assumed that the chromosomal basis of parthenogenesis in rotifers is
as indicated in Figure 1: amictic females
have oocytes which undergo a single cquational maturation division ("mitosis"), so
that continued amictic reproduction gives
rise to clones of genetically identical females. The oocytes of mictic females undergo ordinary meiosis; the resulting
haploid eggs are either fertilized, producing diploid resting eggs, or mature as
haploid males, which produce haploid
sperm by an equational maturation division. These assumptions have formed the
basis of the little work that has been done
on the genetics of rotifers, and are also
important in the design and interpretation
of many physiological experiments. But
the evidence, or lack thereof, for the pre-
PARTHENOGENESIS IN ROTIFERS
0 E G G AMICTIC
M
(2N) ? <2N>
MITOSIS
(2N1 '
(f >
d*<N>
FIG. 1. Life cycle of the monogonont rotifers. "Mitosis" and "meiosis" refer to the oocyte maturation
divisions in amictic and mictic females, respectively. (Reprinted from Birky, 1964).
r
r
•
v
sumed cytological and genetical events
bears careful examination, especially as it
is crucial to the questions which we will
raise later concerning the structure and
evolution of the rotifier gene pool.
It was first noted by von Erlanger and
Lauterborn (1897) that the eggs of mictic
female Asplanchna priodonta have two polar bodies, while those of amictic females
have only one. This observation has been
extended to many different monogonont
rotifers, and suggests that amictic oocytes
either undergo only a single maturation
division or that one polar body nucleus
fuses with the egg nucleus. The latter possibility can be ruled out, for Asplanchna at
least, by direct observation of oogenesis in
living females. Subsequent cytological
studies by Shull (1921) on Epiphanes senta, by Whitney (1909a, 1924, 1929) and
Tauson (1924) on Asplanchna species of
the brightwelli group, and by Storch
(1923, 1924) on A. priodonta showed that
amictic oocytes undergo a single matura-
247
tion division, with no indication of synapsis of homologous chromosomes or of reduction in chromosome number. Mictic
oocytes, on the other hand, undergo classical meiosis.
With respect to male haploidy and the
nature of the spermatogenic divisions, confusion reigns. Whitney (1924) claimed
that the spermatocytes of male A. intermedia have a haploid number of 26 chromosomes, and develop directly into spermatozoa, with the exception of some which divide to produce cells with 13 chromosomes, which then develop into "rudimentary" spermatozoa (See Koehler, 1965, for a
description of these cells). Later, Whitney
(1929) reversed his earlier opinion on the
basis of studies with A. amphora ( = A.
sicboldi), and stated that primary spermatocytes contain a haploid number of 13
chromosomes; they undergo an equational
division to produce haploid secondary
spermatocytes which differentiate into
sperm. Tauson (1924, 1927&) found a
haploid number of 12 chromosomes in A.
intermedia. She claimed that unfertilized
haploid eggs diploidize before the first
cleavage, so that the mature male is diploid but homozygous. The diploid primary spermatogonia undergo an equational
division to produce diploid secondary spermatogonia, which then undergo a reductional division to produce haploid spermatogonia; the latter undergo a further equational division to produce spermatids
which differentiate into spermatozoa. The
interpretation of this work is further confused by the taxonomic difficulties involved with these species of Asplanchna.
It might be expected that bdelloid rotifers, in which reproduction is exclusively by
female parthenogenesis, would show the
same sort of single, equational reduction
division as is found in amictic monogonont
females. But the studies of Hsu (1956a,
b) on Philodina roseola and Habrotrocha
tridens show two equational maturation
divisions, and a chromosome number of
13. An odd number of chromosomes is perhaps not too surprising in an organism in
which synapsis of homologues may never
248
C. WILLIAM BIRKY, JR. AND JOHN J. GILBERT
be required. The occurrence of two equational maturation divisions suggests that
the bdelloids did not evolve from monogonont forms by the simple loss of ability to
produce mictic females.
It is important to note that all of these
studies have utilized sectioned, paraffinembedded material taken from females
fixed at random, and that rotifer chromosomes, in the species studied, are extremely
small. What is needed is a rotifer with
nice large chromosomes; if, as we suspect,
this is too much to hope for, then the
natiu-e of the maturation divisions can
probably still be clarified by the use of
modern squash techniques and plasticembedded sections. Since the maturation
divisions in AspJanchna, at least, can be
easily seen in living females (all except the
chromosomes!), the cytogenetical studies
should be made on oocytes fixed at known
stages in the maturation division (s).
Ideally, the presumed nature of the rotifer life cycle should be verified by genetic
studies in which the behavior of specific
marker genes is followed. Only a beginning has been made in this direction. We
can ask, first, if chromosome segregation
and recombination actually do occur during mixis. They do, as has been shown in
studies on the male-sterile mutant in Asplanchna brightwelli (Birky, 1965). The
results of a series of matings, including
crosses, test-crosses, and selfing, were compatible with the hypothesis that the malesterile trait is controlled by a simple Mendelian gene. The mutant allele (ms) is
sex-limited, having no effect on female fertility. Homozygous mutant females produced only mutant, sterile male progeny.
Heterozygous females produced two kinds
of male offspring, in approximately equal
numbers: those carrying only the mutant
allele, and those carrying only the wildtype allele. Both kinds of males were fertile, indicating a maternal effect (control
of the male phenotype by the maternal
genotype).
The above results verify the assumptions
about the mictic portion of the life cycle,
except that they say nothing about wheth-
er males are haploid or diploid, but homozygous. This question is largely immaterial from the standpoint of the geneticist
and population geneticist, for in either
case one would expect recessive genes to be
expressed at once in males. It is important
from the standpoint of the mechanism of
sex determination, which would be most
easily understood if, simply, all haploid
individuals are male and all diploids are
female. An alternative mechanism would
be that suggested for the wasp Bracon ( =
Habrobracon), in which all individuals
heterozygous for any one of a number of
sex-determining alleles are females. This
mechanism is rendered unlikely for rotifers
because mictic females from highly homozygous strains produced by repeated
selfing (Birky, 1967a) do not produce mixed
broods of male and female offspring.
We are left with the question of the
extent to which any sort of segregation or
recombination may occur in the oocytes of
amictic females. In other words, is a
"clone" of amictic females really a clone?
The following facts are available: there is
only one maturation division, which probably leaves the egg nucleus diploid, and
there is no fusion of polar body and egg
nuclei, or of cleavage nuclei. This rules
out most possible mechanisms of parthenogenesis (Suomalainen, 1950), but still
leaves three:
(1) Apomixis, by an equational maturation division (inhibition of mciosis I). If
synapsis of homologues is absent, it is generally believed that crossing-over will not
occur, so that this sort of maturation division should result in no change in the
genome. This is a weak argument. The
cytological studies favor this mechanism,
but can scarcely be regarded as conclusive
(See Marinelli, 1925). '
(2) Automixis, by the complete suppression of meiosis II. This sort of maturation
division should produce homozygous eggs
from heterozygous oocytes, unless crossingover occurs between the gene and centromere.
(.8) Automixis, by a single maturation
division with random segregation of all
< i
i
•4 •
H
PARTHENOGENESIS IN ROTIFERS
centromeres. Here, a heterozygous female
should produce a 1:4:1 ratio of homozygous and heterozygous oocytes, and it is
assumed that no crossing-over occurs.
We wish to emphasize two points. First,
with only a single locus it would be difficult or impossible to distinguish between
these three modes of parthenogenesis, except to rule out the already unlikely third
mechanism. More marker genes are needed! Second, some degree of crossing-over
and recombination can occur with any
mechanism. It may not be completely safe
to assume, as we have all been doing, that
a "clone" is really a clone, even ignoring
mutation. Unfortunately, finding marker
genes has proved difficult, at least in Asplanchna. Intraclonal variations in phenotype are known in rotifers. It has been
shown in A. brightwelli, for example, that
some amictic females are much more likely
to produce mictic female offspring (Gilbert, 1968) or are more responsive to the
morphogenetic effects of vitamin E (Birky,
19G9) than are other females from the
same clonal culture at the same time. The
source of this variability and its heritability are not known, except that it occurs at
such a rapid rate as to make mutation an
unlikely explanation.
Ruttner-Kolisko (1968, 1969) has strongly questioned whether mictic reproduction in rotifers does, in fact, involve effective fertilization and genetic recombination. In an earlier paper (1948) she reported that females of a population of
Keratella quadrata produce resting eggs
without fertilization; unfortunately, neither these nor fertilized resting eggs could
be hatched in the laboratory. In further
supjDort of her argument, she carried out
an interspecific cross with remarkable results. Matings of female Brachionus urceolaris by male Brachionus quadridentatus,
easily distinguishable by morphology of
the lorica and by the fact that resting eggs
produced by female B. quadridentatus cannot be hatched in the laboratory, produce
an Fx generation identical to the female
B. urceolaris parent in all respects. Six successive generations of selfing, starting with
249
Fx animals, again produced only animals
of the B. urceolaris type, as did backcrosses
of the Fx with male and female B. urceolaris. These results are interpreted as a
case of "cryptoparthenogenesis" in which
the sperm in the original cross induced the
synthesis of resting egg shells but failed to
contribute chromosomes to the F 1; which
developed by parthenogenesis. It is, however, difficult to understand how this parthenogenetic development took place if the
female parents were ordinary mictic females producing haploid oocytes. If they
were amictic females, it is surprising that
resting eggs should have been formed at
all; impregnated amictic Asplanchna do
not synthesize shell precursor. Thus, these
experiments raise the possibility that true
fertilization and mixis never occur in B.
urceolaris, and possibly never occur in other species as well.
Ruttner-Kolisko also found regular
changes in the ability of resting eggs, produced by the successive generations of
selfing, to hatch. To explain this, she has
devised a complex hypothesis of centriolar
segregation coupled with lethal interactions between centrioles and cytoplasm.
She also shows that this hypothesis can
explain the results of Birky's (1965) crosses with the ms gene. We believe that the
more conventional explanation of these
crosses is preferable; this is especially true
since Birky (1967a) showed that the
hatchability of resting eggs of Asplanchna
brightwelli is strongly dependent upon environmental factors and the physiological
state of the female, while the ability of
newly-hatched females to give rise to
clones, which does not vary significantly in
Ruttner-Kolisko's data, does seem to be
primarily under genetic control.
CONTROL OF THE ROTIFER LIFE CYCLE
IN THE LABORATORY
We have already mentioned that Maupas
(1891) found that the frequency of
mictic offspring could be greatly increased
in Epiphanes senta by rearing cultures at a
higher temperature. Nussbaum (1897),
250
C. WILLIAM BIRKY, JR. AND JOHN J. GILBERT
working with the same species, concluded
that starvation of amictic females induced
the production of mictic daughters and
suggested that Maupas' temperature effect
was indirect, with starvation occuring at
higher temperatures. There follows in the
literature a long series of papers by Whitney (1907, ]909«A 1910, 1912«A 1914,
1916aA 1917, 1919), Hertel (1942),
Shull
(1910, 1911, 1912, 1913aA
1915, 1918, 1925), and Shull and
Ladoff
(1916) in which the two
major workers and their students sought to
identify the environmental stimulus (or
stimuli), often with contradictory results.
Most of this early work, and a good deal of
that which followed, is difficult or impossible to interpret, for a variety of different
complex culture media was used; food supplies were poorly controlled quantitatively
and qualitatively; temperature, photoperiod, and population density were often
uncontrolled; and appropriate controls
were frequently omitted. A striking and
typical example of the latter fault is seen
in a paper by Punnett (1906), in which he
concluded that starvation and changes in
food supply did not induce mictic female
production, but failed to show that his
strain of E. senta was capable of producing
mictic females at all (it probably was
not!). Two conclusions can be drawn from
this work, however: (1) A variety of factors, including genotype and age of females, quantity of food, and certain chemicals, can influence the production of mictic
offspring in E. senta; and (2) the most
consistently effective inducer of mictic female j:>roduction is a switch of major food
organisms from the colorless alga Polytoma
to the green alga Chlamydomonas.
The genus Brachionus has also been
used extensively in studies of the environmental control of mictic female production. The literature has been reviewed by
Gilbert (1963); additional references will
be found in Pourriot (1965). Again, many
expeii meats aie difficult to interpret due
to failure of the authors to control (or to
specify the control of) important variables
(Morn, 1915; Whitney, 1916&; Luntz,
1929; Pourriot, 1957, 1965). This criticism
also applies to the paper of Laderman and
Guttman (1963), whose experimental organisms have in any event been reidentified as Monostyla quadridentata.
Ruttner-Kolisko (1964) has shown that
the percentage of mictic female offspring
produced by B. rub ens grown at 20° can
be consistently increased from nearly zero
to 55% by exposing the amictic mothers to
a cold shock (two hours at 6° or 10°).
Lowering the culture temperature to 10°
and holding it there did not induce the
production of mictic offspring. The applicability of this finding to natural populations is doubtful, since it is highly unlikely
that animals would be exposed to such
brief and extreme temperature shocks, except possibly during the course of very unusual vertical migrations.
The other consistent, clear-cut effect on
mictic female production is that of population density. Gilbert (1963) has shown
that amictic female B. calyciflorus produce
significantly greater percentages of mictic
female offspring when cultured at a density
of 4.0 females/ml than at 0.66 females/ml. These experiments confirmed
the earlier work of Buchner (1941) and
Ito (1957, 1960) and further demonstrated
that the effect of population density is
independent of the actual numbers of females per container, i.e. does not depend
upon social effects. Recent experiments
(Gilbert, unpublished) using monoxenic
cultures of B. calyciflorus (Gilbert, 1970)
have shown that mictic females are rare
when the cultures are initiated and become maximally abundant after about a
week when the population density is about
7 to 10 females/ml. In contrast to
Epiphanes senta (see above), B. calyciflorus produces about the same proportion of
mictic females whether fed on green Euglena or on colorless Euglena or Polytoma
(Gilbert, 1963). The mechanism of the
density effect in B. calyciflorus is not
known. It has some specificity, in that mictic ieniale production cannot be induced
by crowding with paramecia, and has been
tentatively attributed to the accumulation
PARTHENOGENESIS IN ROTIFERS
of some compound which is at least specific for rotifers (Gilbert, 1963).
Less extensive studies have been made of
the role of particular environmental variables in controlling the production of mictic daughters in several other species of
rotifers. According to Tauson (1925),
studies by Zawadovsky (1916) on Diglena
volvocicola implicate a role of food quantity in the control of mixis. With the carnivorous Eosophora najas, Pourriot (1960)
found a striking difference with the use of
different food organisms. For three other
species (Nolommata copeus, N. codonella,
and Trichocerca rattus), Pourriot (1963,
1965) concluded that photoperiod exerts
control over mictic female production;
male eggs and resting eggs were found in
cultures exposed to 15 or 24 hours of light
per day, but not in cultures exposed to
nine hours of light per day or reared in
continual darkness. All cultures were fed
algae, but the effect was shown not to be
attributable to the photoperiod under
which the algae were reared. Each of these
studies, unfortunately, is again difficult to
interjDret unambiguously due to failure to
report important information, to control
other variables, or to use appropriate measures of mictic female production. They
are of interest primarily in indicating the
diversity of environmental factors which
may exert some degree of direct or indirect
control over the occurrence of mixis.
This diversity, coupled with apparent
differences in controlling factors between
species or even between different laboratories working with the same species, have
unfortunately led a number of investigators and an even greater number of authors of text books to the conclusion that
virtually any change in environmental
conditions may induce rotifers to produce
mictic offspring. This conclusion is unfortunate, for a number of reasons. It is, first
of all, directly contradicted by the results
of a number of studies which have shown
that many types of environmental changes
are completely ineffective as inducers,
while consistent and continuous differences
in the production of mictic females are
251
seen between cultures exposed to certain
different environmental factors. It is essentially a defeatist attitude which discourages
further research. Our own procedure has
been to attempt to identify specific controlling factors which are of overriding importance, and to proceed from there examining both the mode of action of these factors at cellular and molecular levels and
also the ecological and evolutionary consequences of the response. With these goals
in mind we (along with several other laboratories) have been intensively investigating the control of mictic female production in the rotifer genus Asplanchna for
several years.
Apart from some observations on A.
girodi which do not permit clearcut conclusions (de Beauchamp, 1935; Lechner,
1966), work has been limited to the taxonomically confusing brightwelli group. This
group of species includes forms identified
as A. brightwelli, A. sieboldi, A. intermedia, A. ebbesborni, and A. amphora. The
early work, already reviewed in detail by
Gilbert (1968), suffers from some of the
problems of interpretation previously mentioned. Briefly, the production of mictic
females was reported to be enhanced by
quantitative changes in feeding (Mitchell,
1913) and by changes in oxygen concentration, carbonate concentration, and, especially, pH (Tauson, 1925, 1926, 1927a).
Mitchell (1913) reared Asplanchna on
what has become the standard food organism, Paramecium. He found few or no mictic females under these conditions, but was
able to induce mictic female production
by the addition of green algae to the diet.
This finding was confirmed under more
rigorously controlled experimental conditions by Birky (1964), Buchner and
Kiechle (1965), Kiechle and Buchner
(1966), and Buchner, Kiechle, and
Tiefenbacher (1969). Gilbert (1968), in a
more thorough analysis, showed that in
an appropriate culture fluid, no mictic females are produced unless algae are
present in the diet; furthermore, he was
unable to confirm Mitchell's results with
quantitative food changes or Tauson's re-
252
C. WILLIAM BIRKY, JR. AND JOHN J. GILBERT
NUCLEAR NUMBER
RESPONSE
More Cell Divisions in
mtnt
elands
and
i t e llarium
ot- totepbc rol }
FECUNDITY RESPONSE
Fewer Off>princg
BWO
RESPONSE
Dif-fercntial Growth of
Syneytial Hypoderwiis
-•Body Vail Outnrou/ths
MIXIS RESPONSE
SACCATE, AMICTIC
FEMALE
Meiofcic Maturation Div
Male Evn
l i r r~«rtWi
HUMPED, MICTIC
FEMALE
FIG. 2. Summary of responses of Asplanchna females to dietary vitamin E. The BWO response is
shown in the most extreme form for A. sieboldi;
A. brightwelli shows a much reduced response. See
text for further explanation.
suits with pH.
Further investigations designed to identify the specific component (s) of green
plant material responsible for induction of
mictic female production (Gilbert, 1967,
1968) led to the identification of a-tocopherol (vitamin E) as the active ingredient (Gilbert and Thompson, 1968). It is
also responsible for changes in fecundity,
in embryonic mitosis, and in the embryonic morphogenetic growth which results in
the production of "humped" females with
lateral and dorsal body-wall outgrowths
(BWOs) (Gilbert and Thompson, 1968;
Birky, 1968). Figure 2 summarizes these
phenomena. Subsequent experience has
demonstrated that a-tocopherol is absolutely essential for mictic female and BWO
responses; in the absence of this molecule,
no combination of environmental factors
can induce mictic female production or
BWOs. These conclusions hold for our
stocks of A. sieboldi and A. brightwelli,
and for a stock identified as A. sieboldi by
Buchner and Kiechle (1965) but which is
actually quite similar to our A. brightwelli
although reproductively isolated from
both species.
in Asplanchna by a-tocopherol is very specific (Gilbert and Birky, 1971). These
studies employed the body wall outgrowth
(BWO) response of A. sieboldi, but both
this and the mictic female response are
approximately equivalent. Of all the authentic compounds tested only tocopherol
isomers were highly active. Many compounds with a-tocopherol activity in vertebrate systems had very little or no activity
in the Asplanchna assay. In fact, the
Asplanchna responses to date appear to be
the most specifically controlled of the
many known tocopherol responses and
provide some of the most convincing evidence for the hypothesis that tocopherol
may act as a catalyst.
Female A. sieboldi embryos may remain
labile, with respect to differentiating into
either amictic or mictic females, until they
are almost completely developed. When
gravid amictic females were transferred to
a medium containing P. aurelia and a suspension of Chlarnydomonas reinhardti
(3-20 X 1°2 cells per mm 3 ), embryos which
at the time of transfer had separated from
the yolk gland-ovary complex and had developed through gastrulation could still be
induced to develop into mictic females by
The control of mictir female production
253
PARTHENOGENESIS IN ROTIFERS
this change in the maternal diet (Gilbert,
1968). Similar but more extensive and
refined experiments using 10~ 6 M d-a-tocopherol showed that embryos were labile
as late as the stage when internal muscle
contraction becomes evident (Riggs and
Gilbert, unpublished). Embryos with extensive organ differentiation and with coronal
cilia having a strong metachronal beat (See
embryo stage 98 of Tannreuther, 1920) were
still inducible. The labile period may actually extend well past this point in development, however, since there must be some
lag from the time that a-tocopherol is
ingested by the amictic mother to the time
that the inducer reaches the embryos in
the uterus.
This very long labile period in Asplanchna embryos is completely different from
the situation found in brachionid rotifers
where the embryos are determined before
the commencement of cleavage (See Gilbert, 1968, for discussion and references).
This discrepancy is probably because embryos develop in utero in Asplanchna and
outside the maternal pseudocoel in
brachionids (Gilbert, 1968).
Before investigations are begun on the
mode of action of a-tocopherol at the molecular level, it is essential to know if this
extrinsic inducing molecule, after being
ingested by an amictic female, acts directly
upon the embryo in utero as an intrinsic
inducer to modify its development. The
alternative hypothesis is that its effects are
indirect, mediated by an altered physiological state or by a specific intrinsic inducing
molecule produced in females fed a-tocopherol. In experiments designed to answer
this question (Birky and Gilbert, unpublished), we made use of the phenomenon
that effects of dietary a-tocopherol are inherited for several generations. When a
saccate, amictic female (Ax generation) is
fed a-tocopherol (about 1 0 - 6 M ) from birth
until just before the birth of her first offspring (0-24 hours), then washed free of
the inducer, her A2 progeny show a
marked BWO response and include a high
percentage of mictic females (Birky and
Power, 1969). Although these A2 females
33x10-'* moles/S
I
18 5% 43 2%
574% 541%
55 31
Aj 8 3%
A2I
A2ir
A2m
132% 112% 79%
3 8KIQ-1*
A2ET
A2Y
3 8%
20% 0 7%
v" i1
»\
A\
\
23%% 84%
19
-J 7 7x 10"
V
A,BI
110%
A2SI
X
A2 7.6%
8.8% 5 6%
V.
FIG. 3. Summary of experiments on uptake, loss,
and transmission of a-tocopherol in female Asplanchna sieboldi. a-tocopherol contents are given
in moles/female; the content of At females is that
immediately following
exposure of females to 10-°
or HH M H3-o-tocopherol for 20-24 hours after
birth. The cumulative percentage of the total maternal label which is lost to the environment is
shown above the line. The percentage which is
transferred to successive offspring is shown below
the line. Underlined values represent the means of
two or more experiments. The a-tocopherol content of the A4 and AE generations is inferred, assuming that 23.3% of the parental label is transmitted
to the first female offspring in each generation
after the first. Dorsal and lateral views of typical
females are shown for each generation.
received no additional extrinsic a-tocopherol, their A3 progeny in turn show the
BWO response and include some mictic
females, though both responses may be
weaker. Detectable responses may be seen
again in the A4 and, rarely, the A5 generation. If a-tocopherol is acting as in intrinsic inducer, it must be transmitted intact
from generation to generation with high
efficiency.
We fed pulses of H3-a-tocopherol to A1
females and determined at intervals the
total amount of radioactivity in the A^
A2, and A3 generations by liquid scintillation counting. Acetone extracts of females
from each generation were subjected to
thin-layer chromatography; from 76% to
100% of the radioactivity chromatographed
as a-tocopherol. The details of these experiments will be published elsewhere; a
summary of the results is given in Figure
3. The results for the A: and A2 generations are similar, but the latter is of
greater interest since some of the Ax label
254
C. WILLIAM BIRKY, JR. AND JOHN J. GILBERT
is adsorbed on the surface of the animals.
A cumulative total of about 40% of the
total label in an A2 female is lost to the
environment. Almost all of the remaining
label is transferred to her offspring, with
successive offspring receiving progressively
smaller amounts (but approximately constant proportions of the parental label, excluding that which is lost to the environment or already transferred to offspring).
It is thus apparent that dietary a-tocopherol behaves in a very different manner
from ordinary metabolites, being specifically protected from oxidation and transferred from parent to female offspring with
high efficiency. Male embryos receive
slightly less a-tocopherol from their mictic
female parents; this is probably attributable to the greater number and smaller size
of embryos produced by mictic than by
amictic females in these experiments. In
addition, the response to a-tocopherol,
measured in these experiments by the
quantitative analysis of the BWO response
and symbolized by the variable growth of
humps in the females in Figure 3, showed
a rough but consistent correlation with the
a-tocopherol content. Autoradiographs
showed that a-tocopherol tends to be localized in the uterine fluid which surrounds
the developing embryos. These results, and
others which will be described elsewhere,
strongly suggest that a-tocopherol serves
not only as an extrinsic inducer but also as
an intrinsic inducer which acts directly
upon developing embryos to determine
whether they will mature as mictic or amictic females.
At this point, we may inquire into the
evolutionary significance of the requirement of high levels of dietary vitamin E
for mictic female production. Here, it
should be noted that (1) vitamin E is
essential for male, but not female, fertility
in some animals, and (2) male rotifers
cannot feed, and thus could obtain vitamin
E only from their mothers. These facts
have led us (Gilbert and Thompson, 19f>8;
Birky, 1969; Gilbeit, 1971) to postulate that \itamm E is essential foi male
fertility in Afpfatifhtifi- If so. n mechanism
would have to be evolved to insure that
dietary vitamin E is protected from degradation and efficiently transmitted from females to their offspring. In addition, the
evolution of an absolute requirement of
vitamin E for mictic female production
would insure that mictic females would
appear only when dietary vitamin E levels
were sufficient to insure that their male
offspring were fertile.
Our hypothesis that vitamin E is required for male fertility has, unfortunately, some of the features of an untestable
hypothesis, since one cannot obtain males
without insuring at the same time that
they contain vitamin E. However, some
indirect evidence for the hypothesis has
been obtained (Gilbert, unpublished).
Males of A. sicboldi, whose mictic mothers
and amictic grandmothers had been given
dZ-a-tocopherol-5-methyl-H3, were embedded
in Epon and sectioned at Ijion. Autoradiographic analysis of these and control sections demonstrated a significant localization of label in the testis. This concentration suggests that a-tocopherol, and perhaps its metabolites, may be required by
the testis for the production of functional
sperm.
THE ROTIFER LIFE CYCLE IN NATURE
AND ITS CONTROL
As mentioned above, laboratory studies
have implicated several environmental factors in the control of mictic females: high
population densities favor, mictic female
production in Brachionus; high levels of
vitamin E are required for mictic female
production in Asplanchna (and in Epiphanes senta as well?); and a number
of other factors, especially photoperiod
and temperature, may also be of importance. We can now ask if any of these
environmental factors may be responsible
for controlling mixis in natural populations.
Numerous relevant studies of natural
populations have been made, almost all on
planktonic rotifers in temperate climates
(See Pourriot, 1965; Gilbert, 1963; and
PARTHENOGENESIS IN ROTIFERS
Hutchinson, 1967, for recent reviews and
references). The following general conclusions emerge from these studies:
(1) Most natural populations overwinter as dormant resting eggs or as a small
number of amictic females with a very
slow reproductive rate, or possibly as both
in some cases. An increase in water temperature in the spring allows resting eggs
to hatch and amictic females to reproduce
more rapidly. During the spring, summer,
and fall, most populations show one or
more marked increases in population density. The resulting population maxima are
generally of short duration, with only a
few days or weeks at the peak, followed by
rapid declines.
(2) Many populations of a number of
species, especially those in the genera
Brachionus and Asplanchna, appear to be
mictic only or primarily during these population maxima (Carlin, 1943) as shown
by the appearance of mictic females and/
or males followed by the appearance of
resting eggs. Generally, the data are not
sufficiently precise to identify the exact
time of the first appearance of mictic females, but it is clear that in some cases
they may appear during the increase in
numbers before peak population densities
are reached.
(3) Many natural populations never
seem to produce mictic females. This same
observation has often been made in the
laboratory and will be discussed further
below. Sexuality and consequent resting
egg production are usually especially common in small ponds, and are often rare or
absent in large lakes (Wesenberg-Lund,
1923).
(4) Some populations show no clear-cut
relationship between population density
and mixis in nature, producing mictic females both at population maxima and
minima.
(5) Different populations of the same
morphological species may show different
patterns of mixis [(2), (3) or (4) above].
Populations of even closely-related species
in the same body of water may show population maxima with associated mictic
255
reproduction at very different times.
(6) The production of mictic females
seems to be suppressed in the winter even
in those forms which overwinter as females
rather than as resting eggs (WesenbergLund, 1898). This suppression may be due
to short-day photoperiod, low temperatures (Buchner and Kiechle, 1966; Buchner
et al.j 1969), or both, but other possible
agents such as changes in food quantity or
quality cannot be ruled out.
During warmer months, there is a
marked tendency for mixis to occur at times
of high population density. But it
should be clearly understood that in no
case are the data detailed enough to rule
out a correlation of mixis with other environmental factors. The field data do, however, coincide nicely with the results of
laboratory studies on Brachionus (see
above). Moreover, we would like to suggest that for two reasons it would be selectively advantageous for a population of
rotifers to produce mictic offspring only at
times of high population density. First,
mixis must lead to the production of resting eggs; this is absolutely essential for
populations which overwinter in the form
of resting eggs. Resting egg production requires fertilization, which may be more
effective at higher population densities.
This is particularly true in rotifers, where
mictic females of most species can only be
impregnated with sperm during the first
few hours after birth (see above). Some
preliminary laboratory experiments with
Asplanchna (Birky, unpublished) do indeed suggest that fertilization is more effective at population densities corresponding
to those of population maxima in nature.
This is presumably due to more frequent
contacts between males and fertilizable
mictic females. But the interpretation of
such experiments is difficult, and much
more work needs to be done.
In addition, the production of large
numbers of mictic females may serve as a
mechanism for population control. The
decline in population following a maximum may be attributable, in part, to a
reduction in the number of rapidly-
256
C. WILLIAM BIRKY, JR. AND JOHN J. GILBERT
reproducing amictic females and to their
replacement by mictic females and males,
and subsequently, resting eggs which require several days at least before hatching.
One can imagine, therefore, that rotifers
which produce mictic females at high population densities might have a marked selective advantage in that they would avoid
intensive intraspecific competition for
resources. We do not propose this as the
only, or even the primary, mechanism of
population control in planktonic rotifers,
but suggest that its importance should be
investigated.
These arguments, which suggest an evolutionary rationale for the natural coincidence of mixis with high population density present a difficulty in interpreting laboratory studies with Asplanchna. The studies described above point to the control of
mictic female production by levels of dietary vitamin E. Data are not available on
dietary vitamin E levels in natural populations. However, some field studies (e.g.
Kofoid, 1908) suggest that algae are available to Asplanchna throughovit most of the
summer. Most natural collections of Asplanchna females which we have made
show evidence of ingested algae or of
ingested rotifers which in turn eat algae,
whether mictic females are present or not.
In addition, Gilbert (1969, 1971) has
found a-tocopherol and a second, unidentified compound with strong inducing activity in dried grasses. He has also demonstrated biological activity—probably a-tocopherol—in detritus particles derived from
the decomposition of fresh grass in distilled water. Thus, detritus-feeding microplankton could pick up these compounds
and deliver them to Asplanchna even in
the absence of large phytoplankton
blooms. We would guess, then, that fairly
high levels of dietary vitamin E are always
available to Asplanchna and we are left
with the problem of explaining the sporadic occurrence of mixis, correlated more
clearly with population density than with
vitamin E levels. It is, of course, possible
that changes in feeding behavior occur as a
direct result of population density changes,
and that these in turn result in changes in
dietary vitamin E levels.
A simpler hypothesis is suggested by the
experiments of Birky (1969). It was shown
that amictic females reared at a population density of 100 females/liter and exposed to a-tocopherol produced a higher
percentage of mictic offspring and offspring which showed stronger BWO and
fecundity responses than did females
reared at a population density of 10 females/liter. These densities correspond
roughly to those of some natural populations at times of maximum and low population density, respectively. It was further
shown that enhanced sensitivity to vitamin
E was due to one or more soluble conditioning factors which are released into the
medium by amictic females and act on the
same or other females.
It is thus possible to reconcile the laboratory and field observations on Asplanchna by hypothesizing that, although there is
sufficient vitamin E to permit mictic female production during most or all of the
year, mictic females can only be produced
at times when high population densities
lead to sufficient concentrations of the conditioning factor (s) which permit a response to vitamin E. Population density,
rather than vitamin E levels, would thus
be the effective factor controlling sexual
reproduction in nature. Attempts to isolate
and identify the conditioning factor (s)
which may be pheromones, have failed.
Our finding that population density enhances the sensitivity of Asplanchna to
a-tocopherol contradicts the conclusions of
Buchner et al. (1969), who state that the
production of mictic females is independent of population density. But their experimental designs, and the conclusions
which can validly be drawn, are completely different from ours. Their experimental
results are, in fact, compatible with ours if
one assumes that (1) at extremely high
population densities (== 50 females/ml),
old culture fluid actually inhibits the production of mictic females (see below), and
(2) in a clone of amictic females, some
are relatively insensitive to a-tocopherol
PARTHENOGENESIS IN ROTIFERS
and cannot be induced to produce mictic
females, although their amictic female offspring will regain sensitivity after one or a
few generations. This physiological variation in ability to produce mictic offspring
has, in fact, been demonstrated in experiments of Birky (1969) and of Buchner et
al. (1969). Thus, one can only conclude
from the work of Buchner et al. (1969)
that the correlation between population
density and sensitivity to a-tocopherol is
not absolute, and that other factors may
interfere.
Two other environmental factors were
investigated by Buchner et al. (1969). The
percentage of mictic female offspring was
higher at higher temperatures (See also
Buchner and Kiechle, 1966); this observation may in part explain the suppression
of mixis in natural populations during the
winter as discussed above. They also obtained evidence that high O2 concentrations stimulate, and high CO2 concentrations inhibit, the production of mictic females. They suggest that these effects may
explain, at least in part, the induction of
mictic female production by dietary algae
and the inhibition by old culture fluid.
These results, together with those of Birky
(1969) on the effects of different culture
media, serve to emphasize the number of
different factors which can affect the transition between sexual and asexual reproduction.
Much of the effort of biologists studying
the control of sexual reproduction in rotifers has been directed toward identifying
the environmental factor controlling the
production of mictic females. It is tempting to extend our hypothesis concerning
the control of sexual reproduction in Asplanchna to other rotifers. It is also plausible: Brachionus species may have a similar
absolute requirement for high levels of
vitamin E, for all lab studies have been
done with animals fed algae, and Epiphanes senta may require high population densities (all lab studies have been
done at fairly high densities, and there are
no field data). However, it has been repeatedly found that the sensitivity of roti-
257
fers to mixis-inducing agents varies greatly
from clone to clone (e.g. Birky, 1969). As
the most extreme example noted to date
Solberg and Dougherty (1959) have described an apparent case of a mutation in
Brachionus variabilis which rendered mutant females completely unable to produce
mictic daughters under their conditions.
This, together with the wide variety of
environmental and physiological factors
which may influence the response, suggests
that each species and even each individual population may have evolved a set of
responses to environmental factors which
will insure that sexual reproduction occurs
at a time appropriate for its own specific
habitat. Thus, a search for the universal
controlling agent, or even set of controlling agents, may be doomed to failure
(Birky, 1969).
Nevertheless, we wish to reiterate what
is probably the most important single conclusion to be drawn from the work described above. The transition from asexual
to sexual reproduction in rotifers is not
simply attributable to change in the environment, or to a deterioration of the environment. Specific controlling stimuli can
be identified, and can be shown to make
sense in terms of the organism's physiology, ecology, and evolution.
RAPID PARTHENOGENETIC DEVELOPMENT
AND ITS PHYSIOLOGICAL BASIS
Amictic rotifer populations are capable
of extremely rapid growth. For example,
the data of Kofoid (1908) on Asplanchna
brightwelli from the Illinois River show
ten to twenty-fold increases in population
density in periods of one to two weeks; this
is not atypical, even though it involves a
river system rather than a lake. In this
section we will ask how this rapid population growth rate is achieved, and in the
next section we will suggest an evolutionary rationale for its origin, in the case of
rotifers.
To begin with, it is reasonable to attribute this high rate of population growth
in part to parthenogenesis itself. To the
258
C. WILLIAM BIRKY, JR. AND JOHN J. GILBERT
extent that amictic females have dispensed
with sexual reproduction, they have eliminated the time required lor contact and
mating with males. This can buy considerable increase in the rate of reproduction,
other factors being equal, as has been
shown mathematically by Tomlinson
(1966). In addition, the effective fecundity
of the population is doubled by the absence of males.
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A second factor in the achievement of
rapid population growth rates may be seen
by examining the reproductive parameters of amictic females. Some representative data for laboratory populations are
given in Table 1. Data on development
times and birth rates for natural populations of several species may be found in
Edmondson (1960). The general picture is
one of very rapid embryonic development, short immature periods, and the
rapid sequential production of a small
number of eggs during a rather brief mature period which is followed very shortly
by death. It is clear that rapid population
growth is achieved by rapid development
from egg to mature adult, as opposed to
high fecundity. How is this rapid development achieved in rotifers?
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Most of the relevant studies have been
done on Asplanchna, but the basic features
are probably common to most or all rotifers. References on rotifer embryology and
techniques are given by Birky (19676); a
more recent investigation at the ultrastructural level is that of Bentfeld (1968). Autoradiographic studies on DNA synthesis
have been done by Birky, Bignami, and
Bentfeld (1967) and Birky (unpublished),
and on RNA and protein synthesis by
Birky (unpublished) and Bentfeld (1968,
1969); synthesis was measured by incorporation of H 3 -thymidine, H 3 -uridine, and
a
semiH 3 -leucine, respectively, in
quantitative manner with the appropriate
en/yme controls.
Oocyte maturation requires about five
1 houib at 2 J
in *l.\[)ltuii !u>i< b> i^Jila.'i!!'.
i During t li is time, tvtoplasm rapidly
streams into the matin ing oocyte irom the
PARTHENOGENESIS IN ROTIFERS
vitellarium, and the oocyle undergoes a
1000-fold increase in volume. No RNA
synthesis is detectable in the nuclei of immature or maturing oocytes; given the
level of sensitivity of autoradiography, this
suggests the complete absence of rRNA
synthesis and at most a low level of mRNA
synthesis. RNA synthesis occurs rapidly in
the vitellarium nuclei; however, these nuclei are characterized by large, lobate nucleoli, and are probably synthesizing both
rRNA and mRNA. Protein synthesis occurs at a rapid rate in the vitellarium and
at all developmental stages. Polyribosomes,
and thus presumably functional mRNA,
are included in the material supplied to
the maturing oocyte from the vitellarium.
These results suggest that the oocyte nucleus is relatively inactive, and that most of
the proteins of the maturing oocyte are
synthesized on maternal templates, in the
vitellarium, and perhaps also in the oocyte
itself.
After separation from the vitellarium,
the mature oocyte enters the second, or
mitotic, phase of development, which requires about six hours. About 10 rounds of
mitotic cell division occur during this
phase, together with morphogenetic movements (including the beginning of gastrulation) and cell differentiation. The first
three cleavages take place at intervals of
about 20 minutes. The 20-minute cell cycle
includes about eight minutes for mitosis, a
Gi period of about one minute or less, an
S period of about six minutes, and a G2
period of five minutes or less. There is
little or no growth of the embryo as a
whole during the mitotic phase of development. There are no nucleoli, and RNA
synthesis is not detectable following short
(10 or 20-minute) pulses of H3-uridine.
RNA derived from the vitellarium during
oogenesis is still present, however, and
there is rapid protein synthesis.
During the final, or postmitotic, phase of
embryonic development, there are no further mitoses or cell divisions, and no DNA
synthesis except in vitellarium nuclei
which are presumably becoming at least
259
partially polyploid. Cell membranes break
down, so that some tissues become syncytial. Gastrulation and organ and cell differentiation are completed. There is little
growth until about the last five hours,
when the body wall (hypodermis) expands
and the pseudocoel begins to fill with
fluid. At the beginning of the post-mitotic
phase of development, nucleoli appear and
RNA synthesis in embryonic nuclei is easily detectable.
The development of the male offspring
of mictic females is very similar, but no
studies of macromolecular syntheses have
been done on male embryos. In Asplanchna, both male and female parthenogenetic embryos develop to reproductive maturity in utero; in most species, development begins in utero and is completed outside the female but with the embryo surrounded by a thick shell. Thus, the bulk of
the molecules used both for energy and
for raw materials for syntheses must be
supplied by the mother.
We postulate that the rapid embryonic
development of rotifers may be attributed
at least in part to the fact that the embryo
is spared the necessity of acquiring its own
raw materials (i.e., in most rotifers, there
is no larval stage and only a brief immature stage), and, perhaps more importantly, the necessity of carrying out RNA synthesis during much of development. It is
reasonable to suppose that, since the machinery for protein synthesis in early development is largely or entirely supplied
by the mother, the young embryo is able to
devote most of its energy resources to
DNA replication, membrane synthesis, and
chromosome movements. Moreover, if the
embryo's genome is not being transcribed,
G1 and G2 periods can be reduced in
length, as they clearly have been, which in
turn will speed up the rate of cleavage
divisions. Use of a maternal tissue for the
transcription of RNA has the further advantage that this tissue, unlike the germ
line, is free to become polyploid or polytene and hence to synthesize RNA more
rapidly.
260
C. WILLIAM BIRKY, JR. AND JOHN J. GILBERT
ROTIFERS AS OPPORTUNISTIC ORGANISMS:
SPECULATIONS ON THE EVOLUTION
OF PARTHENOGENESIS
Sexual reproduction, with its obvious
advantage of providing in every generation
new combinations of genes for adaptation, is so much the rule among higher
organisms that biologists are forced to
provide special explanations for exceptions
to the rule. How, then, are we to explain
the obvious success of rotifers, which have
managed to acquire an eminent, and sometimes pre-eminent, place among the
zooplankton in habitats ranging from eutrophic waters of the tropics to the antarctic even while abstaining partly or entirely
from sex?
The answer may well be that organisms
which adopt a certain life style can afford
to give up sexual reproduction in favor of
other adaptations. We begin by referring
to the general subdivision of organisms
into equilibrium and opportunistic species.
Equilibrium species, exemplified by many
vertebrates, maintain relatively constant
population sizes, in part by being adapted
to reproduce, at least slowly, in most of the
environmental conditions which they
meet. Opportunistic species, on the other
hand, show extreme population fluctuations; they are adapted to reproduce only
in a relatively narrow range of conditions,
but make up for this by reproducing extremely rapidly in favorable circumstances.
At least in some cases, opportunistic organisms can also be categorized as colonizing
organisms. Bacteria and other microorganisms provide the extreme examples
within this category.
Many or most rotifers clearly are opportunistic and colonizing organisms, as well.
Recall that they undergo rapid fluctuations in population during warm months,
and, in temperate climates, the first resting
eggs to hatch in the spring or the few
surviving amictic females have a whole
lake, pond, or stream to colonize. The potential for rapid reproduction is the essential evolutionary ticket for entry into the
opportunistic life style. In rotifers, we sug-
gest that this ticket has been purchased in
two payments (without specifying the order of payment):
(1) By the evolution of parthenogenesis. This, as noted above, eliminates
the time required for mating and doubles
the effective fecundity.
(2) By the evolution of the use of a
polyploid maternal nurse cell tissue, the
vitellarium, to carry out syntheses in advance for the developing embryo. This, as
suggested above, can be expected to materially enhance the rate of embryonic development.
The second point requires further comment. What is important here is that the
intrinsic rate of natural increase (the
Malthusian parameter, r,) be increased for
the population. This, in turn, can be increased by any of several methods: increasing fecundity, decreasing time to peak
fecundity, decreasing time to the end of
the fecund period, and decreasing the immature period, as well as by increasing the
rale of embryonic development (Birch,
1948). Lewontin (1965) has shown, mathematically, that for a colonizing species
the most efficient method of increasing rt is
to increase the rate of embryonic development, i.e., a given change in r{ requires a
smaller percentage change in developmental rate than for example, in fecundity.
Note that rotifers in general do not show
strikingly high fecundity.
Apomictic parthenogenesis and the pattern of macromolecular syntheses described
here for rotifers are seen singly and in
combination, in other groups as well {e.g.,
Drosophila is a well-known example of the
use of nurse cells). They may not always
be correlated with rapid development or
with the opportunistic life style. We do
not, of course, wish to imply that these
adaptations are the only mode of entry
into this life style, or that they are by
themselves sufficient for entry (See Birch,
1918, for a discussion of this sort of analysis). We suggest only that in the specific
case ot rotifers, and perhaps some other
organisms, selection lor rapid population
growth rate provides an evolutionary ra-
261
PARTHENOGENESIS IN ROTIFERS
tionale for the origin of both iJarthenogenesis and the use of nurse cells.
Our suggestions could be tested, though
perhaps with some difficulty. It needs, for
instance, to be shown that the mating process does, indeed, require sufficient time so
that, if amictic females had to be fertilized,
the rate of increase of the population
would be significantly reduced. Further, selection for rapid embryonic development
should have substantially reduced the genetic variance for that parameter without
having had so great an effect on fecundity
(Lewontin, 1965). Further selection in the
laboratory should be unable to reduce development rates further, but might succeed
in increasing fecundity. Perhaps most interesting of all would be comparative
studies of rotifer populations which are,
relative to each other, more in equilibrium
and more opportunistic. The latter
should show more rapid developmental
rates and, as species, a greater tendency to
dispense entirely with sexual reproduction.
IS SEX NECESSARY, AND IS IT REALLY SEX,
IN ROTIFERS?
There seem to be two extreme views on
the evolutionary implications of parthenogenesis and sexual reproduction in rotifers.
The first was expressed to one of us in
several conversations with the late
Ellsworth Dougherty, who argued that sexual reproduction was so essential for the
evolutionary success of any organism that
even bdelloids must indulge in some form
of exotic sex, perhaps analogous to
transduction in bacteria, which has thus
far escaped detection. At the opposite extreme, Ruttncr-Kolisko (1968, 1969) has
argued that sexual reproduction is important primarily because it results in resistant resting eggs, and that it is ineffective
genetically either because true recombination does not take place (see above) or
because it consists primarily of selfing. The
strongest support for her argument is that
sexual reproduction is obviously dispensable, since many successful monogonont rotifer species or populations have lost sex-
ual reproduction and bdelloids may never
have had it.
We would like to argue for a more balanced view point. To begin with, Dougherty may well have been correct in principle, though not in detail. Let us assume for
the moment that the mechanism of amictic parthenogenesis is the one most widely
accepted, i.e. an essentially equational maturation division. During amictic reproduction, recessive mutations will accumulate in the genome, providing heterozygosity and new genotypes. This accumulation
may be considerable, even in forms which
have relatively short periods of amixis interrupted by mixis, due to the large number of germ line cell divisions which occur
during rapid amictic reproduction. The
exact rate of accumulation will depend, of
course, upon mutation rates, which are in
turn subject to genetic control and can be
optimized to fit an organism's requirements.
The rate of accumulation of new genotypes will also depend upon selection.
Crow and Kimura (1965), following an
argument proposed by Muller (1932),
have compared the rate of incorporation
of new favorable mutations into populations of sexual and asexual organisms. Figure 4 summarizes the problem (using
haploids for simplicity): in a sexual population of constant size, new mutant genes
A, B, and C which appear at roughly the
TIME
SEXUAL
POPULATION
SIZE
ASEXUAL
FIG. 4. The incorporation of new mutations into
asexual and sexual populations. The abscissa is
time; the ordinate is number of individuals. Total
population size is assumed to be constant (Modified from Crow and Kimura, 1965).
262
C. WILLIAM BIRKY, JR. AND JOHN J. GILBERT
TIME
POPULATION
SIZE
~BC|
FIG. 5. Hypothetical diagram o£ the appearance of
new mutations in an exponentially growing amictic
rotifer population. The abscissa is time; the ordinate is number of individuals. See text for further
explanation.
same time will all be incorporated into the
population by sexual reproduction, in all
possible genotypic combinations. In an
asexual population, on the other hand, selection will eliminate B and C, and the
population must wait until these occur
again to acquire genotype AB, and must
wait still longer to acquire the genotype
ABC.
This analysis, however, may not apply to
many rotifers. Recall that these are opportunistic organisms, which under favorable conditions are reproducing asexually
(by apomixis) at exponential or nearly
exponential rates. This implies that during
such periods reproduction is taking place
under conditions of essentially unlimited
resources (Lewontin, 1965) and, consequently, of reduced or no intraspecific
competition and of greatly reduced selection. The situation may, then, be as diagrammed in Figure .">, in which most or
all new mutant genes will persist in the
exponentially growing population. Sexual
reproduction will still result in a moie rapid production of new genotypes if it occurs
early (at time tx), but not if it occurs late
(af time t..).
Counteracting the tendency toward
heterozygosity will be crossing-over, which
can provide still more genotypes by making individuals homozygous for the newly
introduced recessive mutations. Such crossing-over may theoretically occur during
the equational maturation divisions of
apomixis, analogously to somatic crossingover. For our purposes, the important
point is that the rate of fixation of genes in
the homozygous condition depends upon
the degree of linkage of the gene to the
kinetochore and upon the strength of selection, if any, for the heterozygote. Again,
we note that linkage can be readily
modified by chromosomal rearrangements,
so that the tendency of each locus to maintain heterozygosity can be adjusted independently. The overall rate of movement
to homozygosity can, of course, be adjusted
by controlling the frequency of synapsis
and crossing-over.
We thus suggest that a "clone" of rotifers can indeed acquire a wide variety of
different genotypes; to the extent that
these are acquired during the apomictic
maturation divisions through crossingover, i.e. recombination, the process is really sexual from the standpoint of the student of genetics and evolution. It must be
emphasized again that component events
are subject to genetic control, and thus the
rate of acquisition of new genotypes can
be adjusted in the course of evolution.
If the argument is correct thus far, what
additional genetic advantages are provided
by sexual reproduction? It is certainly
true that the production of new genotypes
can be further enhanced by sexual reproduction. But this contribution will lie reduced by the relatively poor success of sexual reproduction; the fecundity of impregnated females is lower than that of amictic
females and the hatchability of resting
eggs, even under laboratory conditions,
rarely approaches 100% (Birky, 1967a).
Sexual reproduction thus may result in the
loss of many genotypes. Moreover, the
contribution of sexual reproduction to
genetic variability, other factors being
equal, will be reduced to the extent that
263
PARTHENOGENESIS IN ROTIFERS
rotifers are inbreeders tending to mate
with close relatives and thus to combine
genomes which are already similar. It is
difficult to evaluate the extent of inbreeding in rotifers on a priori grounds. The
formation of resistant resting eggs provides
an ideal mechanism for dispersal and thus
for outbreeding. The wide distribution of
many morphological species provides concrete evidence for outbreeding; but this
evidence must be interpreted with caution.
Birky (unpublished) has attempted without success to cross two American stocks of
Asplanchna hrightwelli with a stock of
morphologically identical and physiologically similar rotifers from Germany (supplied by and incorrectly identified as A.
sieboldi by Kiechle and Buchner, 1966).
Many of the different populations of morphologically identical rotifers may thus actually be reproductively isolated.
Several factors, in contrast, argue strongly for inbreeding. These include the inability of females to be fertilized following
the first few hours after birth, the reproductive maturity of newborn males, and
the short life span of the males. All of
these factors favor mating soon after birth,
before much migration has occured, and
thus with close relatives. In addition,
Ruttner-Kolisko (1968) has pointed out
the possibility that, due to the presumed
small number of resting eggs which hatch
and the rapid colonizing ability of the first
egg to hatch in a pond, many small bodies
of water may be populated with one or a
few clones which in turn would impose
extreme inbreeding or selfing.
Few data relevant to these questions are
available (See Birky, 1967, for reference to
the limited evidence for heterozygosity in
rotifer populations). The point we wish to
make is that, theoretically, rotifers can acquire and maintain a considerable degree
of genetic heterogeneity without recourse
to sexual reproduction. The additional advantages of sexual reproduction may not
be great and must be balanced against the
advantages of eliminating sex entirely,
which would further improve the reproductive rate. From this point of view, then,
it is not surprising that so many rotifer
populations and entire species have dispensed with sex entirely.
In a broader context, we note that is has
often been suggested that sexuality is so
important for organisms in general that
"asexual" parthenogenetic forms must represent evolutionary dead ends. The chief
argument for this viewpoint is the supposed more rapid production of genotypic
variability, and thus of evolutionary flexibility, in sexual forms. Nevertheless, parthenogenetic forms have achieved continuing success by counterbalancing their limitations through the development of certain
advantages. These include more rapid
reproduction, elimination of the segregation load, etc. We would like to suggest
that the time has come to tackle the basic
argument itself and to re-examine the
question of relative rates of genotypic variation in sexual and asexual populations.
The difference, as suggested above for rotifers, may not be so great. We are pleased
to see that a start has been made in this
direction (Asher and Nace, 1971).
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