Conjugation and autogamy in ciliated protozoans

Paul R. Earlpearl@dsi.uanl.mx

Facultad de Ciencias BiológicasUniversidad Autónoma de Nuevo León

San Nicolás, NL 66450, Mexico

A little bit of historySexual processes in ciliates have been known since they were discovered by the Dutch lens maker Antoni van Leeuwenhoek over 300 years ago. The Danish naturalist O. F. Müller (1786) was the second observer of the sexual event conjugation. Others like Balbiani (1858, 1861) followed, including Hertwig (1889) and Maupas (1889). Giant steps in the study of ciliate conjugation were taken by Gary N. Calkins (1869-1943) with Paramecium caudatum as in Calkins & Cull (1907). Herbert S. Jennings (1868-1947) in Paramecium had an analogous role to that of Thomas H. Morgan (1866-1945) in Drosophila. Autogamy was discovered around 1934 in P. aurelia by William F. Diller (1902-1984), and Tracy M. Sonneborn (1904-1981) discovered mating types in 1938 in Paramecium.

Ehrenberg (1838) suggested immortality for the ciliates, and Maupas (1889) found that they had the periods of youth, maturity and senescence. The rejuvenation of old clones follows a sexual event. Without conjugation, autogamy or cytogamy, the cell line will die out as found by the remarkable French-Algerian protozoologist Maupas (1842-1916).

Sexual processesAutogamy is like conjugation, except that the entire process occurs in single cells. Ralph Wichterman (1904-1999) discovered cytogamy. See his 1986 book on Paramecium, and the book edited by Görtz (1988). Cytogamy is like conjugation, except that there is no nuclear exchange between the partners. It is independent double autogamy.Ciliates have several features of chromosome segregation that are unique among eukaryotes, including their maintenance of 2 nuclei: the germline micronucleus (MI), which undergoes conventional mitosis and meiosis, and a somatic macronucleus (MA) with a great number of minichromosomes that divide by a poorly understood amitotic process. Ciliates can propogate without the MI !

Each cell contains an MI with conventional chromosomes and a transcriptionally much more active polyploid MA that controls the cell. The MA genome is com-posed exclusively of short linear minichromosomes that typically contain single coding regions. The MA DNA molecules are derived from a copy of the MI genome during conjugation through an extensive DNA reorganization process that includes the fragmentation of the MI chromosomes and the new addition of telomeres to the ends of the formed MA minichromosomes via reverse transcriptase subunit of telomerase or TERT.

P. aurelia does not really exist as a species any longer since Sonneborn (1975) divided the aurela complex into 14 syngens = species. A species, following Mayr (1957), is a sexually isolated population. It cannot cross with any others. How different species are morphometrically, or in their DNA or RNA sequences does not involve the species definition that is only concerned with crossability.

The sequences of base pairs from several genes like the histone centromere gene can be most helpful for phylogenetic analyses. The polymerase chain reaction, its wonderous results, blotting and other molecular techniques are still in their protozoologic infancy, although new insights are appearing at a fast pace.

Know your Darlington ! NeoDarwinism combines Charles Darwin's theory of evolution by natural selection and Gregor Mendel's theory of genetics with random mutation as the source of variation. Cyril D. Darlington (1903-1981) discovered chromosomal crossover and outlined the stages of meiosis in his 1937 textbook updated from his 1931 text.

A reduction division MUST OCCUR in meiosis. Reduction before fertilization is gametic, whereas reduction after fertilization is zygotic meiosis. Meiosis reduces the number of sets of chromosomes by half, so that when gametic nuclei recombine the life cycle diploidy of their parents will be reestablished.

In mitosis, 2 new chromosome sets segregate to 2 new cells. In meiosis, homologous pairs of chromatids (bivalents) segregate to become the gametes that can fuse to become the synkaryon (fertilization nucleus).

Macronuclear regeneration. Reciprical fertilization in the synkaryon produced a genetically new MA. Most commonly the old MA is degenerated and resorped. However when the new MA fails to develop, fragments of the old MA are able to grow and resume control of the exconjugant cell. Macronuclear regeneration was discovered by Sonneborn in Paramecium in 1940.

The MA genome has only a small % of the MI genome (5–10%); thus, the chromatin elimination involves as much as 95% of the DNA. Two types of DNA processing form the MA chromosomes: 1) deletion of DNA sequences that are ISEs and retention of the MA genomic sequences--the MDSs; and 2) fragmentation of chromosomes 5' and 3' of genes coupled with the new addition of telomeres.As we move along, we find new directions leading still farther from textbook cytology.

General conjugation with emphasis on Tetrahymena

In the crescent soon after pairing, the 10 diploid chromosomes of Tetrahymena are reduced to 5 haploid MIs in meiosis and are later exchanged between the conjugants. In each cell of the pair, the migratory MI fuses with the stationary MI to form a new zygote which is the synkaryon.

The fertilization nucleus proceeds through 2 mitotic divisions, usually, resulting in 4 daughter nuclei. Two of the nuclei can serve as the germ line MI for the daughter cells following the next round of cell division. One of the nuclei will segregate to the new MA, and one to the new MI. The old MA will be degraded unless needed to salvage a malfunctioning new MA. One of 2 MI in the 1st exconjugant is eliminated so that the 2nd excongugant regains 1 MI + 1 MA.

Soon after mitosis, chromosomes within the nucleus are destined to differentiate, undergoing multiple rounds of replication to form polytene chromosomes. The chromosomes are radically fragmented to minichromosomes, telomeres are added to sequences of the MDSs, and the DNA of the remaining non-MAs and the old MA fragments are degraded.

The illustration above fits classic meiosis.

The stages of the crescent after Sugai & Hiwatashi (1974)

First prize goes to the people of the Yao laboratory

The best interpretation of conjugation is by Marcella D. Cervantes, Xiaohui Xi, Danielle Vermaak, Meng-Chao Yao and Harmit S. Malik (J Biol Cell 17: 485-497, 2006), mainly because they have used the most recent technology which is that of immunofluorescence with centromeric antibody comple-mented with DAPI fluorescent dye specific to DNA as in their 3 figures given here.

Remember that the somatic MA is highly polyploid and consists of amplified, highly rearranged segments of the MI genome. The MA is transcriptionally active and is responsible for much of the gene expression in ciliates, whereas the MI is active at special times like meiosis.

The completion of meiosis sets up a cascade of events that leads up to the formation and diversification of the germline MI & MA. One of 4 meiotic MIs is selected as a gamete and undergoes 1 round of mitosis. The paired cells exchange MIs, and then the 2 gametic nuclei, one from each parent cell, fuse forming the diploid zygotic nucleus--the synkaryon or zygote.

These MIs are the stationary and the migratory nuclei and usually identical. Nevertheless, different allopolyploids might well be encountered.

Tetrahymena thermophila has 10 diploid chromosomes. They are represented by the brilliant green staining of the appropriate antigen, centromeric histone.

Stage III of the crescent and the distribution of Histone 3. Credit to Cervantes et al, 2006

Destruction of the old MA. Credit to Cervantes et al, 2006

Let's review conjugation again !

When conjugation is induced, the germ MI enters meiosis and forms a synkaryon after reciprocal exchange of gametic nuclei between the mates. The synkaryon divides 3 times--usually--and differentiates into new macro- and micronuclei. The newly developed macronucleus (MA anlagen) begins to function, the old MA degenerates and the cells separate to be exconjugants. Eliminate extras like 2 not 1 MIs in excongugants to fit the condition. That's it !

FPD first prezygotic division, TPD third prezygotic division, PZD post zygotic division.

Exconjugant of Paramecium with 4 MA anlagen. The Mis are lost among MA fragments. Basic fuchsin, fast green.

An older exconjugant of Paramecium with 2 new MAs.