Cellular innovations at the origin of new reproductive modes: the case of pseudogamy in nematodes Marie Delattre Plasticité et Evolution de la Division Cellulaire ENS Lyon Marie-Anne Félix Evolution des Caenorhabditis ENS Paris Manon Grosmaire (PhD) Caroline Launay (IE CNRS)
Diversity of reproductive strategies Sexual/ Amphimixy Asexual/ Parthenogenesis Genetic mixing + Cost associated with the production of males and search for males No genetic mixing + No Cost associated with the production of males and search for males
What are the cellular and developmental innovations that allow the emergence of new reproductive strategies? Amphimixy Parthenogenesis Oocytes Spermatozoa At fertilization, meiosis resumes The sperm provides the centrosomes. (polarizes the embryos)
What are the cellular and developmental innovations that allow the emergence of new reproductive strategies? Amphimixy Parthenogenesis Oocytes Spermatozoa At fertilization, meiosis resumes In the absence of sperm: -oocyte activation? -cell polarization -de novo formation of centrosomes -deal with ploidy The sperm provides the centrosomes. (polarizes the embryos)
Soil nematodes represent a diversity of reproductive modes o Non parasite, non pathogenic o Short life cycle o Feed on E. coli in the lab o Can be frozen
Soil nematodes represent a diversity of reproductive modes o Non parasite, non pathogenic o Short life cycle o Feed on E. coli in the lab o Can be frozen
Soil nematodes represent a diversity of reproductive modes Rhabditidae family (wormbook.org) Male/female species (gonochoristic) Self-fertilizing hermaphrodites Parthenogenesis C. elegans Pseudogamy
In pseudogamous species, fertilization is necessary but the male DNA is not inherited= sexual parasitism Amphimixy Pseudogamy Oocytes Spermatozoa Spermatozoa At fertilization, meiosis resumes Sperm cells of other species are used for oocyte activation (and provide centrosomes?) The sperm provides the centrosomes. (polarizes the embryos)
Pseudogamy in soil nematodes: the case of Mesorhabditis belari
Pseudogamy in soil nematodes: the case of Mesorhabditis belari o o o o Population with females and males The sex ratio is extremely biased: ~ 10% of males in the population Males are necessary for reproduction, but their DNA is not systematically utilized Partial pseudogamy
Pseudogamy in Mesorhabditis belari 2 categories of embryos are produced Amphimixy Pseudogamy Oocytes are blocked in prometaphase of Meiosis I Homologous chromosomes are prematurely separated Meiosis I : homologous chromosomes separate Sister chromatids separate Meiosis II : sister chromatids separate? Second Meiosis aborts or only one division of meiosis 2 polar bodies, 2 haploid pronuclei 1 polar body, 1 single diploid pronucleus 50% males, 50% females 100% females
Pseudogamy in Mesorhabditis belari The sex ratio reflects the execution of female meiosis Amphimixy Pseudogamy 20% 80% Oocytes are blocked in prometaphase of Meiosis I Homologous chromosomes are prematurely separated 2 polar bodies, 2 haploid pronuclei 1 polar body, 1 single diploid pronucleus 10% males, 10% females 80% females At low temperature: More males Ageing of the mother: Fewer males
Pseudogamous species of the Mesorhabditis genus o Males are necessary for reproduction, but their DNA is not systematically utilized o The sex ratio is extremely biased: ~ 10% of males in the population o Combines the disadvantage/advantages of sexual and asexual reproduction? o Mesorhabditis species (gonochoristic and pseudogamous) are very abundant in the soil!
Questions o What are the cellular mechanisms: o At the origin of the stochasticity of meiosis/ and the influence of the environment? o Allowing the control of male DNA in response to female meiosis progression? -> New mechanisms of chromatin regulation
Questions o Evolution of pseudogamy within the Mesorhabditis genus: o Single or multiple origin of pseudogamy? o Different optimization of the sex ratio in different pseudogamous species? o Preconditioning in male/female closely related species? Male/female species 2 Male/female species 1 Pseudogamous species 1 Pseudogamous species 2 Male/female species
Questions o Evolution of pseudogamy within the Mesorhabditis genus: o Single or multiple origin of pseudogamy? o Different optimization of the sex ratio in different pseudogamous species? o Preconditioning in male/female closely related species? Male/female species 2 Pseudogamous species 2 Pseudogamous species 1 Male/female species 1 Male/female species -> evolution of new reproductive strategies
Projects o Objective 1: Establishment of a collection of species and phylogenetic reconstruction
Projects o Objective 1: Establishment of a collection of species and phylogenetic reconstruction o We have established a collection of 22 strains ( 20 new ones, isolated in the past few months), 14 pseudogamous and 8 true male/female
Phylogenetic relationship between Mesorhabditis strains Sequencing of ITS, 18S and 28S RNA Genetic crosses between strains Morphological description JU2846; JU2765 JU2871; JU2755 JU2854 DF5017 JU2855, JU2887, JU2888, JU2889 JU2870 JU2847 JU2848 PS1179 JU2864 JU2890, JU2858 JU2817, JU2856, JU2859 C. elegans N2 Male/female species Pseudogamous species
Phylogenetic relationship between Mesorhabditis strains Sequencing of ITS, 18S and 28S RNA Genetic crosses between strains Morphological description JU2846, JU2765 JU2871, JU2755 JU2854 M. longespiculosa DF5017 Short tail M. monhystera JU2855, JU2887, JU2888, JU2889 M. franseni JU2870 M. litoralis JU2847 Long tail M. vernalis JU2848 M. microbursaris PS1179 M. sp. JU2864 M. paucipapollata JU2890, JU2858 Short tail M. belari JU2817, JU2856, JU2859 C. elegans N2
Phylogenetic relationship between Mesorhabditis strains 8 pseudogamous species, one common ancestor We isolated 3 strains of M. belari (Cambridge, Orsay, Angles/Anglin) Pseudogamy allows speciation JU2846, JU2765 JU2871, JU2755 JU2854 M. longespiculosa DF5017 Short tail M. monhystera JU2855, JU2887, JU2888, JU2889 M. franseni JU2870 M. litoralis JU2847 Long tail M. vernalis JU2848 M. microbursaris PS1179 M. sp. JU2864 M. paucipapollata JU2890, JU2858 Short tail M. belari JU2817, JU2856, JU2859 C. elegans N2
Phylogenetic relationship between Mesorhabditis strains Males from pseudogamous species have a reduced size, reduced copulatory apparatus, reduced sperm size and reduced sperm motility JU2846, JU2765 JU2871, JU2755 JU2854 M. longespiculosa DF5017 M. monhystera JU2855, JU2887, JU2888, JU2889 M. franseni JU2870 M. litoralis JU2847 M. longespiculosa DF5017 M. vernalis JU2848 M. microbursaris PS1179 M. sp. JU2864 M. paucipapollata JU2890, JU2858 M. belari JU2817, JU2856, JU2859 C. elegans N2 M. belari JU2817
Phylogenetic relationship between Mesorhabditis strains Males from pseudogamous species have a reduced size, reduced copulatory apparatus, reduced sperm size and reduced sperm motility JU2846, JU2765 JU2871, JU2755 JU2854 M. longespiculosa DF5017 M. monhystera JU2855, JU2887, JU2888, JU2889 M. franseni JU2870 M. litoralis JU2847 M. longespiculosa DF5017 M. vernalis JU2848 M. microbursaris PS1179 M. sp. JU2864 M. paucipapollata JU2890, JU2858 M. belari JU2817, JU2856, JU2859 C. elegans N2 M. belari JU2817
Characterization of M. belari JU2817 Influence of the age of the mother and of the temperature on the sex ratio 25 20 20 C, n=3884 15 10 5 0 D1 D2 D3 D4 D5 D6 D7 D8 Adult Day 8
Characterization of M. belari JU2817 Influence of the age of the mother and of the temperature on the sex ratio 25 20 25 C, n=3575 15 20 C, n=3884 10 5 16 C, n=1916 0 Adult D1 D2 D3 D4 D5 D6 D7 D8 Day 8 young females produce more males in JU2817 at lower temperature, the sex ratio is not increased in JU2817
Characterization of M. belari JU2817 Influence of the temperature and of the abundance of females on the mating success 120 100 37 18 15 31 32 19 80 60 28 15 40 15 20 9 8 0 20 C -> 20 C 25 C -> 25 C 16 C -> 16 C 20 C -> 20 C 25 C -> 25 C 16 C -> 16 C 20 C -> 25 C 20 C -> 16 C 20 C -> 20 C 25 C -> 25 C 16 C -> 16 C 1 female x 2 males 5 females x 2 males 10 females x 2 males Crosses rarely occur when females are singled on plates with males
Characterization of M. belari JU2817 Amphimixic embryos Pseudogamous embryos 10 embryos gave 100% males 49 embryos gave 100% females ~16% of amphimixic embryos at 20 C Amphimixic embryos may produce only males (in contrast to Nigon s results)
Characterization of M. belari JU2817 Analysis of meiotic figures in female and male gonads Distal gonad: proliferative germ cells Uterus(fertilized embryo) Maturing oocytes Spermatheca Vulva
Characterization of M. belari JU2817 Analysis of meiotic figures in female and male gonads (analysis of chromosome pairing, segregation etc. by analogy of mechanisms described in C. elegans) Mitotic zone Pachytene Transition zone Mitotic zone Transition zone Diplotene Pachytene Diplotene 10 Bivalents
Perspectives Analysis of known markers of DNA decondensation and premature separation of homologous chromosomes in JU2817 (by analogy with C. elegans) Identification of other factors influencing the sex ratio in JU2817 (food, starvation, abundance on plates ) Is there different optimization of the sex ratio in different genetic background? Measure of the sex ratio (depending on age and temperature) in the two other strains of M. belari, and other pseudogamous species Are Mesorahbditis male/female species also characterized by an incomplete meiosis (preadaptation in the genus)? Analysis of meiotic figures in non-pseudogamous species of Mesorhabditis
Projects o Objective 2: Establishment of genetic tools to study the cellular origin of pseudogamy Our goal is to identify genes involved in the control of sex ratio Control of female meiosis progression Control of paternal DNA
Projects o Objective 2: Establishment of genetic tools to study the cellular origin of pseudogamy Mutagenesis on JU2817 and screen for high/low incidence of males QTL mapping between two strains of M. belari displaying different sex ratio Comparative transcriptomic on both categories of embryos (easy to sort out) Amphimixic, 2 pronuclei Pseudogamous, 1 pronucleus
Projects o Objective 2: Establishment of genetic tools to study the cellular origin of pseudogamy DNA and RNA sequencing of JU2817 and de novo assembly of the genome (sequencing in progress- collaboration with Blaxter lab/edimburgh) Re-sequencing of other M. belari strains, to identify SNP markers (will be used to identify the causative mutations, once mutants are isolated)
Strategy to identify causative mutations: detection of SNP by NGS SNP based strategy EMS based strategy Review in Zuryn and Jarriault 2013
A pilot screen in JU2817 to identify visible markers EMS mutagenesis of an entire population 400 F1 males Isolation of single F1 males Cross with WT females Mating between F2 siblings Isolation of F3, homozygous mutants
Conclusion: Evolution of pseudogamy in the Mesorhabditis genus New mechanisms of chromatin regulation? Unique mechanisms to control the sex ratio? Pseudogamous species Male/female species C. elegans What is the consequence of this reproductive mode on the genetic structure of the populations?