1. Autogamy, or self-fertilisation in the marine ciliate Euplotes minuta
has been studied by genetic and cytological means. The naturally occurring,
between stock variation in four characters was used to contrast the genetic
consequences of both conjugation and autogamy.
2. The existence of a number of different mating types allows crosses to be
made, and the inheritance of the various characters to be analysed. All
mating type alleles behave in crosses as if they are alternatives at a single
locus, and as if there is a serial system of dominance - whereby an allele
assigned a number high in the series is dorninant over all alleles lower in
the series. Stock A25, previously described as being heterozygous at the
mating type locus (Nobili ana Luporini, 1967) , was found to have the genotype
mt⁷/mt⁷.
3. In this species, two types of stock are available; one type possesses the
ability to pass through autogamy, the other type does not. The F₁ clones
derived from crossing autogamous and non-autogamous parents were all capable
of autogamy. The fact that the ratio of autogamous to non-autogamous clones
was not significantly different from, firstly a 1 : 1 ratio in backcross to one non-autogamous parent, and secondly a 3 : 1 ratio in an F₂ derived by
crossing two F₁ clones, means that this trait could be controlled by a single
locus, at which an allele which permits autogamy is dominant to an allele which
does not allow this process to occur. Non-autogamous stocks must be homozygous
for the non-autogamous alleles, and autogamous stock A25 must be homozygous for
the allele which confers autogamy.
4. Antisera were prepared against two strains of E.minuta. when prepared
against an autogamous strain, the antiserum is efficient at immobilising the
homologous and heterologous autogamous stocks, but gives a weak reaction
against the non-autogamous stocks. An antiserum against a non-autogamous
strain is also capable of discriminating between the two classes of stocks.
Serotype was not inherited according to simple Mendelian rules. It is
suggested that several loci may interact in the determination of this
character.
5. By the use of electrophoresis of cell extracts, followed by detection of
localised enzyme activity on a starch-gel, stocks were found to differ with
respect to an esterase termed Es-l. This enzyme was characterised by
inhibitor studies. Whereas the non-autogamous stocks used for genetic
purposes possess an active form of Es-l, and are said to carry the allele
Es-lᵇ, the autogamous stock A25 has no Es-l activity and carries only the
silent Es-lᵃ alleles. The presence of esterase Es-l is controlled by a single gene; the allele Es-lᵇ being dominant to the silent allele Es-lᵃ. The results are consistent with the enzyme being a single polypeptide chain.
6. There was no genetic linkage between any of the four characters studied.
7. F₁ clones heterozygous at the loci controlling the four characters were
passed through autogamy. Although after conjugation the segregation ratios
for those traits which are certainly controlled by single genes, never deviated
significantly from expectation, the phenotype of the F₁ was in most cases
retained in the F₂ from autogamy. At the mating type and Es-l loci the
recessive homozygote appeared in only 1 in 30 exautogamous clones.
8. Nuclear events at conjugation and autogamy were studied by Feulgen and
Giemsa staining. The micronucleus first divides mitotically and the two
products undergo meiosis. Two of the 8 haploid products of the second meiotic
division persist and undergo a further division. Two of the four products
are the gametic nuclei. After synkaryon formation there is normally one post-zygotic division; one product becomes the new micronucleus, the other
enlarges into the macronuclear anlage. In the exconjugants this anlage
differentiates to produce the sickle-shaped adult macronucleus. After
autogamy the macronucleus rarely develops beyond the anlage stage; the
anlage disintegrates and most animals which survive autogamy do so by
regeneration of one or two fragments of the old macronucleus which are still
present in the cell.
9. Knowledge of the sequence of nuclear events preceding conjugation and
autogamy makes it possible to compute the expectations for the various genotypes after fertilisation. In P.aurelia, only 1 haploid nucleus survives
beyond the second meiotic division, and produces gametes; therefore,
exconjugants are alike, and exautogamous animals are homozygous. Due to the
persistence, in E.minuta, of 2 haploid nuclei which appear to be the products
of different meioses, gametic nuclei may be sisters, or non-sisters.
Cytological data and genotypic ratios support the conclusion that in a cross Aa x aa the two exconjugants shculd be alike in 2/3 of the pairs, and unlike
in 1/3. Crosses with E.vannus, E.crassus (Heckmann, 1963, 1964) and E.minuta
fit this expectation. The expected ratio for synkarya after autogamy of
animals of genotype Aa, is lAA : lAa : laa. The genetic evidence shows that
this expectation is clearly not fulfilled.
10. Macronuclear regeneration produces heterocaryons in other ciliates. The
possibility that exautogamous E.minuta are heterocaryons is discussed. It is
concluded that macronuclear regeneration is not an artefact of laboratory
culture, but has an advantage over genuine autogamy in that maximum heterozygosity is retained. Autogamy starts a new phase of the life cycle in E.minuta; animals initially being immature. The, fact that autogamy can counteract
the effects of senescence enables the nature of the rejuvenation process
and theories of ageing to be reconsidered.
