History of research on C. elegans and other free-living nematodes as model organisms^*

Until 1920, most authors employed culture methods based on those of Maupas (1900). The container was a hollow slide, a watch glass or a Petri dish, maintained in a humid chamber or under glass sealed with paraffin oil. The medium was a layer of water, thin enough to allow for oxygenation. Food was provided by pieces of decomposing meat, the nematodes feeding on the bacteria that grew on them. Some authors tried peptone solutions with variable success. Haven Metcalf was the first to employ agar media (Metcalf, 1903).

When comparing the culture methods used for free-living nematodes with those in practice in Drosophila genetics, Nigon noticed major differences (Nigon, 1943). In Drosophila crosses, progenitors were isolated by pairs and transferred every day to new containers. They thus received constant and reproducible conditions. For nematodes, transfer of isolated progenitors could be achieved more readily by replacing the liquid medium with an agar medium. Nigon thus poured an agar medium in large drops on preparation slides that were maintained in humid chambers (Figure 1). The medium was seeded with micro-organisms that served as food: first yeasts, easily obtainable anywhere, which were later replaced by defined bacterial cultures by Dougherty and Brun. The animals developed in a confined medium because those venturing outside the drop onto the glass surface were threatened by rapid desiccation. Every day the adult(s) were moved to a new drop, leaving the progeny produced during 24 hours on the previous drop. In some difficult research settings (Bergerac), the animals were followed with the naked eye, transported with a wood needle, with a possible a posteriori confirmation of their number and sex using a low-magnification compound microscope (Nigon, 1943) (Figure 1). In more comfortable laboratory conditions, all manipulations were carried out under the dissecting microscope (Nigon, 1949a).

Following procedures established for C. briggsae by Margaret Briggs in her 1946 Master thesis at Stanford University, Dougherty and Calhoun (1948b) also used an agar medium, seeded with a culture of E. coli. By treating isolated animals with antiseptic solutions, they achieved monoxenic cultures.

Dougherty and his collaborators devoted the greatest part of their activity to seeking a chemically defined culture medium. Their plan was to look for nutritional mutants, following the work of George Beadle and Edward Tatum with Neurospora (Dougherty and Calhoun, 1948b). A first step towards this goal was to succeed in employing an axenic liquid medium containing either a chick embryo or a liver extract (Dougherty, 1950; Dougherty et al., 1959), but the composition of these media still remained largely undetermined.

The next objective was thus to create a fully chemically defined medium. In their last publications on the subject (Dougherty et al., 1959; Nicholas et al., 1959), the authors described a fully defined medium (GS-25), which could sustain the development of larvae into adults, with, only once, the production of an F1 generation. The addition to this medium of 1% of a protein fraction of liver (LPF-C) allowed them to obtain up to eight successive generations. They concluded with the following sentence: “The axenic cultivation of C. elegans on a chemically defined medium still presents challenges. Undoubtedly, more workers are needed.” (Dougherty et al., 1959).

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Nigon succeeded in setting up strictly controlled culture conditions, using an agar medium and regular transfers of single animals, where the free-living nematodes could grow and mate (Nigon, 1943). Culture conditions and nematode transfer methods have not greatly changed since, but have tended to converge to one standard agar medium (called Nematode Growth Medium or NGM agar, Maintenance of C. elegans), now poured into plastic Petri dishes. Following Brenner (1974), C. elegans is fed monoxenically a growth-restricted Escherichia coli strain called OP50, which allows for better observation of the animals on the E. coli lawn (Maintenance of C. elegans). Strains can now be kept frozen, a major improvement in terms of strain maintenance and genotype stability.

The serious efforts called for by Dougherty et al. (1959) have been made: the nutritional requirements of C. elegans and C. briggsae are now understood and a chemically defined axenic culture medium has been devised. An excellent analysis of subsequent studies performed by Dougherty's and other teams is available in Nicholas (1975), including those by W.F. Hieb who had worked in Dougherty's laboratory (Hieb and Dougherty, 1966). Specifically, C. elegans was found to require some external sterol source (Hieb and Rothstein, 1968; now added as cholesterol in the agar medium, because E. coli does not provide it), as well as heme (Hieb et al., 1970; Rao et al., 2005). In modern standard culture conditions, the heme is provided by E. coli, but in axenic conditions, heme must be provided, together with a carrier protein (Buecher et al., 1970; Vanfleteren, 1974). A chemically defined medium could finally be devised, where the worms could proliferate, albeit more slowly (Lu and Goetsch, 1993; Szewczyk et al., 2003). This medium is rarely used, although it is famous for space shipping and C. elegans survival after the Columbia space shuttle disaster (Szewczyk et al., 2005). Nutritional requirements of C. elegans have been studied by Nancy Lu and her team in Berkeley and San Jose, California (Lu et al., 1977; Lu et al., 1983; Lu and Goetsch, 1993; Perelman and Lu, 2000; Balachandar and Lu, 2005; Zhao and Lu, 2008; Xiong and Lu, 2011). The genetic screens for nutritional mutants that Dougherty dreamt of are still to come, but other physiological studies are promising.

Earlier authors, such as Maupas, were able to observe live animals in the microscope. To visualize chromosomes, Eva Krüger was the first in 1913 (after Boveri on Ascaris) to embed animals in paraffin, section and stain them with hematoxylin. This technique did not allow an easy analysis of the succession of cellular structures during gametogenesis of a given individual. Nigon (Nigon, 1946; Nigon, 1949a) then established a method that overcame this difficulty. A microknife was built by breaking the edge of a razor blade with forceps. This small blade was inserted into the chuck of a watchmaker tool. An individual, male or female, was transported onto a dry preparation slide into a small waterdrop. A strike of the microknife skillfully aimed at the nematode's flanks made it expel its internal organs. A drop of Carnoy fixative was immediately added, ensuring at the same time the fixation of the animal and the coagulation of the expelled substances. After this surgery, the dissected animal was stuck on the glass surface, with its internal organs spread out. The preparation was subjected to the Feulgen reaction, which colored DNA bright red. After a brief drying step, a drop of mounting medium and a coverslip were added, with no need for sectioning. Except for the Feulgen reaction, which dates from the 1930s, this method was derived from a technique that had been in common use in protistology for a long time. It is thus somewhat surprising that protistologists such as Maupas and Bělař did not apply it to nematodes. Using this method, one can follow the successive steps of gametogenesis as they occur along the genital tract of a single individual (Nigon and Brun, 1955) (see Section 4.3). A few such preparations were sufficient to capture all steps of gametogenesis and fertilization. Phase contrast microscopy further helped visualization. As was already achieved by previous authors, one could follow and film the developmental process in different focal planes, on isolated eggs or on the live animal without prior dissection (Nigon et al., 1960).

Later, the same razor blade technique was applied to animals after incubation with radioactive (tritiated) thymidine for a few hours. The animals were dissected and covered with a sensitive film in the dark room. DNA synthesis could thus be followed during gametogenesis (Nonnenmacher-Godet and Dougherty, 1964).

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The razor blade technique described by Nigon is still being used for gonadal and intestinal staining, as well as for egg and early embryo isolation. Advances in microscopy have been crucial. Nomarski microscopy allowed the nucleus of each cell to be followed in a live animal and rendered possible the determination of the entire cell lineage by John Sulston et al., from egg to adult (Sulston and Horvitz, 1977; Sulston et al., 1983). 4D-microscopy movies (with different focal planes over time as in Nigon et al., 1960) are now achieved through automatic recording, which further facilitates the determination of cell lineages. The development of electron microscopy was also crucial, especially to determine the wiring of neurons (White et al., 1986). Fluorescent Hoechst staining or labeling of histones with Green Fluorescent Protein are now used to observe DNA in live animals. Specific staining of a given sequence of nucleic acids or of a protein is now possible by in situ hybridization (Single molecule fluorescent in situ hybridization (smFISH) of C. elegans worms and embryos), immunostaining (Immunohistochemistry), and/or transgenic fusions of endogenous proteins with fluorescent proteins (Reporter gene fusions).

Many free-living nematodes have separate sexes (gonochorism). The gonad of females produces oocytes, that of males spermatozoa. After mating, spermatozoa are stored in the spermatheca of the female gonad, usually located between the site of oocyte maturation and the uterus. When oocytes mature, they pass one after the other through the spermatheca where they are fertilized.

A key observation of Maupas (1900) was that in nematodes, the hermaphrodite individuals display a female body morphology. In their germ line, gametogenesis begins in early adulthood with the production of sperm, which is then stored in the spermatheca (Figure 2). Oogenesis then occurs in a chain of successive maturations along the gonadal axis, as in true females. This mode of reproduction is thus called protandric hermaphroditism. As in true females, the most mature oocyte passes through the spermatheca and becomes fertilized by self-sperm. In sum, the hermaphrodites are similar to the females as far as the soma is concerned and only differ in germ line behavior. One must thus distinguish in these species between sexual differentiation of the soma and that of the germ line (Maupas, 1900; Nigon, 1949a).

In most hermaphroditic species, male individuals are also produced at a frequency of around 0.1-1%. These males produce sperm of identical shape as those produced by hermaphrodites. According to Maupas, however, these males (here of C. elegans) appear to have little sexual instinct: “I actually doubt that any male ever occurs in nature. With animals so little lustful as the males of our Rhabditis, the very special conditions of sequestration in which I maintained them must be required to induce some of them to mate.”. They often show variability at the level of the male tail and spicules. Some intersex males that form oocytes in their testis have been observed (Maupas, 1900; Nigon, 1949a) (Figure 2E).

Maupas tested the ability of C. elegans to reproduce for many generations through self-fertilization without outcrossing. He cited Charles Darwin on the fact that outcrossing seemed favored in nature: “Nature thus tells us, in the most emphatic manner, that she abhors perpetual self-fertilisation” and “No hermaphrodite fertilises itself for a perpetuity of generations”. Testing Darwin's suppositions, Maupas transferred 20 virgin C. elegans hermaphrodites per generation, during 50 generations. He observed no degeneration. (His experiment was interrupted by summer, the high temperature likely causing sterility of the line.) He concluded that outcrossing is not necessary, at least in the short term.

Maupas’ article compared reproduction of several hermaphroditic nematode species (Maupas, 1900). Different species displayed at the level of the sexual structures and of the mode of reproduction a considerable variability of modalities, sometimes within the same species:

1) Maupas found Reiterina viguieri to be the closest to the reproduction of gonochoristic species, with about 5% males. These males were active and often found mating. Among females (the term included all somatically female individuals, including hermaphrodites), about one in ten was without sperm. This species thus included three sexual forms: hermaphrodite (most common), female and male. Maupas did not systematically study the progeny of different types of females, crossed or not with males.

2) In Rhabditis marionis, Maupas observed a lower frequency of males, 0.76%. Many hermaphrodites of this species laid fertilized and unfertilized eggs at the same time. Maupas hypothesized that the presence of unfertilized eggs (also observed in Rhabditis duthiersi) may be due to the fact that spermatogenesis only occurred in one of the two ovaries. This remains unclear as Maupas did not directly assess the absence of sperm with his microscope and this has not been studied since.

3) In Rhabditis guignardi, Maupas observed the smallest proportion of males (0.015%). Hermaphrodites produced many sperm and on average 520 progeny per individual–thus, much more than other hermaphroditic species (usually 300 or less).

After Maupas, F. A. Potts (Potts, 1910), based in England, studied new hermaphroditic species, which he described succinctly (Pristionchus linstowi, Pristionchus maupasi, and Rhabditis gurneyi). By culturing P. maupasi unmated hermaphrodites on different peptone solutions in different instances and locations, Potts noticed in their progeny large variations in male frequencies, ranging between a very low value and 30%. He did not control several factors, such as temperature, which may have caused variations in observed male frequencies. Potts reported that R. gurneyi females first produced unfertilized oocytes, then fertilized ones. He interpreted these results to mean that some individuals displayed several successive phases of spermatogenesis. Like Maupas’ hypothesis above, Potts’ hypothesis on R. gurneyi was never verified by a direct observation of spermatogenesis, and the laying of unfertilized eggs may be explained by factors other than the lack of sperm. His results, thus, remain unclear and have not been reproduced.

At the time of Maupas’ work, a chromosomal basis for sex determination was not known. Maupas (1900) attempted matings between the hermaphrodites and the rare males that they produced via self-fertilization. To avoid interference by hermaphrodite sperm, Maupas used old females, presumed to have exhausted their own sperm, as inferred by the fact that they had ceased to lay developing embryos for at least 24 hours. Maupas mostly used C. elegans for his experiments. He set up eight cultures by mixing several hermaphrodites and several males. Two did not yield any progeny, while the six others produced 127 males and 147 hermaphrodites, thus approximately an equal number. Some resulting males were intersex, with oocytes in the testis (as shown in Figure 2E). When isolated, the F1 hermaphrodites gave rise mostly to hermaphrodites, with only 0.23% males. The F1 males were associated with other old hermaphrodites in five cultures (each with 4-11 males). In this second generation, no progeny were obtained. Seven other hermaphroditic species tested by Maupas in the same manner were infertile in the first generation of mating pairs. In R. marionis and R. duthiersi, the old hermaphrodites placed with males yielded only further hermaphrodites. In summary, Maupas succeeded in mating C. elegans for one generation, which indicated that outcrossing with males may increase male production. But his results could not be repeated at the next generation. He concluded that the F1 males were degenerate and sterile. However, as explained below, his culture conditions may not have been optimal for mating.

Hikokuro Honda tried to reproduce Maupas’ experiments (Honda, 1925). He isolated C. elegans (likely this time in North America where he worked). He noticed that his C. elegans strain, even after having stopped producing fertile eggs for 24 hours, may still sometimes produce progeny–thus possibly explaining why Maupas’ mating experiments with old R. marionis and R. duthiersi hermaphrodites did produce progeny but only hermaphrodites. In C. elegans, Honda did not obtain any males in his mating attempts. However, with O. dolichura he obtained males in the first generation. He correctly observed six bivalents in the oocytes and spermatids of C. elegans.

Nigon (Nigon, 943; Nigon, 1946; Nigon, 1949a) was the one who succeeded in carrying out crosses reproducibly in species with hermaphrodites and rare males–C. elegans and O. dolichura. Nigon isolated C. elegans from garden soil in Bergerac, France (see Appendix 2, Section 17 and Appendix 3, Section 18.4). In contrast to his predecessors, he maintained his cultures by isolating animals individually at each generation, on a drop of agar on a slide (Section 2.1, Figure 1). When obtaining a rare male from an isolated hermaphrodite, he placed it with one of its sisters. In some cases, the offspring was exclusively hermaphroditic–the males may have been sterile. In other cases, the progeny contained approximately the same number of males and hermaphrodites. Most males of the second generation were fertile (93%) and again gave rise to both hermaphroditic and male progeny. From the third generation, almost all males were fertile. Males could thus be propagated through crosses at will.

In summary, Maupas used group cultures of several (5-20) males with as many aged hermaphrodites. His spontaneous males obtained from hermaphrodite self-fertilization were often fertile, while all males of the second generation seemed to be sterile. By contrast, in Nigon's experiments, young males were associated with young hermaphrodites. The spontaneous males were often sterile, but from the third generation, almost all were fertile.

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Why were the mating experiments successful in the hands of Nigon and not of his predecessors? Several factors may have contributed. First and perhaps most importantly, the culture and mating conditions that Nigon set up were highly controlled and allowed reproducible experiments, including crosses with single hermaphrodites and males, on an agar medium incubated in a humid chamber (Section 2.1). In contrast, Maupas was culturing large populations in depression slides, in water to which rotten meat was added. Honda also used a liquid medium, in which C. elegans males could perhaps not mate. Second, Maupas used aged hermaphrodites, which may have been too old to reproduce. The first point is likely the main factor. The control of mating of males with hermaphodites is the basis for modern C. elegans genetics (Dougherty, 1963; Brenner, 1974).

The odd three-sex situation described by Maupas in Reiterina viguieri has been found again in ”Rhabditis“ sp. SB347, related to Reiterina viguieri (see Section 8.2).

The use of crosses between males and hermaphrodites allowed the testing of cross-fertility between isolates from nature. The lack of males in the progeny of a cross between males of strain A and hermaphrodites of strain B (or reciprocally) indicated that the two strains were not cross-fertile. An absence of cross-fertility indicated the presence of two different species, provided within-strain controls were positive. Maupas had attempted such hybridization tests between morphologically diverse species, without success. Some matings between morphologically diverse species occurred with the deposition of a copulatory plug, while a few yielded arrested embryos or gonadless adults (Maupas, 1919). Conversely, using a strain that was morphologically difficult to distinguish from the Bergerac isolate, Dougherty and Nigon discovered through crosses a new species, which they named C. briggsae.

The new strain had been collected in 1944 by Margaret Briggs, then a student at Stanford University, using a peanut butter enrichment procedure to initiate the culture (Dougherty and Nigon, 1949; Nigon and Dougherty, 1949; Briggs Gochnauer and McCoy, 1954; procedure described in Dougherty and Calhoun, 1948b). This culture was passed to E. Dougherty, who first called it ”R. elegans“ because of its close morphological similarity (Dougherty and Calhoun, 1948b). Briggs continued to work on C. briggsae culture conditions, including its response to antibiotics (Briggs Gochnauer and McCoy, 1954). This strain is still available as DR1690 (Fodor et al., 1983; McGrath et al., 2011).

By crossing the Berkeley strain to the C. elegans Bergerac strain, Nigon and Dougherty found no males in the progeny. In addition, using a micro (dumpy) mutant (see Section 9.2) in the heterozygous state in males, they did not see the mutation segregate in the grandprogeny of hermaphrodites (Dougherty and Nigon, 1949; Nigon and Dougherty, 1949). Some phenotypic features of the two cultures were noticed: the Berkeley strain displayed a lighter color of intestinal granules, a smaller number of embryos in the uterus, fusion of sensory male rays 3-4, and a slower growth at 12 °C. Comparing morphological measurements with those of Maupas for C. elegans, they concluded that the California strain was the new species and they named it Rhabditis briggsae (Dougherty and Nigon, 1949), now Caenorhabditis briggsae. They concluded that there may be many cryptic species that cannot be distinguished morphologically in the genus Rhabditis.

The same procedure was used by Nigon to assign a new Caenorhabditis isolate from Bristol to C. elegans. The Bristol strain was isolated in 1951 from mushroom compost by Warwick Nicholas during a nematology course organized by L. N. Staniland in Bristol (Nicholas, 1975; Hodgkin and Doniach, 1997; McGrath et al., 2011). This strain passed through the hands of nematologists such as Osche (Nicholas et al., 1959) and B. G. Chitwood (Hansen et al., 1960), and was definitively identified as C. elegans by Nigon, through positive hybridization to the Bergerac strain (Nicholas et al., 1959).

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As many more morphologically similar species of Caenorhabditis have been found, the biological species concept for species definition has been further implemented in the genus by Sudhaus and Kiontke (2007) and Félix et al. (2014). A gonochoristic Caenorhabditis species that is morphologically similar to C. elegans and C. briggsae was described in 1974 as C. remanei (Sudhaus, 1974). Dozens of new gonochoristic species have been found since, as well as a third hermaphroditic species, C. tropicalis (Kiontke et al., 2011; Félix et al., 2014). The finding of closely related species with partial hybridization properties has already allowed studies of genetic incompatibility and speciation in Caenorhabditis to start (Woodruff et al., 2010).

Hermaphroditism in nematodes appeared to be independently derived several times from the gonochoristic mode via evolutionary modification of the female germline (Maupas, 1900). This suggested that the evolutionary transition from one mode to another was easy.

This idea was reinforced in later experiments by Hedwig Hirschmann on the gonochoristic Pristionchus lheritieri (then Diplogaster lheritieri) (Hirschmann, 1951). Hirschmann established parallel lines of P. lheritieri through inbreeding (brother x sister) of a strain she had isolated from nature; these lines rapidly decreased in fertility, a case of inbreeding depression. In the F4-F5 generations, she observed some self-fertile animals as well as other types of intersex individuals. With the self-fertile animals, she started a line that reproduced through self-fertile, protandrous hermaphrodites and rare males. Four replicates of inbreeding confirmed this transition in reproductive mode.

She described these selfing strains under a new species name, “Diplogaster biformis”. She also repeatedly isolated from nature selfing strains with a similar morphology. She tested a selfing strain for reproductive isolation with P. lheritieri, and found that they did not produce cross-progeny. But it is unclear whether she used the selfing laboratory descendants of P. lheritieri or the morphologically similar selfing species that she had found in nature (in fact likely P. maupasi, Osche, 1954; Triantaphyllou and Hirschmann, 1964). Thus, it is unclear whether the selfing species obtained by inbreeding was reproductively isolated from its gonochoristic parent.

Yet, her inbreeding experiment suggests that the evolutionary transition from obligate outcrossing to selfing may be obtained in the laboratory, at least in some cases, when starting with natural populations of gonochoristic species.

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These findings have not been reproduced so far, but we now know that in the gonochoristic C. remanei, hermaphroditism can be obtained through targeted mutational change in just two independent pathways (Baldi et al., 2009).

Cultures of gonochoristic free-living nematodes generally contain the two sexes in approximately equal proportion based on an XX/XY or XX/X0 chromosomal sex determination. The cytological study of chromosomes of free-living species by Nigon showed either no difference in chromosome number between males and females (Panagrolaimus rigidus, Nigon, 1949a), suggesting a XX/XY mechanism, or one less chromosome in males (Pellioditis pellio, Hertwig, 1920; Pelodera strongyloides, Nigon, 1949a). During P. strongyloides spermatogenesis, all primary spermatocytes were observed to bear a single X and “reduction” of the X chromosome occurred at the second meiotic division (Nigon, 1949a). XX/X0 sex determination had been found previously in some parasitic nematode species, such as Angiostomum nigrovenosa (now called Rhabdias bufonis; Boveri, 1911; Schleip, 1911). Note that some male-female plant parasitic nematodes display environmental sex determination (Triantaphyllou and Hirschmann, 1964).

In hermaphroditic species as in gonochoristic species, mating of males with hermaphrodites gave rise to males and hermaphrodites in similar proportions (Nigon, 1949a). Cytology, which required destroying the animals, was best performed on young males before they mated. Males could, however, be examined in the following generations and were shown to carry one less chromosome than hermaphrodites (Figure 3). These results indicated that sex determination in hermaphroditic species had a chromosomal basis, with an XX/X0 system, as in the related gonochoristic species. This XX/X0 system was observed for both O. dolichura (Nigon, 1946) and C. elegans (Nigon, 1949a).

What about the chromosomal content of the rare males coming from self-fertilization of hermaphrodites? Nigon (1949a) found it impossible to both examine chromosome number in a male and test its fertility. Fertile males were likely X0 because when crossed to hermaphrodites they gave rise to progeny of which half carried 11 chromosomes (X0) and the other half 12 chromosomes (XX). What about the sterile males that were produced by selfing hermaphrodites, and to some extent by hermaphrodites, crossed to a fertile male? Several experiments attempted to answer this question, including trying to visualize spermatogenesis in the hermaphrodites and to obtain more males in the progeny of selfed females. Both are detailed below.

A special behavior of one chromosome pair, likely the sex chromosomes, was observed during hermaphrodite spermatogenesis in O. dolichura (Nigon, 1946; Nigon, 1949a). In some spermatocytes, a standard symmetrical separation of all bivalents (homologous chromosomes) took place at anaphase, while in some others, separation of one bivalent was delayed. This delay could possibly result in this bivalent entirely segregating into one daughter cell, or being lost in the residual body discarded during sperm maturation. Assuming that the delayed bivalent corresponded to the X chromosome, one could thus obtain nullo-X spermatozoa. Their fusion to a normal oocyte would give an egg with a single X, producing a male. To be sure of the identity of this delayed chromosome, a marker on this chromosome would have been useful. A similar delay of one chromosome was also observed in C. elegans at anaphase of meiosis I, but to a lesser extent (Figure 3C).

Several methods were employed to induce a hermaphrodite to produce a higher incidence of males: submitting the hermaphrodites to a heat shock (for example 25°C for several hours) or treating them with a colchicine solution (Nigon, 1949b). When these treatments were applied during the spermatogenesis of hermaphrodites, they led to various chromosomal aberrations that could be observed cytologically. The animals thus treated were often sterile, or had diminished fertility. The progeny included many irregular cases (Nigon, 1949a), including males, some of which were intersexual, a form already described by Maupas (1900), or true female individuals with no trace of spermatogenesis. It may be hypothesized that these abnormalities resulted from abnormal chromosomal contents. The absence of morphological differences or of gene markers among chromosomes was here again the obstacle to further studies.

Nigon and Brun studied chromosomal behavior in meiosis, using Feulgen staining (Section 2.2) and live material. Early meiotic stages could be conveniently studied in adult females and hermaphrodites, as successive stages are aligned along the ovary in a single animal (Nigon, 1949a; Nigon and Brun, 1955) (Figure 4). Nigon and Brun observed that at the tip of the gonad, oogonia underwent mitotic divisions. The oocyte nuclei then entered meiotic prophase I and transited from synapsis to diakinesis along the arm of the gonad (Nigon and Brun, 1955). Fertilization then triggered the two meiotic divisions and polar body formation.

Chromosome behavior during pairing and recombination was studied during oogenesis in C. elegans (Figure 4) (Nigon and Brun, 1955). Chromosomes were observed to synapse (pair) at the onset of meiotic prophase. One of them often appeared hypercondensed (the X chromosome). After pachytene, the chromosomes entered a dispersed stage (now called the diffuse stage), most easily seen in older adults. During diakinesis, the recombined homologs started separating, usually adopting a V-shape. The chromatids then partially separated (tetrads). Further condensation resulted in two bivalents, which then migrated to opposite poles of the first meiotic division. This scheme was somewhat different from that proposed for species with a distinct rather than diffuse centromere (Darlington, 1937).

The study of spermatogenesis in X0 males was performed by Nigon (1949a), mostly using O. dolichura. As in C. elegans, the diploid number of chromosomes was 12 in hermaphrodites, 11 in males. In the O. dolichura male, the first meiotic division was observed to be most often reductional for autosomes, but equational for the (haploid) X chromosome, with each chromatid migrating to a different daughter cell at meiosis I (as in Pelodera strongyloides, Section 4.1) (Figure 3C). Yet the anaphase migration of the X chromosomes was delayed compared to that of the autosomes–so much so that in some cases, both chromatids were found in the same spermatocyte after the first division, in which case the equational segregation occurred at the second division (Nigon, 1949a).

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Studies of meiosis in C. elegans have largely developed in the last ten years (Rog and Dernburg, 2013; Lui and Colaiacovo, 2013). The dynamics of chromosome synapsis can be followed live (Rog and Dernburg, 2015). A single crossing-over occurs for each pair of homologous chromosomes in C. elegans meiosis (Meneely et al., 2002), usually on a chromosome arm rather than in the center of the chromosome; the site of the unique crossing-over defines a long and a short chromosomal arm. Specific protein complexes associate and regulate double-stranded breaks, crossovers, short and long arms of chromosomes, and apposed homologs versus chromatids (Rog and Dernburg, 2013; Lui and Colaiacovo, 2013). The V shape (Y shape in Nabeshima et al., 2005) is transient in early diplotene, when the long arms but not the short arms have separated. When the short arm chromatids separate, it becomes a tetrad (Chi or X shape; Nabeshima et al., 2005). Further condensation, especially of the short arm, results in the Phi shape described above, and finally a bivalent, where the long arms are located outside and will attach to the spindle microtubules. Chromosomes in C. elegans are holocentric, meaning that centromeres are diffuse and the microtubules contact a large part of the chromosome (Albertson et al., 1997; Albertson and Thomson, 1993; Schvarzstein et al., 2010; Melters et al., 2012).

In true parthenogenesis, no sperm is involved. Among the species studied by Maupas (1900), this mode of reproduction was found in Rhabditophanes schneideri, Plectus cirratus, and Aphelenchus agricola, as well as four new species he described: Cephalobus dubius, Cephalobus lentus, Alaimus thamugadi, and Macrolaimus crucis (Maupas, 1900). Maupas noted the absence of sperm in the ovary and the absence of males, which made it likely that these species were true parthenogens. He did not further study the cytology of meiosis.

In the animal kingdom, two subtypes of true parthenogenesis have been described, depending on the presence of chromosome pairing. In both cases, the oocyte nucleus undergoes a single maturation division and generally emits a single polar body. In the first subtype, the chromosomes are paired during oogenesis, and recombination can occur between them. In the second subtype, pairing does not occur, and the single maturation division is equational, but apparently without recombination. Both meiotic and mitotic parthenogeneses have been described in plant parasitic nematodes, such as Meloidogyne spp. (Triantaphyllou and Hirschmann, 1964). Karl Bělař (see Section 18.3) described the cytology of meiosis in free-living nematodes with true parthenogenesis, using a single, undescribed, species, Rhabditis XIX (Figure 7). This species displayed diakinesis and pairing, followed by separation of sister chromatids after anaphase. Here, in contrast to usual female meiosis, focused centrosomes remained during meiosis (thus alleviating the need for a spermatozoon to provide them) (Bělař, 1923; Bělař, 1924).

The second major mode of parthenogenesis requires a spermatozoon to activate the oocyte. The sperm chromosomal material then degenerates without contributing to the genome of the progeny. This type of parthenogenesis, known as merospermy or pseudogamy, can be further divided into three subtypes.

Nigon et al. (1960) observed the early stages of development on living embryos in several free-living species: C. elegans, M. belari, R. anomala, and R. maupasi. Using a camera mounted on the microscope, the behavior of the egg was recorded in time lapse. The first events following sperm entry had to be studied on eggs that were still in the female uterus, because at this stage the egg shell is under formation and does not sufficiently protect the embryo. Observations become easier later in development when the eggs can be isolated. The use of an incubator case for the microscope made it possible to vary the temperature (see Figure 22). Although embryo development was analyzed until the four-cell stage, we will here mostly report on events from sperm entry until first cleavage.

In free-living nematodes, the anatomy of the ovary and uterus made it possible to distinguish in the elongated shape of the mature oocyte a proximal or external pole, towards the vulva, and a distal or internal pole, towards the distal germ line. [These poles were called anterior and posterior, respectively, in the article by Nigon et al. (1960). Because the modern reader may be used to the reverse nomenclature, we use here proximal and distal in reference to the orientation in the gonad and restrict anterior and posterior to their current use, which corresponds to the anterio-posterior axis in embryonic development (animal-vegetal).] A key question concerned the appearance of cells of different developmental fates and the control of embryonic cleavage patterns during the early embryonic divisions. Each species was seen to elaborate specific variants on a background of mostly common events. In gonochoristic and hermaphroditic species, sperm entry occurred most often at the proximal pole (the future posterior embryonic pole). We will first describe the case of C. elegans in some detail, then examine the observed particularities of other species more briefly.

The early cell lineage and the adult cell content of specific organs were shown to be invariant in a given species, first when mostly parasitic nematode species were studied (Martini, 1923), and later on the free-living species Anguillula aceti (now Turbatrix aceti, family Panagrolaimidae; Pai, 1928). After a first phase of fast cleavage divisions during embryonic development, cell divisions stop. Then comes a phase when cell types differentiate and organs are formed, without production of new cells. Individuals of the same species have an invariant number of cells in a given organ; this stability in cell number has been called eutely (Martini, 1923).

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That the entire somatic cell lineage is mostly invariant has been unequivocally demonstrated by following the entire cell lineage from zygote to adult (Sulston and Horvitz, 1977; Kimble and Hirsh, 1979; Sulston et al., 1983). Development is mostly reproducible in terms of cell division patterns and cell fates. The reproducibility of the cell lineage suggested to Brenner, Sulston, et al. that each cell computed its fate according to its position in the lineage rather than its spatial position in the embryo (Brenner, 2001). Yet this was soon found to be only partially the case since, by the 4-cell stage, cell fates turned out to be specified via signaling between cells. Cell fate specification is now known to occur through a combination of: 1) asymmetric divisions, as that initiated by sperm entry; and 2) intercellular signaling between cells. Reproducible patterns of cell contacts render the cell lineage basically invariant (Schnabel and Priess, 1997; Schnabel, 1997). The mechanisms behind the reproducibility of cell fates are thus relatively well understood, but the regulation of cell cycles in the different sublineages less so.

Although quite invariant in a species, the early cell divisions, the cell lineage, and the partitioning of cell fates (e.g., gut) varies among species (Sternberg and Horvitz, 1981; Sternberg and Horvitz 1982; Sulston et al., 1983; Embryological variation during nematode development; Houthoofd et al., 2008; Schulze and Schierenberg, 2011; Schiffer et al., 2014; Lin et al., 2009).

The first important studies dealing with the chromosomal determination of sex and the alternation of generations were conducted by Boveri (1911) and Schleip (1911) on Rhabditis nigrovenosa (now called Rhabdias bufonis), a nematode species that parasitizes amphibians. The parasitic generation of this species is composed of protandrous hermaphrodites, who give birth to a free-living gonochoristic generation, which in turn produces eggs that develop into infective larvae. Thus, with the alternation of parasitic and free-living forms, the modes of reproduction also alternate, from hermaphroditic to gonochoristic. Given an apparent chromosomal sex determination, two problems arise: how can a hermaphrodite give rise to both females and males, and conversely how can females and males give rise to hermaphroditic “females” only?

The parasitic hermaphrodites of Rhabdias bufonis display a diploid number of 12 chromosomes in their mitotic germ line. After two normal maturation divisions, the oocyte contains a standard haploid content of 6 chromosomes. Spermatogenesis in hermaphrodites is more complex, producing spermatozoa with 6 or 5 chromosomes. How can a hermaphrodite with 12 chromosomes give rise to these two kinds of sperm? According to Boveri, spermatogenesis may entail the loss of one chromosome (as in O. dolichura, see Section 3 and Section 4.2). In contrast, according to Schleip, the maturation of spermatocytes proceeds normally, giving rise to spermatids with 6 chromosomes, and it is only during the transformation of spermatids into spermatozoa that one chromosome would be degraded in some spermatids, producing spermatozoa with 5 chromosomes. In any case, fusion of these gametes gives rise to the free-living generation: those with 12 chromosomes are females, while those with 11 are males.

Concerning the second question, the authors stated that in the free-living generation, gamete formation occurred normally, with two maturation divisions. They thus gave rise to oocytes with 6 chromosomes in the females and sperm with 5 or 6 chromosomes in the male. The development of hermaphroditic “females” only in the next generation (parasitic) would derive from the fact that spermatozoa bearing 5 chromosomes were inactive. This last conclusion was hypothetical.

Studies on species with alternating generations continued, but few of them dealt with the determination of the succession of generations and their reproductive mode. Nigon and Roman (1952) collaborated on a first work in this field on Strongyloides ratti, a rat parasite. Roman maintained one line of Strongyloides ratti for several years in the laboratory, while Nigon performed cytological investigations. In S. ratti, the parasitic generation is parthenogenetic, while the free-living generation is gonochoristic (Figure 15).

In parasitic females, the mitotic zone of the ovary shows a diploid stock of 6 chromosomes, which do not pair in early meiosis. The oocytes develop in a parthenogenetic mode without fertilization, with a single maturation division. Parasitic parthenogenetic females give birth to rhabditiform larvae that escape through the host feces (stercoral generation).

Nigon and Roman (1952) found that the development of the next generation depended on external factors:

1). If the larvae were cultured on an agar medium, only males reached the adult stage. They represented approximately 0.5% of the population. The other larvae developed into filariform infective larvae, which might infect rats when applied to their skin. Some of them would reach the intestine and further develop into parasitic females.

2). If the larvae were raised in coproculture (on feces), the production of males was not modified, but adult females were also formed in a number similar to that of males. This free-living generation was composed of two sexes. The remainder (i.e., 99% of the progeny of the parasite) developed into infective larvae.

In the free-living generation, females displayed a diploid stock of 6 chromosomes, and the males only had 5. Non-disjunction of the sex chromosome in the parasitic female parent was hypothesized but not actually observed, which was not surprising given that non-disjunction should only take place in 0.5% of the meioses.

In the oocytes of free-living females, Nigon and Roman (1952) observed 3 bivalents at diakinesis. The oocytes were fertilized by sperm from the free-living males, and a fertilization membrane was formed. The oocyte nucleus migrated to the plasma membrane, apparently preparing for its first reductional division, with homolog pairing. But at metaphase, the bivalents separated and formed an equatorial plate with 6 chromosomes. The unique maturation division splitted the 6 chromosomes, and a polar body was extruded. Thus the maturation division started like a reductional division but ended in an equational division. During this time, the sperm was condensed into a small DNA mass that could later be found in one cell of the cleaved embryo. This behavior thus corresponded to a pseudogamous parthenogenetic reproduction, as described above for M. belari (Section 5.2.3). The absence of true fertilization would explain why only females were formed in the next infective generation.

Some observations of Nigon and Roman suggested the presence of rare alternative behaviors. In one egg, they described the presence of two pronuclei, each containing 3 chromosomes; no condensed sperm was found in this egg. This configuration thus corresponded to a normal fertilization, with two haploid pronuclei. In the same animal, another egg displayed the most usual pseudogamous behavior. Thus, as in M. belari, in the same individual two modes of meiosis and fertilization could co-exist. Some eggs also appeared to contain polyploid nuclei, with up to 24 chromosomes. In these eggs, the sperm remains were identifiable, but no polar bodies were seen, suggesting the occurrence of abnormal maturation divisions. Thus, next to the known parthenogenetic development, variant behaviors could be observed in free-living females, with unclear consequences.

In free-living males, spermatogenesis occurred with many irregularities. A detailed study was not undertaken. Observations of the development of the eggs showed that in most cases, the male genome did not participate to reproduction. However, in some cases, at an unknown frequency, amphimixis could be observed, which sugested the existence of a spermatozoon with 3 chromosomes.

In conclusion, the free-living gonochoristic generation from the strain under study in uniform culture conditions reproduced mostly via pseudogamous parthenogenetic reproduction, more rarely through amphimictic reproduction, as in M. belari. The development of amphimictic eggs has not been studied.

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More recently, alternative lifecycles of Rhabdias bufonis have been studied by Spieler and Schierenberg (1995). They showed that while at hatching, larvae of both free-living and parasitic generations display a similar number of nuclei, the parasitic adult (of larger size) contains many more nuclei than the free-living generation. This is in part due to the presence of over 2000 nuclei in the intestine, versus about 20 in the free-living generation as in C. elegans.

Concerning Strongyloides ratti, excellent recent reviews are those of Streit (2008) and Viney and Lok (The biology of Strongyloides spp). Viney studied many strains of S. ratti, with a well-controlled culture technique, and found that coproculture gave rise to a high proportion of free-living females. Development in free-living adults versus infective larvae was governed by many factors including the strain, the culture conditions and the immunological status of the host rat (Viney et al., 1992; Viney, 1996; Harvey et al., 2000).

The reproduction mode of the free-living generation of S. ratti remains controversial and it is possible that the character varies among closely related species (Streit, 2008). Through studies of the segregation of molecular genetic markers, Viney et al. confirmed that the parasitic generation of S. ratti reproduced parthenogenetically (Viney, 1994) and that males carried a single copy of one chromosome, consistent with XX/X0 sex determination (Harvey and Viney, 2001). However, in contrast to the cytological studies by Nigon and Roman (1952), they found, using genetic markers, that the free-living adults reproduced through true fertilization (amphimixis) (Viney et al., 1993; Nemetschke et al., 2010b). The discrepancy between the cytological studies and the later genetic studies remains unexplained (Streit, 2008). The occurrence of pseudogamy versus amphimixis, or their relative frequency if they coexist, may depend on external factors as well as on the genetic composition of different S. ratti lines (or cryptic Strongyloides species). Studying the cytology of meiosis and fertilization on lines where amphimixis seems prevalent appears useful at this point. Ongoing research indicates an exciting future for studies on this species and closely related species (Nemetschke et al., 2010a).

A new convenient model system to study the alternation of generations with different modes of reproduction is now available, without an obligate parasitic stage. The species provisionally called Rhabditis sp. SB347 develops on agar plates seeded with E. coli, either through a generation that adopts the dauer stage and develops into hermaphroditic adults, or through a gonochoristic generation that does not pass through the dauer stage (Félix, 2004). The ecology of the species is unknown but it is possible that the dauer stage corresponds to passage in a host. Morph and sex determination in this species are being studied in the laboratory of Andre Pires daSilva (Chaudhuri et al., 2011). The paucity of males in the progeny of the gonochoristic generation has been explained by the asymmetry of spermatocyte division and the selection of the X-bearing sperm spermatocyte, vindicating the hypothesis of Boveri and Schleip for R. bufonis (Shakes et al., 2011).

Dougherty and Hermione G. Calhoun were the first to suggest the potential relevance of species of the rhabditids for studies in formal genetics (Dougherty and Calhoun, 1948a). Dougherty foresaw the advantages that the reproductive modes (Dougherty, 1949) and the developmental speed could bring to genetic studies. The selfing mode of reproduction appeared particularly convenient to produce homozygous mutant animals. Dougherty hoped that the possibility of culture on defined media could lead to studies in physiological genetics inspired by those of Beadle and Tatum on Neurospora.

While the first genetic variant in C. elegans was the tetraploid line (Nigon, 1949b; Nigon, 1951a) (see Section 4.4), the first description and analysis of a recessive mutation was in C. briggsae (Nigon and Dougherty, 1950). This mutation appeared in a culture that had been exposed to a temperature higher than 23°C. In the adult stage, this mutant was approximately a quarter shorter than the wild-type, hence its designation as micro. Its width was identical to that of the wild type, giving it a stocky appearance (Figure 16). Its fecundity and motility were reduced.

Crosses of micro hermaphrodites with wild-type males gave rise to heterozygote progeny of wild-type phenotype, both males and hermaphrodites. When the F1 hermaphrodites were selfed, the F2 generation showed a segregation of wild-type and mutant phenotypes. The F3 progeny from F2 individuals of wild-type phenotype were further followed (Figure 16C). The values for the F2 genotypes were close to the 1:2:1 ratio expected for the F2 progeny of a F1 hybrid between wild-type and homozygous recessive mutant parents. In addition, F1 males backcrossed to micro hermaphrodites yielded hermaphrodites and males, both of wild-type and mutant phenotypes (Figure 16D). The F1 males were thus heterozygous, showing that the mutation is not sex-linked. It was concluded that the micro character depended on an autosomal recessive mutation.

Several attempts were made by Dougherty and Nigon to study the effect of chemical mutagens. The first experiments, performed in Paris in 1949, made use of yperite and acriflavine (Nigon and Dougherty, 1953; Dougherty and Nigon, 1953). C. elegans did not provide a variety of obvious mutant phenotypes. Treatment with yperite resulted in a C. elegans strain with variable phenotypes, named “pygmy", which differed from the micro mutants (Nigon and Dougherty, 1953). These animals grew with a variable rate, ranging from normal speed to very slow growth. For those reaching the adult stage, their shape and size were close to those of normal individuals of this species, and some died before reaching the adult stage. This form was maintained for hundreds of generations by selfing, without modification of the phenotypic variability. Nigon pursued studies on the variable expression of the phenotype, trying to select phenotypic extremes in the pure line (Nigon, 1954).

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The spontaneous micro mutant in C. briggsae corresponds to a common class of mutants found in C. elegans and other rhabditids, now called dumpy (abbreviated dpy) following Brenner (1974). The slow-growing pygmy mutant with variable expression (Nigon and Dougherty, 1953) was likely what is now called gro (abnormal GROwth rate) or clk (CLocK, biological timing abnormality) mutants, which display reduced growth rates. Nigon was interested by the variability of its phenotype, which may have reflected the variable expressivity of such mutants.

Together with the tetraploids, these mutants marked the beginning of C. elegans and C. briggsae genetics. Reasons why Nigon and Dougherty did not carry on with mutageneses are discussed in Section 12.1.1.

The sensitivity of C. elegans (and other species) to high temperature was noted by Maupas (1900). In Bergerac, Nigon collected C. elegans twice (Appendix 2, Section 17). The first strain, sampled in 1941, could multiply well at 25 °C. The second strain (known today as Bergerac) isolated in March 1944, displayed abnormalities already at the temperatures of 23-24 °C (Nigon, 1949a). Only this second strain was studied by morphologists (Dougherty, Osche, etc.) and confirmed by them as C. elegans. It is possible that the first isolate might have been another species, such as C. briggsae, or a C. elegans with normal resistance to temperatures of 23-25 °C.

The C. elegans strain from Bristol is indeed still fertile at 25°C. The transmission of the genetic difference in heat tolerance between the 1944 Bergerac and the Bristol strains of C. elegans was studied by Helene Fatt and E. Dougherty (Fatt and Dougherty, 1963). At that time, crosses between the Bergerac and Bristol strains could not be made in the axenic liquid media used in the Dougherty lab, so they used agar plates seeded with E. coli for the crosses and, after antibiotic treatment, brought them back to axenic liquid culture. The heat tolerance of Bristol was found to be dominant and to segregate in an approximate 3:1 ratio. Analysis of individual heat-tolerant F2s yielded a ratio of 1 homozygous to 1.5 heterozygous, not too distant from the expected 1:2. Following a first cross of Bergerac hermaphrodites with Bristol males and a backcross of F1 males to Bergerac, some F2 progeny were heat-tolerant–thus the transmission was not X-linked but autosomal. In conclusion, heat sensitivity of Bergerac vs. Bristol was transmitted as a recessive autosomal trait. This is the first example of genetic analysis of a natural variation in C. elegans.

This genetic study was followed by an attempt by Fatt to characterize factors of the culture medium that participated in inhibiting progeny production of the Bergerac strain at high temperature. These environmental manipulations were performed by changing the composition of the axenic medium. Fatt found that at high temperature Bergerac individuals produced an inhibitory substance, whereas Bristol did not (Fatt, 1967).

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The major autosomal locus responsible for the difference in heat tolerance between the Bergerac and Bristol strains of C. elegans has been mapped to chromosome II and the locus called zyg-12 by Wood et al. (1980); it was mapped again by Shook et al. (1996). The gene and molecular lesion were identified by Malone et al. (2003). The Bergerac allele is a thermosensitive reduction-of-function missense mutation (ct350, an A to C change) in the zyg-12 gene. This gene encodes a transmembrane protein of the conserved Hook family that links the centrosome to the nucleus (Sato et al., 2009), and through the nuclear envelope also participates in chromosome pairing in early meiosis as well as in the overall architecture of this part of the gonad (Penkner et al., 2007; Penkner et al., 2009; Zhou et al., 2009). The temperature-sensitivity of the Bergerac allele appears to originate in the binding inability of the Bergerac ZYG-12 protein at high temperature (Malone et al., 2003).

The presence of this temperature-sensitive zyg-12(ct350) allele in the C. elegans Bergerac explains several temperature-dependent phenotypes that were observed by Nigon and colleagues, such as unpaired chromosomes in early meiosis and polyploid nuclei in the gonad (Nigon, 1951b; Brun, 1955), centrosomes detaching from the male pronucleus after fertilization, and the two pronuclei staying apart resulting in embryonic cleavage defects (Nigon et al., 1960). Note that independent of the zyg-12 mutation, chromosome pairing in meiosis is also temperature-sensitive in the Bristol/N2 background, which may explain the increased male production at high temperature (Bilgir et al., 2013). Thus, although the specific zyg-12 mutation makes some cellular reactions particularly sensitive to high temperature for Bergerac animals, other processes are similarly temperature-sensitive in the different wild strains of C. elegans.

Jean-Louis Brun was a student of Nigon in Lyon (see Appendix 3, Section 18.6). Brun started classical genetic experiments with C. elegans, after performing the high temperature experiments described in Section 10, at a time when Nigon himself had left the worm and gave Brun the chance to conduct his own research. With Michel Dion, Brun first isolated and analyzed two spontaneous dwarf (dumpy) mutations in the C. elegans Bergerac background (Dion and Brun, 1971). These mutations were found to be recessive, autosomal and unlinked. One of them is available at the CGC as FF1, of genotype dpy-5(f1) (M. Whistler, D. Riddle, P. Swanson, personal communication, The Worm Breeder's Gazette, 7 (1): 50). Cadet and Dion, informed by Brenner (personal communication in Dion and Brun, 1971), then reported on the use of ethyl-methane sulfonate (EMS) as a mutagen. After mutagenesis they isolated seven other dwarf mutants, as well as one mutant with a defect in locomotion (Cadet and Dion, 1973). X-ray mutagenesis was also used (Ouazana and Brun, 1975).

Complementation and linkage studies were performed (Dion and Brun, 1971) (Figure 17). Using five alleles of what is likely dpy-1 III (Ouazana, 1975), intragenic recombination could be detected and measured (Ouazana and Brun, 1975).

We will be brief on this part that came after the first publication on C. elegans by Brenner in 1974. After this, the Brun team published on a variety of mutant phenotypes, such as a high incidence of males (called Him) (Béguet, 1978), a transformation of the hermaphrodite tail towards a male tail (Béguet and Gibert, 1978) (identified as a weak tra-2 allele by Hodgkin (1985)), temperature-sensitive lethality and sterility (Abdulkader and Brun, 1976; Abdulkader and Brun, 1978, 1980; Abdulkader et al., 1980), as well as unconditional sterility (Mounier and Brun, 1980; Gibert et al., 1984). Brun passed away in 1981, but the team continued until the end of ongoing PhD theses. Overall, one focus was the dumpy mutants (Ouazana and Brun, 1978; Ouazana, 1978) and collagen structure (Ouazana and Gibert, 1979; Ouazana and Herbage, 1981; Ouazana et al., 1985), because many dumpy mutations affect collagen genes. The second focus was gametogenesis and the sterile mutants, because of the central importance of gametogenesis in reproduction (Abi-Rached and Brun, 1975; Abirached and Brun, 1978; Abirached and Brun, 1979; Starck et al., 1983; Starck, 1984). The sterile mutants were, however, more difficult to maintain and study than the classes first chosen in the Brenner lab, such as uncoordinated mutants or cell lineage mutants with egg-laying defects.

Other than the tra-2 allele that was crossed into the N2 background (Hodgkin, 1985), these later mutants, all in the Bergerac background, do not seem to have been preserved.

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Genetic complementation and linkage were developed in the team of Brun. Although Dougherty and Brun agreed that they needed to develop genetics, their attempts were dwarfed by the extent (number of mutants and genetic map), the phenotypic diversity, and the biological relevance of the first mutageneses performed by Brenner et al. The Brun team later started isolating a variety of mutant phenotypes, possibly following contact with the budding C. elegans community (particularly David Hirsh). A strength of Brenner's work was to find many locomotion-defective (“uncoordinated”) mutants that allowed him, crossing them with the dumpy mutants, to build a genetic map (Brenner, 1974). Using dwarf mutants alone, linkage analysis was more arduous (Dion and Brun, 1971). This is an example of the awkwardness in the use of genetics by the French team, which was isolated in Lyon from geneticists in France and elsewhere.

Another problematic issue was the divergent use of the Bergerac and Bristol strains. While the rest of the world adopted Bristol, which developed better at temperatures above 20°C, the team in Lyon had naturally carried on with mutagenesis of the Bergerac strain. This may have contributed to their mutants having been little used and forgotten.

In an experiment running over thousand generations and several years, Brun progressively adapted the Bergerac line to higher temperatures, using the following protocol. At each generation, 6-10 young adult progeny from the same parent were isolated (each in a separate culture), in the high temperature lines (cultured at temperatures ranging from 22-24.5 °C) and in an 18 °C control line. Selection was applied in the sense that: 1) the chosen brood was that with the most abundant progeny at this (early) time, and without aberrant-looking individuals; and 2) the individuals were chosen among the first descendants, so as to avoid crossing with males.

Brun first defined four types of behavior of the Bergerac line when brought to a range of high temperatures (Brun, 1966a) (Figure 18). The Bergerac line was routinely kept at 18 °C, without signs of sterility. Above 24 °C, the strain was immediately sterile. At 23 or 23.5 °C, the strain only became sterile after several generations, with a progressive decrease in brood size. This multigenerational phenotype resembles that now called Mortal germ line (Mrt) (Ahmed and Hodgkin, 2000). At 22°C, the strain remained fertile, and after 100 generations, was able to sustain 23 °C without a Mrt phenotype: the line had thus adapted to a higher temperature regime.

Brun then continued using this method of acclimatization, from 22 °C to 23 °C, then using 0.5 °C steps of 100-450 generations each, until, after over 1000 generations, the culture could be maintained at 24.5 °C, a temperature at which it was initially sterile. This acclimatization was reproducible when parallel lines were followed, with a similar timecourse: a first rapid decrease in brood size when the strain was shifted to the higher temperature (over 2-10 generations–potentially leading to a Mrt phenotype), a phase of rapid recovery (over ~100 generations), followed by a late phase with a slower increase in brood size (up to 500 generations). During this last phase, the line became finally able to withstand the next 0.5 °C increase.

When brought back to 18 °C, high-temperature adapted lines showed a higher brood size than the control 18 °C line for several generations. The effect was cumulative: brood size upon return to 18 °C increased as a function of the adaptation temperature and the duration of culture at this temperature. This effect of long exposure to high temperature was reversible and brood size then gradually decreased over 10-100 generations of return to 18 °C.

Upshifting the temperature again after a few generations at 18 °C, Brun further showed that the adaptation to high temperature was rapidly lost after as few as 7-10 generations at 18 °C (Figure 19). He repeated these observations with several lines and several adaptation and reversion temperatures in a reproducible and apparently progressive fashion (Brun, 1966b). Thus, adaptation to high temperatures was slow–over hundreds of generations–but reversible over a few generations of selfing at 18 °C.

Cytologically, high-temperature shift of the control line caused defects in oogenesis in young adults, such as a lack of oocytes in the synapsis and pachytene zone, and the presence of oocytes with multiple nuclei or giant polyploid nuclei in the diakinesis zone. These phenotypes were absent in the adapted line.

The final paper of the series (Brun, 1966c) discusses possible factors underlying the observed temperature adaptation. First, evolution in the bacterial community used as food and uncontrolled variation in agar medium composition were ruled out by control experiments. Second, the presence of sufficient initial genetic variation that could be selected upon was also ruled out by the selfing nature of C. elegans and the maintenance regime with a single individual prior to the experiment. Third, de novo mutations surely occurred over hundreds of generations and could have been selected for; some temperature steps may possibly have been achieved by such incoming genetic variation. Yet Brun disfavored the role of genetic evolution by mutation in his experiment for several reasons: 1) the overall continuous adaptation over the five temperature steps, 2) the reproducibility of the adaptation schedule in different lines and at different temperatures, and 3) the rapidity and reproducibility of the reversion in adaptation upon return at a lower temperature.

Having ruled out that the above factors were sufficient to explain the adaptation dynamics in his selfing lines, Brun proposed that the observed heritable adaptation to high temperature occurred through non-genetic changes (“genetic” or “genic” is here used to mean based on DNA sequence, whereas “heritable” refers to transmission between generations, which is not necessarily DNA-sequence based, e.g., through small RNAs and histone modifications). Brun envisioned on one hand heritable non-genic sterilizing processes (called S) that would decrease fertility across generations, and on the other hand a slower acclimatization process (called A) that would slow down the fertility decline. He hypothesized that the acclimatization could work either through variation and selection on the heritable sterilizing processes and/or of a physiological regulation induced by the environment that would inhibit these processes or their heritability. If the modifications were originally induced by the environment, they then became heritable and partially independent of it. The Science article ended with: “Comprehensive observations support the assumption that adaptive transmissible cytoplasmic states are produced gradually and are responsible for the production of fertile high-temperature strains” (Brun, 1965).

The Brun 1966 articles were further summarized in Nigon and Brun (1967), yet hardly cited outside of Brun's group. They inserted themselves well in the contemporary scientific context of some adaptive heredity literature (Lansing, 1954; Jinks, 1964), and were indeed cited on this behalf (Davitashvili, 1969) and more recently (Pal and Miklos, 1999; Tal et al., 2010). Yet they remained unexplained in terms of possible mechanisms and did not fit well with the increased use of standard genetics in both laboratory and evolutionary biology at that time.

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Almost fifty years later, the progressive Mortal germ line (Mrt) phenotype observed by Brun in the Bergerac strain needs yet to be reproduced and explained. This phenotype has not been detected in C. elegans N2, but has been observed in many temperature-sensitive mutants derived from it, affecting : 1) telomere formation and DNA repair (Smelick and Ahmed, 2005; Kirienko et al., 2010), or 2) histone modifications and small RNA synthesis in the germ line (Buckley et al., 2012; Simon et al., 2014). Because germ line small RNAs and histone modifications are transmissible across generations in C. elegans (Grishok et al., 2000; Burton et al., 2011; Gu et al., 2012; Buckley et al., 2012; Ashe et al., 2012; Shirayama et al., 2012; Conine et al., 2013), they could perhaps explain a non-genic transgenerational effect or adaptation.

A feature that is now known to differ between the Bristol (N2) and at least some Bergerac-derived strains is the level of germ line transposition–much higher in the latter–although it greatly varies among derivatives of the Bergerac strain (Emmons et al., 1983; Liao et al., 1983; Moerman and Waterston, 1984) (see Appendix 2, Section 17). Interestingly, some of the mutants with a Mrt phenotype in the N2 background also have an increased transposition rate, through the lack of matching small RNAs (Das et al., 2008; Bagijn et al., 2012). It is so far unclear whether the high level of transposition in both Bergerac and these mutants is a/the process leading to sterility after several generations. Other processes, such as progressive deregulation in gene expression, have also been proposed in the case of germ line small RNA pathway mutants (Buckley et al., 2012).

A few years after Brun's experiments, Hansen, Lower, Yarwood et al., past collaborators of Dougherty (Lower, 1966), adapted large populations of C. briggsae and P. redivivus (Lower et al., 1968; Lower et al., 1970) to high temperatures, with an interesting axenic culture design resembling Drosophila population cages (Hansen and Cryan, 1966). In this work, adaptation was presumed to be genetic and Brun's work was not cited. The authors did not test for the stability of the adaptation.

Early on, Nigon was fascinated by the molecular biology of nucleic acids and was convinced that molecular genetics was the future of genetics. Inspired by the works by Jean Brachet and Oswald Avery, he knew that a relationship between the nucleic acids and a product of their activity must be sought. But it was unclear how to apply these notions to the genetics of eukaryotes, and likely it was much too early. Robert Delavault, a colleague and collaborator in Paris, tried to stain and visualize nucleic acids of several species of free-living nematodes (Delavault, 1952a; Delavault, 1952b; Delavault and Nigon, 1952). Later the Nigon team in Lyon succeeded in radioactive labeling of nucleic acids of various organisms. Jacqueline Nonnenmacher-Godet, after initial work in Nigon's laboratory, went to Dougherty's laboratory and applied the radioactive labeling technique to C. briggsae DNA (Nonnenmacher-Godet and Dougherty, 1964).

The molecular biology of the 1950-60s was built on methodological approaches such as crystallography, bacterial and phage genetics, and biochemistry, and not on what we now call “molecular biology” techniques. These techniques only came in the 1970s with the discovery of restriction enzymes and the possibility of cloning DNA. Nigon participated in this work by studying greening of bleached Euglena and thalassemic hemoglobin mutations in humans, leading him to the evolution of primate hemoglobins and the study of aviary viruses carrying erythroblastosis genes. Brenner, despite being a main actor in the development of molecular biology, embraced the genetics of C. elegans in the 1960s without yet knowing how molecular biology could be brought to development and neurobiology studies. The cellular level was the first anchor, but Brenner aimed at the molecular level for the future. Together with the first genetics paper of 1974 (Brenner, 1974), Sulston and Brenner published a lesser known article where they quite correctly assessed the size and complexity of the C. elegans genome using DNA reassociation kinetics (their estimation was 80 Mb for what we now know is 100 Mb) (Sulston and Brenner, 1974). But the way forward was not obvious. Brenner had considered the possibility of isolating nutritional mutants, as advocated by Dougherty, as a possible route to clone genes (Brenner, 2001). He also considered complementing Bacillus subtilis mutants with C. elegans DNA to identify conserved genes (which we now know would not have worked because of introns) (Brenner, 2001). In the end, the first molecular identification of a C. elegans mutation used a biochemical approach: the abundance of muscle proteins allowed the identification of the unc-54 gene, as encoding a myosin heavy chain (Epstein et al., 1974).

Brenner reported: “Jim Watson, in particular, said he would not give me a penny for this work, complaining that I was 20 years ahead of my time. What changed everything was the advent of cloning and sequencing. It opened up all of genetics and gave us direct access to genes.” (Brenner, 2009). As Watson had supposed, molecular biology indeed only developed fully 20 years after Brenner started, when mutated genes could be identified and rescued. Nigon's work was thus 40 years too early. Ironically, the way towards molecular biology is what the Bergerac strain became most known for to C. elegans scientists of the 1980-90s. The first transposon type, called Tc1, was discovered through a restriction length polymorphism between Bergerac and Bristol in Southern blots hybridized with different probes (Emmons et al., 1979). The high number of transposons and the transposition activity in the Bergerac strain then served as tools for gene identification (Emmons et al., 1983; Liao et al., 1983; Moerman and Waterston, 1984; Eide and Anderson, 1985; Plasterk and van Luenen, 1997; Transposons in C. elegans). The hundreds of Tc1 transposons were used for genetic mapping and identification of mutated genes (Finney et al., 1988; Ruvkun et al., 1989; Williams et al., 1992). Moreover, the high transposition rate, at least of the RW7000 subclone of Bergerac, could be used as a mutagen that rendered gene identification easier (Moerman and Waterston, 1984; Greenwald, 1985; Moerman et al., 1986).

In the spirit of the cell lineage or the nervous system connectome, a physical map of the whole genome was built through a new heroic enterprise by Sulston, together with Alan Coulson and Robert Waterston. They cloned the C. elegans genome in cosmids and yeast artificial chromosomes, building a physical map, which was progressively aligned with the genetic map (Coulson et al., 1986; Coulson et al., 1988; Coulson et al., 1995). This led C. elegans to be the first animal to have its genome fully sequenced, from ordered clones (The C. elegans Sequencing Consortium, 1998) as a way towards the human genome sequence to which Sulston, Coulson, and Waterston then contributed (Sulston and Ferry, 2002).

The initial interest in nematodes of early researchers, such as Boveri, Bütschli, van Beneden, Maupas, Krüger, P. Hertwig, and Bělař, was triggered by the diversity of reproductive modes, based on the cytological diversity of meiosis and fertilization events. After Nigon determined the XX/X0 chromosomal basis of sex determination in C. elegans and correctly inferred that the ratio between X chromosomes and autosomes was the key, sex determination mechanisms were further studied in C. elegans using genetic screens, initially by Hodgkin and Brenner (Hodgkin and Brenner, 1977; Hodgkin, 1988; Hodgkin, 2002b). Single mutations could either transform XX animals into males (tra), or X0 animals to females (fem) or hermaphrodites (her), or be lethal specifically for X0 animals (xol). The analysis of these mutants showed that a low ratio of X chromosomes to autosomes in males activates expression of the xol-1 gene and downstream production of the HER-1 intercellular signaling molecule, which represses the TRA-2 receptor and starts a cascade of signaling events in each cell throughout the animal. The ratio between X chromosomes and autosomes is now known to be assessed by several X-linked and autosomal elements that were identified by Barbara Meyer et al. (Somatic sex determination).

Concerning the partial independence between soma and germ line, some sex determination mutations called fog only feminize the hermaphrodite germ line. They transform XX hermaphrodites into true females without spermatogenesis, thus dissociating the mechanisms of sexual differentiation of the gonad from that of the gametes, as predicted by Nigon. Analysis of these mutants led to the understanding that the wild-type hermaphrodite germ line differentiates successively as male and female through a temporal molecular switch from spermatogenesis to oogenesis: the action of the TRA-2 receptor is first inhibited by FOG-2 (sperm formation) before being reactivated (Sex determination in the germ line).

From the knowledge of sex determination pathways, it became possible to manipulate at will the sex determination mechanisms of C. elegans using mutants. Transition between a hermaphrodite selfing mode of reproduction and a gonochoristic reproduction can be easily achieved: mutants in a single gene, such as fog-2, reproduce through females and males (Schedl and Kimble, 1988). Hodgkin could further produce all kinds of situations, including maternally or environmentally determined sex or other forms of chromosomal sex determination (Hodgkin, 2002a).

Once molecular details became clearer in C. elegans, in the last 20 years the evolution of sex determination and of the modes of reproduction in nematodes became again a center of interest (Pires-daSilva, 2007). As inferred by the older authors, the transformation of a male-female species into a hermaphroditic one may be easy in terms of mutational steps. In the gonochoristic C. remanei, mutations in only two independent pathways are sufficient to create hermaphroditic nematodes (Baldi et al., 2009). The Caenorhabditis genus is presently known to harbor three independent transitions to selfing (Kiontke et al., 2011; Thomas et al., 2012). An evolutionary consequence of these transitions is a great reduction in molecular diversity and the appearance of outbreeding depression (the progeny or grand-progeny is less fit than the parents), contrasting with the observed inbreeding depression in gonochoristic nematodes (Dolgin et al., 2007; Gimond et al., 2013). Recessive deleterious mutations can indeed be maintained at low frequencies in gonochoristic species, whereas they become rapidly exposed in a homozygote combination in selfing species and are then efficiently purged by natural selection. The evolutionary maintenance of males remains a key question under active study (Anderson et al., 2010). Spontaneous changes in mode of reproduction from the male-female mode (such as in P. pellio, with pseudogamy through males, Section 5.2.2; or in P. lheritieri, Section 3.5) have not been observed again to our knowledge. They could be looked for.

In a thread running from Boveri, Maupas, and Nigon to recent work in C. elegans (Rog and Dernburg, 2013; Lui and Colaiacovo, 2013), nematodes have been particularly instrumental in studies of meiosis. Now chromosomes or chromosome segments can be individually marked, not only through genetic markers, but also using fluorescent probes (Nabeshima et al., 2011). Homologs find each other at the onset of meiosis through pairing at foci on the nuclear membrane, with different proteins assembling the different chromosomes (Phillips and Dernburg, 2006; Rog and Dernburg, 2013). These pairing centers are anchored at the nuclear membrane through the ZYG-12 protein (that with a missense mutation in Bergerac) (Sato et al., 2009). The sites of cross-overs can be marked and the regulation of cross-overs after double-stranded break formation is increasingly understood (Rog and Dernburg, 2013; Vader and Musacchio, 2014). How these meiotic processes change during evolution is now a promising field of investigation.

Among easily culturable members of the family Rhabditidae alone a wide variety of modes of reproduction with variations in meiosis and fertilization are known: hermaphrodite-male, female-male, parthenogenetic (Diploscapter spp.), alternating generations (Rhabditis sp. SB347), pseudogamous hermaphrodite (Rhabditis anomala and R. aberrans, to be found again, possibly in frozen collections as “hermaphrodites”), and partially pseudogamous females and males (Mesorhabditis belari; Mesorhabditis sp. PS1179). This diversity is a great tool to study genetic mechanisms underlying such evolutionary switches, and their evolutionary consequences. In contrast to the evolutionary transition to selfing, transitions towards parthenogenesis or pseudogamy affect basic aspects of meiosis and fertilization, which remain to be studied. How do the cellular events leading to the evolution of pseudogamy, parthenogenesis, or alternate generations evolve? How is the alternative between pseudogamy and true fertilization controlled in the egg of M. belari?

A further open field concerns the partial stability of tetraploids in C. elegans. Questions concern: 1) the problem of dosage compensation between chromosomes in unusual chromosome formulas, and 2) meiotic pairing and progression in a tetraploid situation.

In conclusion, the diversity in modes of reproduction and meiosis is a historical legacy of research in nematodes, which can be explored in terms of molecular regulation and evolution of cell biological processes, as well as for its impact on evolutionary dynamics.

Brun's experiments of laboratory adaptation of C. elegans to high temperature (Section 10) were unconventional adaptation experiments because he used a very low population size–1-10 individuals per generation. Natural selection is efficient at high population size. In addition, when starting, as Brun did, with a population harboring no starting genetic diversity, a large population size also allows for incoming genetic variation through new mutation. Thus, the use of a low population size may be appropriate in the case of non-genetic acclimatization but not a design for standard genetic adaptation experiments.

The use of C. elegans in experimental evolution has started anew in the last 15 years, along the lines of standard population genetics (Gray and Cutter, 2014). The mode of reproduction and freezing ability of C. elegans are great assets. Experiments of adaptation to novel environments have included pathogens (Morran et al., 2009; Schulte et al., 2010), high salt (Theologidis et al., 2014), as well as high temperature for C. elegans (Gainutdinov et al., 2007) and C. remanei (Sikkink et al., 2014). Experiments at a small population size have also been performed. A population size of one corresponds to the standard design of mutation accumulation experiments, aimed at assessing the rate and distribution of mutational effects, most of them deleterious. Mutation accumulation at a population size of one indeed results in a fitness decay (Vassilieva et al., 2000; Keightley et al., 2000). However, a population size of 10 basically alleviates deleterious effects of mutation accumulation (Estes et al., 2004). A reexpansion at high population size of low fitness lines allows for surprisingly rapid recovery (Estes and Lynch, 2003).

A first exciting prospect concerns the mechanistic and evolutionary aspects of the progressive temperature-sensitive sterility observed by Brun. Although it may differ from that in Bergerac, progressive sterility at 25 °C is common among C. elegans wild isolates (M.-A. Félix, unpublished). The molecular and cellular basis of this progressive sterility and its evolutionary significance in the regulation of transgenerational memory and in adaptation remain to be studied.

A second question concerns the importance of prior non-genetic acclimatization in evolution, as opposed to direct genetic (DNA-based) adaptation. Brun concluded that the adaptation he observed was of a non-genetic nature. Although this possibility has been discounted by the synthetic theory of evolution in the mid-20th century, recent research on C. elegans and other organisms has uncovered molecular mechanisms whereby gene expression changes (potentially induced by the environment) could be passed on to the progeny for several generations. This includes small RNAs in C. elegans, which have been shown to be involved in physiological (same-generation) response to the environment (Félix et al., 2011; Ashe et al., 2013; Juang et al., 2013), and may be passed on to the progeny (Grishok et al., 2000; Alcazar et al., 2008; Burton et al., 2011; Gu et al., 2012; Ashe et al., 2012). Recent demonstrations of multigenerational effects in a physiological wild-type context (Sterken et al., 2014; Rechavi et al., 2014; Remy, 2010; Schott et al., 2014) are interesting and remain to be reproduced by others. Their possible role in adaptation to the environment and their importance and interplay relative to genetic variation under different circumstances are exciting open questions.

The original strain and its descendant lineage in Lyon were temperature-sensitive, displaying abnormalities from 23-24 °C. Males mated well and were used for crosses (Nigon, 1949a; Dion and Brun, 1971). The strain was maintained in Lyon at low temperatures and remained healthy. It was sent to the CGC in 1980, and frozen under the name of Bergerac FR. Liao et al., (1983) state that “The Bergerac FR strain has male fertility levels and brood sizes comparable with Bristol N2, while the Bergerac LY strain has low male fertility and brood sizes.” The strain is no longer available at the CGC and was kindly provided to M.-A. Félix by Mark Edgley and Donald Moerman. It is indeed healthy, compared to the other sublineages mentioned below (observations by M.-A. Félix.).

Nigon shared the Bergerac strain with Dougherty in 1948. After several years of culture (perhaps in part in liquid), males of the California strain could no longer mate well (Fatt and Dougherty, 1963). After Dougherty's death, Eder Hansen gave a subclone to David Hirsh (University of Colorado, Boulder) who used it for further studies of heat sensitivity (Wood et al., 1980). William (Bill) Wood built a strain (BW54) where the heat-sensitive locus from Bergerac was introduced by repeated crossing into the N2 background; this strain was later used for the identification of the zyg-12(ct350ts)II mutation by Malone et al. (2003). The zyg-12 mutation is also present in FR, RW7000 and CB4851 (genotyping by A. Richaud and M.-A. Félix), indicating that the nucleotide substitution was likely already present in 1944 (or appeared shortly thereafter). This temperature-sensitive allele of zyg-12 explains the temperature sensitivity of the Bergerac strain (see Section 9.3). The Bergerac Be strain in Johnson and Hutchinson (1993) and the Bergerac DO mentioned in Levitt and Emmons (1989) seem to correspond to this sublineage via Dougherty in Berkeley.

The Bergerac strain was sent by Nigon to Brenner a first time in 1966 and again in 1969 when the first one was lost (Nigon, 1966/1970). Brenner called it N62. Once the strain nomenclature was standardized (Horvitz et al., 1979), this strain was renamed CB4851 (Hodgkin and Doniach, 1997). CB4851 is phenotypically “sick” (more so than FR) and has a high spontaneous male production (Hodgkin and Doniach, 1997). CB4851 also has a high Tc1 transposon content (Hodgkin and Doniach, 1997).

The BO/RW7000 Bergerac subclone is the best known to C. elegans scientists of the generation that was active in the 1980-90s, when it was used for genetic mapping using transposons and for mutagenesis using transposase activity (see Section 12.2). This BO sublineage originated from a gift of a Bergerac culture by Jean Brun to David Hirsh in Boulder, CO in 1977 (Moerman and Waterston, 1984). This sublineage is a very sick derivative, which bears various names as it moved among laboratories and as strain nomenclature evolved: LY (“Lyon”), BO (“Boulder”), RW7000, DR1344, EM1002, and DP13 (Liao et al., 1983; Moerman and Waterston, 1984; Emmons and Yesner, 1984; Williams et al., 1992). While Hirsh's laboratory used the Dougherty Bergerac strain for zyg-12(ts) isolation, they used that originating from Brun in 1977 (BO) as a source of transposons. The BO subclone was found to contain ~ 200-500 copies of the Tc1 transposon, compared to ~30 for N2, and these differences between BO and N2 were used as polymorphisms for genetic mapping (Emmons et al., 1983; Egilmez et al., 1995; Korswagen et al., 1996). In addition, the BO sublineage evolved an increased Tc1 transposition activity in the germ line, at least 100-fold over that of C. elegans N2 and Bergerac FR (Moerman and Waterston, 1984), resulting in the appearance of phenotypic mutants in the culture (Mutator phenotype). This transposon activity was used for mutagenesis and tagging (see Section 12.2). This increased transposon activity likely occurred through the activation of a transposon copy, forming the mut-4(st700) mutator allele on chromosome I, which then hopped to other chromosomes, giving rise to mut-5(st701)II and mut-6(st702)IV (Mori et al., 1988). These mutator alleles have not been molecularly identified. The Tc2 transposon also has an increased copy number compared to Bristol in Bergerac subclones, including FR (Levitt and Emmons, 1989), while Tc3, Tc4, and Tc5 do not (Collins, 1989; Yuan et al., 1991; Collins and Anderson, 1994). Since CB4851 also has a high Tc1 transposon content (Hodgkin and Doniach, 1997) and BL1/SH, a later derivative from the Lyon lineage, has some detectable transposition activity (Moerman and Waterston, 1984), it appears likely that the ancestor of CB4851, BO/RW7000, and BL1 and maybe the original isolate already had a somewhat elevated level of Tc1 transposon and transposition activity, which further increased in a dramatic fashion in the BO/RW7000 sublineage. In sum, the high transposition activity and extreme strain sickness only apply to the BO/RW7000/etc. derivatives, not to the FR strain that was kept in Lyon.

Note that the high-Tc1 content strains DH424, TR403, and CB4555/DR1349 that were considered to be wild isolates of C. elegans turned out to be recombinants between N2 and a Bergerac subclone (apparently the same recombinant, likely a lab contamination from the start; McGrath et al., 2009).

This account leaves several pending questions:

1) How was the Tc1 transposase activity activated in the BO sublineage?

2) Is there a relationship between the zyg-12 mutation and the transposon activity?

3) Whether wild strains of C. elegans with elevated transposon activity exist remains to be studied.

Emile Maupas studied at the Ecole des Chartes in Paris to become an archivist, thus quite far from biology; his thesis concerned legislation during the wars of the Middle Ages. He first worked as an archivist in Cantal in the center of France, and then moved to Algiers (a French territory at the time) in North Africa, where he stayed from 1871 until his death in 1916. He became the Head of the National Library of Algiers. His afternoons were devoted to the Library, and the rest of his time to his activities as an “amateur” biologist. For the latter, he worked at home, a modest 3-room dwelling in the neighborhood of Bab el Oued, overlooking the Mediterranean Sea. The microscope was in front of the window; the rest of the “laboratory” (a few kitchen plates with his cultures) could fit on the fireplace shelf (right picture; see Caullery, 1937).

Maupas’ curiosity focused on the biology of small organisms such as ciliates, nematodes, rotifers, and oligochaetes, which he isolated from soil or freshwater. He developed culture methods to observe their development, reproduction, and sexuality–a quite unusual endeavor at a time when the study of morphology predominated. He thus can be considered as one of the first biologists who, with rudimentary equipment, applied the experimental method to basic biological questions, similar to that developed in physiology by Claude Bernard.

Maupas is perhaps best known for his studies of lineage degeneration in ciliates, where he showed that a given mitotically reproducing clone had a finite lifetime, unless sexual reproduction occurred. He carefully observed their sexual reproduction and correctly interpreted the exchange of micronuclei. In the context of the 1880s, observing sexuality in a single-cell organism, and the role of the nucleus therein, was of major significance in attributing to the nucleus a role in heredity. Maupas also studied sexuality in rotifers, and turned, in the last 20 years of his life, to using nematodes as model organisms. His interest for nematodes concerned their lifecycles and the evolution of their diverse modes of reproduction (see main text). As an aside, he described and named the several new species that he isolated and observed, including C. elegans (then Rhabditis elegans), clearly his favorite species in the 1900 opus.

Despite Maupas’ institutional and geographical isolation, the meticulousness of his experiments and the relevance of his interpretations attracted the attention of great biologists, such as Otto Bütschli (another “protistologist” with an inclination for nematodes), then Professor in Heidelberg, who obtained for Maupas the title of Doctor Honoris Causa of the University of Heidelberg in 1903. The French Academy of Sciences elected Maupas as a Correspondent in 1901 and awarded him the Grand Prize for the Physical Sciences for his work on development and reproduction of nematodes. In 1932, the French zoologist Maurice Caullery erected a plaque in his honor on his house in Algiers. He then gave a laudatory speech (Caullery, 1937), mentioning that he had never met Maupas, but that Bütschli had expressed to him in 1891 his admiration for Maupas. Caullery was a key figure of French zoology at the time, and the founder and head of the “Laboratoire d'Evolution des Êtres Organisés” (Laboratory of Evolution of Organized Beings) at the University of Paris. He was succeeded in this role by Pierre-Paul Grassé, who hosted Victor Nigon in his first years in Paris in this same (still standing) building on Boulevard Raspail (Caullery was then retired but still around).

A high school bears Maupas’ name in Vire, Normandy (his birthplace) and in Skikda (formerly Philippeville, in Algeria). A Maupas medal is still being awarded for great achievements in the ciliate scientific community.

Paula Hertwig grew up in a family of famous biologists: Oscar Hertwig was her father and Richard Hertwig her uncle. Throughout her life, she was also close to her brother Günther Hertwig, with whom she shared some scientific projects. Paula Hertwig studied in Berlin and obtained her doctorate in 1916. Her first publication in 1911 reported on the abnormal divisions of Ascaris eggs upon exposure to radium. She studied many organisms. Among them, she isolated free-living nematodes to study the cellular basis of meiosis and fertilization and variations in modes of reproduction. In particular, she studied a spontaneous mutant observed in Rhabditis pellio (see main text). Most remarkably, based on this work, in 1919 she was the first woman in Germany to obtain the Habilitation. She became Privat-Dozent at the University of Berlin, despite a law barring women from obtaining faculty positions. Another law also excluded married women from state employment, and she did not marry.

In 1921, she became Assistant at the Institute of Genetics of the Agricultural College, which was directed by the plant geneticist Erwin Baur. In particular, she studied the genetics of a hermaphroditic variant of Melandrium (Silene dioica). She also had a long-standing interest in the hybridization of animal species.

She later focused on mouse genetics and induced mutagenesis. The work that made her famous concerned the mutagenic effects of radiation; she was one of the first voices warning about their adverse effect. She isolated and studied many induced mouse mutants, including some relevant to human disease, such as microphthalmia, oligodactyly, or the Hertwig-Weyers syndrom, named after her.

From 1927 to 1945, she was Extraordinary (Adjunct) Professor of Genetics at the Medical School of the University of Berlin. She was politically active, elected in 1932 as representative of the liberal German Democratic Party in the Prussian Parliament. After the war, she and her brother chose to live in the German Democratic Republic (GDR = East Germany). In 1948, she obtained a Chair in Genetics at the Medical School of the University of Halle (GDR), where she also became the Dean. She headed a new Institute of Genetics in this university. When Lysenko obtained political support in his rejection of genetics, which resulted in the persecution of geneticists in the Soviet Union, she expressed herself against him. She was elected a member of the Leopoldina, the German Academy of Sciences (then in East Germany), in 1953. After the death of her brother, she retired in 1972 to Villingen in the Black Forest in West Germany and, in 1973, became Doctor Honoris Causa of the University of Heidelberg.

Bělař (pronounced Bielarch) grew up in Vienna, in an intellectual environment. He studied zoology, had access to his brother-in-law's private laboratory for his research, and defended his doctorate in protistology in 1919. From 1915 to 1918, he served in the Austrian army. After his doctorate, he was invited to Berlin by Max Hartmann, director of a section at the Kaiser Wilhelm Institute and protistologist, and became his assistant. In 1925, he obtained the title of Privatdozent of the University of Berlin. His main work remained centered on protists, although he became interested in the use of nematodes. In 1929, he took an invited professorship at The California Institute of Technology (Caltech) in the new Division of Biology headed by Thomas H. Morgan, and spent nearly two years in Pasadena, CA, USA. His main material there was the embryo of Urechis, a marine annelid. Just when he was due to return to Germany, he died in a car accident on a trip to the Mojave desert, where he painted, one of his favorite hobbies.

Like Bütschli and Maupas, Bělař mostly worked with protists, studying nematodes and other animals on the side. Although being best known for his work in protistology, he had more general interests and was able to link his work in protistology to general cytology matters. In protists, such as Actinophrys sol, he studied cell division and could draw parallels with the events of mitosis observed in animals. His later work concerned the mechanistic aspects of cell division, including spindle mechanics. He was well-known for the quality of his drawings, and was described as a rigorous and critical mind with wide-ranging intellectual interests, and especially as a gifted amateur artist.

His interest in consistent and varying features of cell division, particularly meiotic divisions and first mitotic divisions, seems to have drawn him to Rhabditis as a model. Following Eva Krüger and Paula Hertwig, he studied several Rhabditis species where the sperm was required for fertilization yet sperm chromosomes degenerated upon entry into the egg (pseudogamy; see Main text).

He wrote several general works concerning cytological questions in genetics, including “Cytologische Grundlagen in Vererbungswissenschaft” (1928; Cytological bases in the sciences of heredity). This book was a reference for V.M. Nigon; in the 1940s, it was the only synthetic work concerning cytological approaches to heredity.

When V.M. Nigon asked Professor Vandel for a subject for his Diplôme d'Etudes Supérieures (Master), which he could accomplish while living 200 km from the university, Vandel referred to Bělař's work. He mentioned a visit to Bělař's laboratory, where Bělař had shown him his nematode cultures and had told him about his nematode work.

Short professional CV: Starting in October 1944, researcher at the Centre National de la Recherche Scientifique (CNRS), working at the Laboratory of Evolution in Paris, directed by Professor Grassé. From October 1947 to September 1949, “Chef de Travaux” (Lecturer) at the Sorbonne. In October 1949, “Maître de Conférences” (Reader) in Lyon, then Professor in 1953. In Lyon, he set up courses in Genetics and Embryology, the Section of General and Applied Biology in 1962 (associated with the CNRS), then a Laboratory associated with the Institut National de la Recherche Agronomique (INRA). This latter was devoted to avian leukemia viruses. These viruses were used as vectors by Nigon et al. to study hemoglobin synthesis in cultures of avian blood precursor cells. From 1970 to 1980, Director of a French-Algerian Programme on thalassemia, financed by the Foreign Affairs Ministery. Emeritus from 1986 to 1989. During retirement, overseas projects from the Foreign Affairs and INRA: Japan in 1991 and 1992, Bolivia from 1994 to 1999. Co-director of the Bolivian Institute for High-Altitude Biology 1995-1999. Honorary Professor of the University of La Paz in 1998.

How did you start working with Rhabditis? This question was often asked of me. It was an exceptional combination of circumstances.

I was born in 1920 in France, in the department of Moselle. Local history played a large influence in my personal life. Europe had been largely united in the Roman Empire, but after Charlemagne's reign, it fell into pieces, each one under the power of local kings or princes. The territory of Moselle was a part of the Dukedom of Lorraine, where the official language was French, but at its Northern border in Moselle a small part of the territory traditionally used a German dialect and was thus known as “bailliage d'Allemagne”. My family had lived in this territory since the 16th century. While speaking at home a German dialect, some family members participated in the French-speaking administration of Lorraine. It took over ten centuries to rebuild large countries like France and Germany. Lorraine was incorporated into France in the 17th and 18th centuries. The unification of Germany occurred later, in the second half of the 19th century, and strove to include all German-speaking regions. After France's defeat in 1871, together with Alsace, Germany thus annexed the Moselle department. However, during years of association with French-speaking Lorraine, the German-speaking Lorraine had adopted French traditions, such as the practice of the Roman Catholic religion (while most regions of Germany were following the Lutheran Reform). For such reasons, the annexation of a part of Lorraine by Germany was not universally accepted and Moselle returned to France after the German defeat of 1918. In 1940, the German Nazi power decided unilaterally to re-annex Moselle. From past experience they knew that annexing a French-speaking part would result in local opposition. They thus decided to expel to France 200,000 French-speaking people in September and October, 1940. My parents, over 60 years of age and partly German-speaking from childhood, could stay. I easily spoke German and had visited a large part of Germany in 1937 and 1938. I appreciated German literature, philosophy, and science. But I gained there a profound aversion to Hitler's politics. Thus, my decision in 1940 was to avoid a German status. My parents approved of my choice. When asked by the German administration they declared that I had been transferred overseas by the French army. They advised me to avoid any contact with the German police, who continued to inquire about me. If I fell into their hands, I would be obliged to choose: either my return to German Lorraine, or the departure of my parents. I thus had to live in the highest discretion, avoiding any contact with Germans. This obliged me to live in the non-occupied zone of France (Note: roughly the South half of France, but the demarcation line run diagonally in the SouthWest, leaving Bordeaux in the occupied zone while Bergerac and Toulouse were in the non-occupied one). This history was a key background, to which I will return below, for why I ended up working on C. elegans, while stranded in Bergerac as a consequence of the war and my Lorraine origins.

From the age of 12, I had decided to become a scientist. At 16 I had entered the University of Paris as a medical student (1936). My plan was to prepare to enter the Pasteur Institute in Paris. I had developed a passion for tropical diseases. Their study would conciliate my passion for scientific research, my social inclinations towards poor people, and my taste for travel. Yet I got rapidly disappointed by the archaic character of medical studies at French Universities. During my second year I met a Laboratory head of the Pasteur Institute, who explained to me that only half of the personnel had a medical degree, while the other half had a science degree. My decision was immediate–the next year, I abandoned the medical curriculum and registered at the Faculty of Sciences, which conformed better to my expectations.

In 1940, my parents, devoid of French money, could no longer finance my studies. I thus had to earn my living, while continuing my studies. At the University of Lyon (in the non-occupied zone), I obtained a low-paid position in Geology, a discipline that did not interest me. I got married, very early. The mother of my spouse died in March 1941. Her father asked my wife, who was the oldest of his children, to settle close to him to help raising his younger children. He offered us a house, a support through his relationships with surrounding farmers and, for me, help in finding private lessons to teach. He lived in Bergerac, a small town that was very appropriate for my need to be discreet. The only difficulty was the distance to the University of Toulouse, the closest one in the non-occupied zone–practically I could not attend the courses. I went to Toulouse to consult Professor Vandel who headed the sector of Animal Biology. He received me with a great understanding and the willingness to help me. I am happy to express here a tribute of gratitude to his memory. Professor Vandel gave me a list of works to study and introduced me to his best students, who gave me their notes on the classes and practicals. I complemented these documents with popular books about genetics and embryology. I passed the exam in October 1941, being the top-ranked student, with the best possible mark.

I could no longer hope to work in the Pasteur Institute, located in German-controlled France. With my wife, we prepared a project for me to teach in French schools in a foreign country, possibly the United States. After the war, I might have been able to obtain such a teaching position and take part in a research lab in the United States. Before this, my next academic step was a year dedicated to work in a laboratory, on a subject that usually implied reproducing data from the literature, with a written dissertation and an oral examination at the end. Unable to pursue work at the University, I had to perform this experimental study at home with rudimentary instrumentation. I proposed this to Professor Vandel, who immediately accepted. He told me about Maupas, of whom he had helped publishing a posthumous article, and of Bělař, whom he had met. He was attracted by the simple requirements of the Rhabditis culture, which I could likely manage. Apparently, he had not grasped the further potential of this material. He likely hoped that, by repeating these experiments, I would confirm, and maybe elaborate on the ambiguous results of Maupas on crosses. I immediately accepted.

The first hermaphroditic nematode that I isolated turned out to be the famous Caenorhabditis elegans described by Maupas and I did not search further. I placed some garden soil in a kitchen plate and maintained its humidity by covering it. On the plate, I added diverse items as traps. I remember using carrot slices and also earthworm pieces from the same garden, cut with scissors! Two or three days later I transferred the trap with some surrounding soil onto a Petri dish with Knop agar. I waited until the Petri dish was swarming with larvae and tried to transport to a new plate the smallest possible number of animals. One day later, they were adults and visible with the naked eye, which allowed me to catch one with the tip of a scalpel and seed a new plate with a single individual. All this without a dissecting microscope at hand: I only had an old Stiassnie microscope offered by my parents at the beginning of my medical studies. By proceeding in this manner, I hoped to select a hermaphroditic species, which I could monitor by observing with my microscope that there were only females. I then needed to hunt for the rare males that would allow me to determine the species. I do not remember having had to proceed through numerous identifications to isolate the C. elegans that I was looking for, based on the initial data by Maupas.

My readings of the prior studies struck me by the rudimentary character of the culture methods that were used. They contrasted with what I knew of the rigorous methods used by Morgan for Drosophila. I thus decided to develop for Rhabditis a more rigorous culture method. Within two months, I invented a method of breeding isolated animals on a drop of agar, bringing nutrients to the culture, with daily transfers of the parents to fresh medium, which, in a few weeks, gave males in great quantity. These results impressed Vandel and I wrote a short article relating them (Nigon, 1943).

After my diploma, I had to participate in “Chantiers de Jeunesse”, which corresponded to practical work that replaced military training in the “free” zone of France. But I soon had to face a new danger: at the beginning of 1943, the French government decided to send those of my age to Germany as compulsory workers. The German police would easily then identify my origin from the German-speaking part of Lorraine and I immediately decided to prevent this. With the help of local medical doctors, I simulated an accident with a broken foot. This resulted in six weeks in the military hospital with my foot in a cast. When I came back to the Chantier, all of those of my age had been transferred to Germany. I had prepared a possible fallback in the maquis in case of danger.

Some time later, I asked Vandel about future possibilities. Naively, I told him of my project to emigrate to the United States. He then informed me about the projects that, with other Professors, doubtless in Resistance networks, he planned for France once liberated. At that time, Germany had lost Stalingrad and was starting to retreat. Many French people were beginning to think that a German defeat was conceivable. Vandel told me that two months after the installation in Paris of a Free French Government, the CNRS would reopen, creating jobs for young scientists. So I should be in Paris at the time of the liberation. He had already talked about me to Parisian colleagues who were ready to host me. After my studies, I had obtained a well-paid job as a chemist in a company in Bergerac, which allowed us to spare the necessary sum for me to spend six months in Paris, while my wife and children would stay in Bergerac, with her father's family. I thus moved to Paris, alone, in April 1944.

I was received by Professor Grassé, who installed me in a laboratory, with dream equipment compared to what I had at home in Bergerac. Paris was liberated in August. On 1^st October, my project on Rhabditis was accepted by the CNRS. I was recruited as a research trainee with a monthly salary of 1000 Francs, a fourth of my salary in Bergerac. It was difficult to support a family with this amount. My wife managed to do so, to the amazement of my friends. In sum, so far my path towards Rhabditis resulted from an encounter with Vandel, with, for me, difficult conditions of living. For Vandel, the lineage was Maupas and Bělař, yet apparently without a deep conviction concerning its interest. It was only through my work in Paris that the richness of the Rhabditis project struck me.

In 1948, I hosted a visit from Ellsworth Dougherty, who spent six months with me, learning to use the experimental method as scientific approach. Our discussions helped us to enlarge the scope of our projects. The misfortunes of the war, the extreme poverty, the cobbled-together equipment, the life with rudimentary comfort, the political climate, etc.: all this deeply marked scientific research in France at that time. Today's scientists cannot imagine the influence of these work conditions and the approaches of researchers in this period. When Ellsworth came to France, he told me of his surprise at this work atmosphere, which he had never experienced. These ascetic conditions, according to him, played an important role in stimulating the creativity of scientists. He had never worked with such intensity and concentration.

I defended my thesis (Ph.D.) in the spring of 1948. On 1st October 1949, I was appointed Lecturer at the University of Lyon. Access to an academic position entailed establishing a research program, with the participation of young researchers. I had reached, at this point, a quite clear view of the potential of Rhabditis. I had some experience with cytology and saw a future with genetics. Yet, in line with a French tradition, the genetics of the time, including the remarkable results of Morgan, appeared to me to suffer from major problems, resulting especially from the abstract character of the gene. It allowed the development of unverifiable theories, such as the theory of polygenes. A way to avoid this abstract theory was through the birth of what has become Molecular Biology; the genetic role of DNA was just proven at this time by Avery's experiments on bacteria. I was thus wondering how to access molecular biology starting from Rhabditis. The only answer I could give was to associate, in one program, several experienced researchers–at least a geneticist and a biochemist. Around me, I searched for candidates and only found young students, without experience. Most experienced researchers considered molecular biology as an unreachable utopia. Nevertheless, I searched for the support of influential Professors. This again was a failure. Evidently, this project could not succeed in France. The choice was limited to continuing on nematodes, forgetting, at least for a while, molecular biology. Or, follow my quest towards molecular biology by testing other material, on which more advanced knowledge could shorten the way towards molecular biology. In 1953, I chose the second path.

At that time, I had already started to train some students in research. Two of them wished to continue on the nematodes: Jean Brun, who worked on this subject until his death, and Pierre Guerrier, tied to his video camera. Later, Guerrier continued to study the first cleavage divisions, this time in marine Spiralia.

In the 1953-1965 period, I had defined an aim–to find transmissible characters in a eukaryotic species, with which one should be able to study the path from the gene to the results of its expression. Following this aim, I tried several species, including Drosophila, the silkworm, and the green alga Euglena. I performed this research with young students. If, after a few years, I could not obtain the desired results on one species, I moved to another. Several of my young collaborators then chose to continue on the material that I had left behind. The sought-after result came with the work on Euglena bleached by culturing them in the dark and brought back to light. One could observe the formation of chloroplasts and perhaps determine the mechanism. We examined ground extracts of greening cells after ultracentrifugation on a sucrose gradient. I immediately recognized the signal of polysomes, already described in bacteria, sea urchin eggs, and erythropoietic cells. This was one of the results I was looking for, as it could result from the expression of the mysterious chloroplastic DNA.

It took us over 13 years to reach this result. During this time, molecular biology had taken shape and established the central molecular mechanisms from DNA, ribosomal, messenger, and transfer RNAs, to proteins. We only reached the result in Euglena through biochemical exploration. This was pursued in parallel by scientists who were qualified in genetics and in biochemistry. Had we known this mechanism earlier, we could likely have reached the sought-after result on all species we studied, including the nematodes! Said otherwise, abandoning the nematodes had been a mistake. During this time, it would have been better to acquire the missing experience in genetics and apply it to Rhabditis... If Dougherty and myself had taken up this direction, the scene would have been changed, for both of us. It was the great merit of Brenner: wiser than us, he attacked the nematode only after sufficient experience in Genetics and Molecular Biology!

For those who may be interested in my later works, they are well documented on PubMed, under my name. Only missing are the studies I conducted from 1995 to 1999, on adaptation to high altitude, in La Paz (Bolivia), at 3700 meters. This work was interrupted by the death of my wife and health problems for me, despite some first results possibly oriented towards the discovery of epigenetic processes (still unpublished).

Text by VM Nigon (translated by by M.-A. Félix).

Ellsworth Dougherty graduated in Zoology at a young age in 1940, and obtained his PhD in 1944 at the University of California in Berkeley. He served in 1944 in the health corps of the US Marines. He then completed his medical studies as a veteran (MD, 1946). As a recipient of a Guggenheim Memorial Fellowship (1947-49), he worked at Caltech and visited France where he stayed in the laboratory of Victor Nigon. After this fellowship, he practiced as a physician in a Kaiser Permanente Foundation hospital in Richmond, CA, and also taught at the University of California in Berkeley.

His first studies concerned systematics of parasitic nematodes. His first work on a free-living nematode, Rhabditis (pellio), took place in Pasadena. He contacted Victor M. Nigon and spent about six months in 1948 working with him in Paris, also attending the International Congress of Zoology that took place in Paris that year. In 1951-52, he came for a second visit to Nigon's laboratory in Lyon for nearly a year. In 1957, he established a laboratory of “Comparative Physiology and Morphology” in the research section of the Kaiser Foundation in Richmond, California. In 1960, he moved to the Department of Nutritional Sciences at the University of California in Berkeley. He joined several scientific trips to Antarctica. He visited Lyon again several times. A last short visit took place in 1961. He had previously suffered from depression that had led him to a first suicide attempt. One of his friends in Lyon, a Professor at the Medical Faculty, organized in his honor an impressive reception. He committed suicide in December 1965.

“We had contracted a deep friendship. Ellsworth had a very imaginative mind and was not naturally an experimenter, yet, by being in contact with me, he got trained and enjoyed the experimental method. In the early 1960s, he hosted in his laboratory Jacqueline Nonnenmacher-Godet, a student from my laboratory (who has served as the head of CNRS Life Sciences in 1999-2002 and is currently President of the French League against Cancer).”–Victor M. Nigon.

E. Dougherty was deeply influenced by George Beadle's results on the biochemical genetics of Neurospora and developed the project to study nutritional mutants, in an animal where the morphology allowed little morphological variation by mutation. He greatly contributed to the nomenclature and development of culture methods of small animals.

He was a strong advocate of using Rhabditis as model organisms (Dougherty and Calhoun, 1948; Dougherty, 1949). Some of his obvious contributions to present-day C. elegans research are: description of R. briggsae with NIgon (Dougherty and Nigon, 1949); axenic cultivation of R. briggsae (Dougherty, 1950); raising Caenorhabditis from a subgenus to a genus (Dougherty, 1953); performing the first genetic analysis of C. briggsae with Nigon (Nigon and Dougherty, 1950) and of C. elegans Bergerac x N2 with Helene Fatt (Fatt and Dougherty, 1963); and sending N2 to Sydney Brenner (Dougherty, 1963).

Jean-Louis Brun (1927-1981)

Jean Brun's early youth was spent in La Chapelle-de-Mardore, a small village in the Beaujolais region in France. He came from a modest family. His father, a farmer, died from an old World War I wound when Jean Brun and his older brother were young boys. His mother remarried and moved to Lyon, where Jean Brun went to school and was found to excel. Pushed by his teachers, and against his mother's wish that he would work, he continued until the high school degree. Then, needing to work, he combined being a school supervisor in Roanne with studying at the University in Lyon, which he reached through a long bike ride. He met his future wife Geneviève at the university and both joined VM Nigon's laboratory in 1949. While she and others worked for their diplomas on Ascaris, Jean Brun started a project on C. elegans, the species he was to continue with all his life. He obtained an assistant position at the University of Lyon in 1949.

Jean Brun first studied gametogenesis of C. elegans, especially chromosome pairing in meiotic prophase. He then described gametogenesis defects at high temperature, including chromosomal abnormalities and cell fusions leading to polyploidy. He experimented with the effects of high temperature on fertility, which led him to the discovery of the progressive decline in fertility over several generations at intermediate temperatures. From there, he patiently adapted the Bergerac strain to 24.5°C, with 0.5°C temperature steps and after hundreds of generations. Through many thorough experiments of temperature reversions, he was led to the conclusion that this adaptation may not be of conventional genetic nature (see main text). Jean Brun was very involved and dedicated to his work. He obtained his “Doctorat d'Etat” in March 1966 with his work on C. elegans temperature adaptation.

Jean Brun and Ellsworth Dougherty were then both interested in developing C. elegans genetics. They had planned for the former to come to Berkeley with his family for the 1966-1967 academic year–a project sadly interrupted by Dougherty's untimely death in December 1965.

From 1967, Jean Brun developed in Lyon an independent research team focused on C. elegans biology and genetics. The team isolated the first mutants of C. elegans (see main text). Jean Brun became an Assistant Professor and became involved in the direction of the academic part of the Biology Department of the University.

The rise of C. elegans through Sydney Brenner, with his scientific aura, was obviously of great importance for the Brun team. They participated in the budding C. elegans scientific community through Worm Breeder Gazettes and the first 1977 Worm Meeting in Woods Hole, MA. Jean Brun and his wife visited David Hirsh in Boulder, CO (in 1976 or 1977). In the later years, Jean Brun developed cancer, yet kept working and following the progress of his team until his death in 1981. Members of the team finished their ongoing studies and published until 1985, then dispersed to other laboratories.