DNA Methylation and DNA Methyltransferases in the Clonal Raider Ant, Ooceraea biroi

DNA methylation and DNA methyltransferase enzymes (DNMTs) are ubiquitous and predate the origin of eukaryotes. In animals, DNA methylation primarily is carried out by DNMT1, which targets hemi – methylated DNA and maintains methylation patterns through cycles of cell replication, and DNMT3, the de novo methyltransferase. These genes are essential to mammalian development, and mutations lead to embryonic (DNMT1 and DNMT3b) or post – natal (DNMT3a) lethality. Studies of DNA methylation and DNMTs in invertebrates have been limited to non – traditional model organisms because Caenorhabditis elegans and Drosophila melanogaster lack the DNMT enzymes, and their DNA has no detectable methylation. In other invertebrates, global DNA methylation levels are generally much lower than those of mammals and largely concentrated in exons of protein coding genes. In social insects, some studies have argued that DNA methylation regulates social behavior or caste differentiation, but others have challenges this idea and it remains controversial. To further understand the DNMT enzymes, we used the clonal raider ant, Ooceraea biroi, a tractable model organism with robust DNA methylation. We utilized CRISPR/Cas9 to mutate the DNMT genes (DNMT1 and DNMT3) and generate four unique mutants (two targeting different regions of DNMT1, one targeting DNMT3 and a DNMT1/DNMT3 double mutant). In the DNMT1 catalytic domain mutant we observed a drastic drop in global levels of DNA methylation as well as reproductive sterility and increased mortality. We did not observe any reproductive or DNA methylation phenotypes in the other DNMT1 mutant or the DNMT3 mutant. Furthermore, in both of these mutants, we observed faithful transcription of the frameshift mutations in aligned mRNA reads, but did not observe any differential gene expression compared to wildtypes. Recently, studies have come to light regarding CRISPR/Cas9 mutagenesis inducing mechanisms of genomic plasticity, such as alternative splicing or translation reinitiating, which ultimately can rescue gene function. It is possible that we did not detect any phenotypes in these DNMT1 and DNMT3 mutants due to such a mechanism, leading to normal gene function despite successful mutagenesis. The sterility we observed as a result of DNMT1 catalytic domain mutagenesis is consistent with growing work demonstrating that DNMT1 plays an essential, possibly methylation – independent, role during oogenesis in insects. To further evaluate this phenomenon, we characterized DNMT1 mRNA and protein in the ant ovary using fluorescence in situ hybridization and immunohistochemistry. We found DNMT1 present in somatic cells within the ovary, in addition to being maternally provisioned into oocytes early in development. Furthermore, we observed developing oocytes in the ovaries of DNMT1 catalytic domain mutants, indicating that this gene is not essential for the initiation or early stages of oogenesis. Our findings demonstrate that unlike in mammals, normal development after DNMT1 inhibition is possible in insects. However, DNMT1 is essential for longevity and progression of insect oogenesis. Further work to understand the precise mechanism of DNMT1 involvement in oogenesis, and potentially meiosis, may shed light on its evolutionary role and why it has been conserved across so many forms of life