Dnmt1 mutant ants develop normally but have disrupted oogenesis

ABSTRACT Although DNA methylation is an important gene regulatory mechanism in mammals, its function in arthropods remains poorly understood. Studies in eusocial insects have argued for its

role in caste development by regulating gene expression and splicing. However, such findings are not always consistent across studies, and have therefore remained controversial. Here we use

CRISPR/Cas9 to mutate the maintenance DNA methyltransferase DNMT1 in the clonal raider ant, _Ooceraea biroi_. Mutants have greatly reduced DNA methylation, but no obvious developmental

phenotypes, demonstrating that, unlike mammals, ants can undergo normal development without DNMT1 or DNA methylation. Additionally, we find no evidence of DNA methylation regulating caste

development. However, mutants are sterile, whereas in wild-type ants, DNMT1 is localized to the ovaries and maternally provisioned into nascent oocytes. This supports the idea that DNMT1

plays a crucial but unknown role in the insect germline. SIMILAR CONTENT BEING VIEWED BY OTHERS CANALIZED GENE EXPRESSION DURING DEVELOPMENT MEDIATES CASTE DIFFERENTIATION IN ANTS Article

Open access 03 October 2022 CASTE-SPECIFIC EXPRESSIONS AND DIVERSE ROLES OF _TAKEOUT_ GENES IN THE TERMITE _RETICULITERMES SPERATUS_ Article Open access 24 May 2023 COMPARISON OF GENE

EXPRESSION PROFILES AMONG CASTE DIFFERENTIATIONS IN THE TERMITE _RETICULITERMES SPERATUS_ Article Open access 13 July 2022 INTRODUCTION How epigenetic processes regulate development and

behavior is a major area of research1. Among the most prominent epigenetic mechanisms is DNA methylation, a covalent modification to cytosine that gives rise to 5-methylcytosine. This

modification is catalyzed by two types of enzymes, de novo DNA methyltransferases (DNMT3) and maintenance DNA methyltransferases (DNMT1). While DNMT3 primarily targets previously

unmethylated cytosines, DNMT1 mostly copies DNA methylation during DNA replication2. In most organisms, the majority of cytosine methylation occurs in a CpG context, i.e., a cytosine base

followed by a guanine3. However, the presence and amount of CpG methylation is highly variable across organisms4,5. The function of DNA methylation has mostly been studied in mammals, where

it is primarily targeted to cis-regulatory elements, transposons, and gene bodies3,6. Promoter and transposon methylation has important functions in gene regulation and usually acts to

downregulate or silence expression, also in the context of genomic imprinting7,8,9,10. However, across the tree of life, DNA methylation can play different roles11. Honeybees and ants

possess full complements of the DNA methylation machinery12,13,14,15, which has been met with considerable excitement for two main reasons6,16,17,18,19,20,21,22,23,24,25,26. First, the

commonly used invertebrate genetic models yeast, _Caenorhabditis elegans_, and _Drosophila melanogaster_, lack parts of the machinery and, therefore, CpG methylation, opening the possibility

that social insects could serve to better understand the role of DNA methylation in invertebrates, which remains poorly known4. Second, social insects show extreme forms of developmental

and behavioral plasticity, and it has been argued that DNA methylation might play important roles in caste development26,27,28,29, the regulation of behavioral roles6,29,30,31, and the

evolution of genomic imprinting as a result of social conflicts32,33. Several studies have reported correlations between DNA methylation patterns and queen vs. worker castes12,34,35, while

others have attempted experimental manipulations and reported effects on caste development or behavior either via changes in gene expression or alternative splicing36,37,38. While these

studies have greatly contributed to our understanding of DNA methylation in social insects, others have failed to find consistent correlations between DNA methylation and reproductive

castes, and some of the core functional results have not been replicated13,29,39. Also, DNA methylation in insects is preferentially found in the exons of constitutively expressed and

evolutionarily conserved housekeeping genes, which seems to contradict the conjecture that it is associated with dynamic gene regulation13,40,41,42. Finally, the few functional studies of

DNA methylation in social insects have been limited to pharmacological manipulations or RNAi knockdowns of DNA methyltransferases36,37,38, calling for additional studies that employ more

definitive molecular genetics approaches. Here, we study the role of DNA methylation in the clonal raider ant, _Ooceraea biroi_. Unlike honeybees and most ants, _O. biroi_ does not have

queens, and all workers in a colony reproduce asexually and clonally43,44. Therefore, mutant lines can be established from any individual with germline transmission of natural or

experimentally induced mutations45. At the same time, there still is phenotypic plasticity along the worker-queen spectrum among workers of this species, with smaller “regular workers” with

two ovarioles and no eyespots, and larger, more queen-like “intercastes” with four to six ovarioles and rudimentary eyespots46,47. Using CRISPR-mediated mutagenesis of _DNMT1_, we show that

ants deficient in DNA methylation still develop normally and do not show obvious alterations to caste phenotypes. However, these ants are sterile, adding to recent studies in other species

suggesting that DNMT1 plays an important yet poorly understood role during insect oogenesis48,49,50,51,52. RESULTS AND DISCUSSION DNMT1 copies DNA methylation patterns during DNA

replication2, and enzyme loss-of-function should result in genome-wide loss of CpG methylation. The _O. biroi DNMT1_ gene (NCBI GenBank, Gene ID: 105286975) is composed of 16 exons (Fig. 

1A), and RNA sequencing (RNA-seq) data show that the short first exon is alternatively spliced (Supplementary Fig. 1). We therefore designed two CRISPR guide RNAs, one to induce frameshift

mutations in the second exon (DNMT1g1), and one to target exon 11 of _DNMT1_, just upstream of a conserved residue in the catalytic domain that is essential for enzyme function in mice

(DNMT1g2) (Fig. 1A)53. EARLY FRAMESHIFTS IN DNMT1 DO NOT LEAD TO LOSS OF FUNCTION We first injected 5152 freshly laid eggs with Cas9 enzyme and the DNMT1g1 guide45, and recovered two unique

stable mutant lines in which both alleles were frameshifted (Supplementary Fig. 1A). Unexpectedly, low-coverage whole-genome sequencing of bisulfite-treated DNA (WGBS) showed that DNA

methylation levels in these mutants were indistinguishable from wild-type ants (Supplementary Fig. 1B), implying that the DNA methylation function of DNMT1 was not affected. Both mutant

strains showed no obvious phenotypic abnormalities, produced viable eggs at normal rates (Supplementary Fig. 1C) and had normal lifespans (Supplementary Fig. 1D). Whole-body RNA-seq of

individuals from one of the two lines (−7bp/−8bp) and matched wild-type controls found no differences in _DNMT1_ expression. Additionally, even though the genomic frameshift mutations were

reflected in RNA-seq reads, we did not observe any major alternative splicing events in mutated _DNMT1_ (Supplementary Fig. 1F). Taken together, this suggests that gene function can be

rescued when _DNMT1_ is frameshifted in early exons. Even though we do not know the exact rescue mechanism in this case, gene rescue is a known problem in functional genetic studies, and can

occur in a number of ways, including translation reinitiation or undetected alternative splicing/exon skipping54,55. These mutants nevertheless provide a useful control in the context of

the current study, because they demonstrate that we can mutate the _DNMT1_ gene with CRISPR/Cas9 reagents without causing adverse fitness effects or other non-specific phenotypes if the

mutation does not measurably compromise the function of the DNMT1 enzyme. DNMT1 LOSS-OF-FUNCTION MUTANTS HAVE REDUCED DNA METHYLATION Across two experiments, we then injected 5643 young eggs

with Cas9 enzyme and the DNMT1g2 guide to generate 33 G0 adults. Because G0 adults have not inherited mutations via the germline, they can be genetic mosaics. However, Sanger sequencing and

subsequent analyses (see below and Supplementary Table 1) showed no evidence of wild-type alleles in seven G0 females and one G0 male, whereas one G0 female had both mutant and wild-type

alleles. We were not able to obtain genotypes for three of the 33 G0s, but one of them was confirmed later as a loss-of-function mutant via immunohistochemistry and WGBS (see below). Because

we could not establish stable lines of DNMT1g2 mutants (see below), this female and the seven females without detected wild-type alleles were used in all subsequent experiments. Individual

details for these eight animals are given in Supplementary Table 1, and we confirmed several mutant alleles with small insertions and deletions at the target site via Sanger sequencing (Fig.

 1B). The remaining G0 adults were wild-type females and served as matched experimental controls. To assess the effect of these mutations on DNA methylation, we extracted DNA from whole

bodies (minus ovaries and brains) of four mutants (Supplementary Table 1, individuals #5-8) and four wild-type controls and conducted low-coverage WGBS. The mutants had greatly reduced

average genome-wide DNA methylation levels (0.26%; 95% CI: 0.21–0.31%) compared to the wild-types (1.02%; 95% CI: 0.90–1.24%) (Fig. 1C). We then conducted high-coverage WGBS for two of these

mutants and two of the wild-types, achieving 14X coverage of the genome on average and greater than 99% sodium bisulfite conversion rates (Supplementary Table 2). The striking reduction in

DNA methylation was constant across all genomic features, regardless of their location in relation to genes, repeats or transposons (Fig. 1D–F, Supplementary Fig. 2). As expected, DNMT1 loss

of function thus results in a substantial reduction in DNA methylation. These data are consistent with the expected function of DNMT1. The remaining low methylation levels in DNMT1g2

mutants could be due to a number of possible mechanisms, including the enzymatic activity of DNMT3 or signal stemming from cells that did not divide after injection of Cas9. No wild-type

reads were detected at the _DNMT1_ target site in the G0 DNMT1g2 mutants with high-coverage WGBS data, providing additional evidence that mosaicism in G0s is low (Supplementary Table 3).

DNMT1 LOSS-OF-FUNCTION MUTANTS ARE NOT MORE QUEEN-LIKE In mammals, DNA methylation is vital to multiple aspects of development, including genomic imprinting56. Accordingly, experiments that

interfere with DNMT function result in DNA replication arrest57,58 and embryo lethality59,60. This contrasts with our findings in _O. biroi_, where both males and females (Fig. 2A) complete

development despite DNMT1 loss-of-function and significantly reduced DNA methylation. This suggests that the roles of DNMT1 and DNA methylation during development differ fundamentally

between mammals and insects. However, the effects of DNA methylation on insect development could be more subtle, and previous work on ants36 and honeybees37 suggested that reduced DNA

methylation modulates caste development and results in larger adults. In contrast to these findings, morphometric analyses of DNMT1 loss-of-function mutants showed that their morphology is

similar to wild-type ants that were reared under identical conditions, and that their overall body size is possibly smaller on average (Fig. 2B, Supplementary Fig. 3). This finding

contradicts the notion that DNA methylation increases in response to a reduced diet or other environmental factors during development27 and then mediates molecular changes that result in

minor worker rather than major worker development in ants36, or worker rather than queen development in honeybees37. However, we currently cannot rule out the alternative possibility that

the role of DNMT1 and DNA methylation in caste development differs between the clonal raider ant, which lacks extreme caste polymorphism, and other eusocial Hymenoptera. DNMT1

LOSS-OF-FUNCTION MUTANTS ARE STERILE To assess the effect of DNMT1 loss-of-function on survival and fertility, we monitored a cohort of eight G0 adults from eclosion onward. By 60 days, we

had collected four of the G0s shortly after they had died, two had likely died but carcasses could not be found (the ants sometimes dismember dead nestmates), and two remained alive. Sanger

sequencing of DNA from whole body extracts showed that the four individuals that had died were DNMT1 mutants with no evidence of wild-type alleles (Supplementary Table 1, #1-4), while the

two surviving individuals were wild type with no evidence of mutant alleles. The survival of DNMT1 mutants was significantly decreased relative to wild-type controls (Fig. 2C). To put this

finding into context, median survival of wild-type _O. biroi_ under similar rearing conditions was 258 days, with a minimum survival of 106 days among 21 individuals (Supplementary Fig. 1D).

This shows that loss-of-function mutations in _DNMT1_ severely compromise longevity. During the 60 days of this experiment, we collected 19 G1 eggs produced by the G0 adults and genotyped

them at the _DNMT1_ target locus. All these eggs were wild type. This is in stark contrast to the genotypes of G0 adults (Fig. 2D, Supplementary Table 1 #1-4) and implies that DNMT1

loss-of-function mutations result in sterility. That sterility is caused by the injection of CRISPR/Cas9 reagents or DNMT1 mutations per se is unlikely, because G0 adults mutated with the

DNMT1g1 guide RNA did not show this phenotype. This opens the possibility that the decreased lifespan of DNMT1 mutants is not a direct consequence of reduced DNA methylation levels, but

rather a result of the compromised reproductive system, for example by disrupting endocrine functions. To better understand the putative role of DNMT1 in reproduction, we characterized

_DNMT1_ mRNA and protein in the ant ovary. Each of the two ovaries of an _O. biroi_ ant is composed of one to three ovarioles (two to six ovarioles per ant), which can be subdivided into a

vitellarium, germarium and the terminal filament (Fig. 3A), similar to ovarioles of other insects61. In the ovarioles, each oocyte originates in the germarium, and travels to the

vitellarium, where it is surrounded by follicular cells and an adjacent bundle of nurse cells (Fig. 3A). Nurse cells are derived from the same progenitor cell as the associated oocyte and

are therefore of germline origin. Using mRNA fluorescence in situ hybridization (FISH), we detected _DNMT1_ mRNA in the germarium, as well as the nurse cells and oocytes within the

vitellarium (Fig. 3B, Supplementary Fig. 4). The broad expression of _DNMT1_ in _O. biroi_ ovaries is consistent with qPCR data from _Solenopsis invicta_ fire ants, showing that _DNMT1_ is

expressed in ovaries62. We then stained ovaries from wild-type and mutant _O. biroi_ using a commercial antibody against the conserved catalytic domain of mammalian DNMT1. Consistent with

the mRNA FISH pattern, we observed DNMT1 protein within the nuclei of cells in the germarium, nurse cells, follicular cells and oocytes (Fig. 3C, Supplementary Fig. 4). Positive and specific

staining was apparent in wild-type ants and DNMT1g1 mutants, but not in the ovaries of three DNMT1g2 mutants (Fig. 3C, Supplementary Fig. 5). The positive staining in DNMT1g1 mutants

confirms that, despite the early frameshifts, they still produce some form of functional DNMT1 protein, highlighting the importance of independently assessing the effects of targeted

mutagenesis on gene function. The lack of staining in DNMT1g2 mutants (Supplementary Table 1, individuals #5-8), on the other hand, validates the specificity of the antibody, and confirms

that mutations in exon 11 indeed result in gene knockout. Furthermore, all mutant ovaries revealed young follicles (Fig. 3C, Supplementary Fig. 5), suggesting that DNMT1 exerts its crucial

function after the initiation of oogenesis. However, even though sample sizes are limited, we never observed fully formed oocytes in mutant ovaries, consistent with the absence of mutant G1

eggs. Given that meiosis in _O. biroi_ is completed only after oviposition44, this suggests that oocyte maturation and meiosis cannot progress without DNMT1 function. The finding that DNMT1

is required for oogenesis in _O. biroi_ is consistent with recent findings in the milkweed bug _Oncopeltus fasciatus_49,52,63 and the red flour beetle _Tribolium castaneum_50, where maternal

RNAi knockdown of _DNMT1_ leads to sterility or early developmental arrest. Interestingly, this opens the possibility of a DNMT1 function independent of DNA methylation26,50. Based on these

findings, we asked whether DNMT1 is present during early oogenesis. Indeed, we observed DNMT1 protein in oocytes within the ovary at multiple stages of development. In very early oocytes,

which are several weeks from maturing, DNMT1 is confined to the nucleus (Fig. 3D, top). As oocytes mature, DNMT1 staining also becomes apparent as large puncta at the periphery of the egg

that do not appear to be associated with DNA (Fig. 3D, bottom). Together, these experiments show that both DNMT1 protein and mRNA are maternally provisioned into the oocyte at an early stage

and support the idea that maternal DNMT1 plays a crucial role in oocyte maturation. While we cannot rule out the possibility that DNA methylation directly regulates gene expression and

alternative splicing in some contexts, our finding that _O. biroi_ workers and males develop normally without a functional _DNMT1_ gene and with greatly reduced DNA methylation levels shows

that, unlike in mammals, this mechanism is not fundamentally required during ant embryogenesis. Instead, the data presented here align with recent evidence that DNMT1 plays a conserved and

crucial, yet poorly understood role in insect reproduction48,49,50,51,52. Importantly, this role might be independent of DNA methylation50,63. DNA methylation-independent functions of DNMT1

have in fact been observed outside of insects. In the African clawed frog _Xenopus laevis_, for example, DNMT1 acts as a direct transcription repressor protein to prevent premature

activation of gene expression before the mid-blastula stage64. Such DNA methylation-independent functions of DNMT1 could help explain why the enzyme is highly conserved over evolutionary

time4,65, and why it has been retained even in some insects that do not methylate their DNA50. Given its potentially broad relevance for reproductive health across the animal tree of life,

future work should aim to better understand the role of DNMT1 during oogenesis. The fact that DNMT1 loss-of-function mutants in the clonal raider ant are sterile and therefore cannot be

propagated led to limitations of the current study. First, with only few mutant individuals available, we had to work with small sample sizes and had to restrict the scope of our

experiments. Second, we had to work with G0 individuals, i.e., individuals that were mutagenized at the egg stage and therefore might have been mosaics of mutant alleles, together with

wild-type alleles at low frequencies. These limitations could be overcome in the future, e.g. by working with genetically modified strains in which loss of DNMT1 function is inducible.

Finally, while we focused our efforts on DNMT1, it remains possible that DNMT3 is involved in regulating caste development via de novo DNA methylation at specific target sites. Studies

targeting DNMT3 for loss-of-function could help resolve this question. METHODS MUTAGENESIS TARGET SELECTION AND AMPLIFICATION The _DNMT1_ (LOC105286975) gene locus was located in the

published _O. biroi_ genome (GenBank assembly accession: GCA_003672135.1)44. Two alternate splice variants (X1: XM_011352329, X2: XM_011352333) were identified, and aligned with MAFFT66,

verifying gRNA sequence presence in both splice variants. Primers were designed to amplify ~200–300 bp fragments surrounding each target cut site (Supplementary Table 4). GENOTYPING AND

MUTANT IDENTIFICATION Two _O. biroi_ clonal lines, Line A and Line B43 were used for this study. All experimental individuals belonged to Line B, and Line A individuals were used as

chaperones to rear eggs or larvae. When necessary, genotyping to distinguish between Line A and B was carried out using a restriction enzyme assay of PCR amplicons of the mitochondrial

cytochrome oxidase subunit 1 (CO1) gene45. Mutants were identified via Sanger sequencing of PCR amplified DNA fragments containing either the DNMT1g1 or DNMT1g2 target locus (Supplementary

Table 4) using a previously described protocol45. Sanger sequencing was outsourced to the company Psomagen. GRNA DESIGN AND REAGENT VALIDATION Guide RNAs (gRNAs) were designed using the

CRISPR Design Tool (Synthego). For each target, four custom gRNAs were purchased from the company Synthego and tested using an in vitro incubation assay with Cas9 enzyme using PCR amplified

and purified DNA from the target region. Reactions were incubated for 4 h at 37 °C, and products were visualized on 2% agarose gels45. All gRNAs successfully digested target DNA, and a

single gRNA was selected for each gene target for further experiments (Supplementary Table 5). CRISPR/CAS9 REAGENT PREPARATION, MUTAGENESIS AND ANIMAL REARING Reagent preparation, egg

collection, injection, incubation and animal rearing followed a previously published protocol for this species45. The CRISPR/Cas9 injection reagent mix included 100 ng/µl Cas9 (PNABio) and

100 ng/µl of the selected gRNA (Synthego) (Supplementary Table 5). DNMT1G1 CRISPR/CAS9 DNMT1G1 MUTANT GENERATION AND LINE PROPAGATION 5152 Line B eggs were injected. From those, 76 larvae

hatched, and 65 of these G0 larvae were fostered. All G0 adults were pooled in a single colony, which was supplemented with adults from clonal Line A. Eggs from this colony were collected

weekly or biweekly and fostered into nests containing 20 Line A chaperones, where they were reared to adulthood. The eclosing callows were individually paint marked45 and pooled into six

units of 16 ants each. All eggs produced by these units were collected weekly or biweekly and fostered with Line A chaperones to propagate the lines. Mutants resulting from these units were

identified via Sanger sequencing of the target region and pooled to generate pure colonies for each unique mutant genotype. DNMT1G1 MUTANT REPRODUCTION AND SURVIVAL From the first four

paint-marked G1 units, eggs were collected biweekly for 2.5 weeks and incubated for 48 h, before they were frozen dry on a glass slide at −80 °C for later DNA extraction and genotyping45.

All eggs produced after this were removed weekly and fostered with Line A chaperones as described above. All carcasses were removed from the units and frozen dry in a PCR tube at −80 °C

until no ants remained. Some carcasses could not be recovered, probably because ants in the colony had dismembered them. For the analysis of reproductive output, only adults (Line A and Line

B) and the first 25 eggs (Line A and Line B) sequenced from each unit where mutant adults were observed were included in the analysis (_n_ = 3 units). For analysis of survival, DNA was

extracted from all frozen adult carcasses for genotyping, and all data from Line A individuals were discarded. Statistical analyses were conducted in GraphPad Prism (v 9.1.1). DNMT1G2

CRISPR/CAS9 DNMT1G2 MUTANT GENERATION, REPRODUCTION AND SURVIVAL We injected 2416 Line B eggs, of which 45 hatched. 39 larvae were fostered to yield 9 G0 adults that survived beyond three

days after eclosion. One of these adults was a male and was excluded from further analyses. All DNMT1g2 G0 animals from this experiment were placed in a single unit, along with paint-marked

Line A chaperone ants to ensure colony stability. All eggs and carcasses were removed from the colony weekly or biweekly and frozen dry at −80 °C on a glass slide or in a PCR tube,

respectively. Four carcasses and 24 eggs were collected, and DNA for Sanger sequencing was extracted from whole carcasses and eggs. Additionally, at 61 days, the two remaining G0 ants were

sacrificed, ovaries were removed, and DNA was extracted from the remaining tissue. Of the collected eggs, 19 were Line B wild type, four were Line A, and one could not be successfully

genotyped and was removed from analyses. No mutant eggs were observed in this experiment. Therefore, unlike for DNMT1g1, we were unable to propagate the mutant lines for DNMT1g2, and all

experiments were thus limited to G0 adults. GraphPad Prism (v 9.1.1) was used for statistical analyses. DNMT1G2 MUTANT GENERATION, MORPHOMETRICS, IMMUNOHISTOCHEMISTRY AND WHOLE GENOME

BISULFITE SEQUENCING We injected 3227 eggs in a separate experiment, of which 109 hatched. 92 larvae were fostered to yield four colonies of G0 adults. A single leg was removed from each

adult, and DNA was extracted for Sanger sequencing. One of these colonies was excluded from analyses because all six G0s were wild-type. The remaining three colonies generated 18 G0 adults,

of which four carried only mutant alleles and 14 carried only wild-type alleles. In parallel, 574 eggs were incubated without injection and reared under identical conditions to yield 17

control adults. At ~1 week of age, all G0 animals (injected and uninjected) were immobilized in a standardized position (Fig. 2A) under acrylic and imaged using a brightfield microscope

(Leica MSV266) for morphometric measurements. A ruler was imaged under identical conditions for accurate calibration. Morphometric measurements of individual body segments were taken using

Fiji67. All mutants and a subset of wild types were dissected, and their ovaries and brains fixed for immunohistochemistry (next section). After the ovaries and head had been removed, the

rest of the body was used for DNA extraction and WGBS (see below). FLUORESCENCE IN SITU HYBRIDIZATION Tissue was fixed, prepared and processed according to a previously published protocol68.

Briefly, to prepare the tissue, ovaries were dissected and fixed in cold 4% paraformaldehyde in PBS for at least 2 h at 4 °C, following an EtOH dehydration and incubation overnight. Tissue

was rehydrated and incubated in 5% acetic acid for 5 min, followed by another fixation using 2% paraformaldehyde for 1 h at 25 °C. The tissue was washed with 0.5% Tween 20 in PBS and 1%

NaBH4. Prepared tissue was rinsed before incubation with the pre-hybridization solution for 30 min at 45 °C, and was then incubated with 2pmol of the probes in probe hybridization solution

for 48 h. Signal was amplified with 30 pmol snap cooled hairpin solution at room temperature. A set of 30 custom probes were designed for _DNMT1_ and purchased from Molecular Instruments

Inc. The probes used a B2 HCR amplifier and were labeled with Alexa Fluor 546. Images were captured with an inverted LSM 780 laser scanning confocal microscope (Zeiss). Images were processed

in parallel with Fiji67 and are shown as maximum projection z-stacks. IMMUNOHISTOCHEMISTRY Tissue was dissected in cold 1xPBS and fixed in 4%PFA for 2 h. Following washes in 1xPBS, samples

were blocked using a solution containing 5% normal goat serum in PBSTx for 1 h. The blocking solution was replaced with a primary antibody solution containing 1:100 DNMT1 antibody (Abcam

ab188453), and incubated overnight at room temperature. Negative controls without primary antibody were incubated overnight in the blocking solution at room temperature in parallel. All

samples were washed and incubated in the secondary solution including 1:500 secondary antibody (Donkey anti-rabbit, AlexaFluor 594, Invitrogen A21207) and 1:500 DAPI for 2 h prior to

mounting in DAKO fluorescence mounting medium. Images were captured and processed as described above. WHOLE GENOME BISULFITE SEQUENCING DNA EXTRACTION, LIBRARY CONSTRUCTION AND SEQUENCING

Genomic DNA was extracted from four mutant and four wild-type replicates of single whole ants (DNMT1g1) or the remaining tissue after brain and ovary dissection (DNMT1g2) using the QIAamp

DNA Micro Kit (QIAGEN). While we were able to establish stable lines for DNMT1g1 mutants and thus extract DNA from whole animals, our sample sizes for DNMT1g2 mutants were necessarily

limited, and we therefore extracted DNA after brains and ovaries had been removed for other analyses. In each case, all replicates were sequenced to evaluate global methylation changes (Fig.

 1C, Supplementary Fig. 1B, 2). Additionally, two of the DNMT1g2 mutant and matched wild-type replicates were selected for more extensive analysis of methylation changes (Fig. 1D–F) using

high coverage sequencing (Supplementary Table 2). MethylC-seq libraries were constructed using the MethylC-seq protocol69. Briefly, genomic DNA was sonicated to around 200 bp using a Covaris

S-series focused ultrasonicator, and end-repaired with an End-It DNA end-repair kit (Epicentre). The end-repaired DNA was subjected to A-tailing using Klenow 3′–5′ exo − (NEB) and ligated

to methylated adapters using T4 DNA ligase (NEB). The ligated DNA was treated with sodium bisulfite reagent using the EZ DNA methylation-Gold kit and amplified using KAPA HiFi uracil + 

Readymix Polymerase. Sequencing was performed on an Illumina NextSeq500 instrument. METHYLOME MAPPING The MethylC-seq data from the Georgia Genomics & Bioinformatics Core were processed

with the “paired-end-pipeline” function in Methylpy70. Reads passing quality control filters were aligned to the _O. biroi v5.4_ reference genome71 using bowtie 2.2.4 (Langmead and Salzberg,

2012), and the uniquely aligned and nonclonal reads were retained. Unmethylated lambda phage DNA was used as a control to calculate the sodium bisulfite conversion rate of unmethylated

cytosines. A binomial test was used to determine the methylation status of cytosines with a minimum coverage of five reads. ANALYSIS OF DNA METHYLATION Protein coding genes (_n_ = 11,868)

and TEs/repeats over 100 bp in size (_n_ = 53,158) were used for the plots. For the metaplots in Fig. 1E, genes or TEs/repeats along with 1 kb upstream/downstream regions were divided into

20 bins, and the average methylation level of each bin was displayed. For Fig. 1F, exons, introns, and 1 kb upstream/downstream regions were divided into 20 bins, and the average methylation

level of each bin was plotted. For the genome-wide methylation analysis shown in Supplementary Fig. 2, the average percentage of CG methylation in each 50 Kb window was calculated and

plotted onto a chromosomal map of the _O. biroi_ genome. For the read level analysis in Supplementary Table 3, the output BAM files generated by Methylpy70 were used to distinguish wild-type

from mutant reads. Each BAM file was visualized with Samtools tview72, and mapped mutant and wild-type reads were counted for each sample. RNA SEQUENCING RNA EXTRACTION, LIBRARY

CONSTRUCTION AND SEQUENCING RNA was extracted and eluted in 30 µl RNAse free water from whole ants dry frozen at −80 °C using the RNeasy Mini Kit (QIAGEN Cat. No. 74104). 1 ng of total RNA

was used to generate full length cDNA using Clontech’s SMART-Seq v4 Ultra Low Input RNA Kit (Cat # 634888). 1 ng of cDNA was then used to prepare libraries using the Illumina Nextera XT DNA

sample preparation kit (Cat # FC-131-1024). Libraries with unique barcodes were pooled at equal molar ratios and sequenced on an Illumina NextSeq 500 sequencer to generate 150 bp paired-end

reads, following the manufacturer’s protocol (Cat # 15048776 Rev.E). ANALYSIS OF RNA SEQUENCING DATA Sequencing reads were trimmed using Trimmomatic73 Nextera PE adapters. Trimmed read

quality was verified using FastQC, and aligned using STAR74 to the _O. biroi v5.4_ genome and converted to BAM files with Samtools72. BAM files were loaded into Integrative Genomics Viewer75

to visualize read alignments at the mutation site and alternative splicing of the _DNMT1_ gene. Gene counts were determined with HTseq76. Normalization and differential gene expression

analysis were carried out in R using DEseq277. STATISTICS AND REPRODUCIBILITY All microscopy images in this paper are representative of multiple examined specimens. The gross anatomy of the

_O. biroi_ ovary shown in Fig. 3A was observed in 12 samples. The FISH staining shown in Fig. 3B and Supplementary Fig. 4A was consistent across 8 experimental animals and 4 negative

controls. The DNMT1 antibody staining shown in Fig. 3C and Supplementary Fig. 4B was consistently observed in 14 experimental animals and 9 negative controls. The DNMT1 antibody staining of

DNMT1g1 mutants shown in Fig. 3C was replicated in 6 animals, including both mutant genotypes. DNMT1 antibody staining is shown for 3 different DNMT1g2 mutants in Fig. 3C and Supplementary

Fig. 5A, B. The DNMT1 antibody staining pattern in oocytes shown in Fig. 3D was consistent across 13 young and 10 old oocytes from multiple animals. Statistical analyses for other

experiments are described in the respective Methods and main text sections, as well as in the figure legends. REPORTING SUMMARY Further information on research design is available in the 

Nature Portfolio Reporting Summary linked to this article. DATA AVAILABILITY Whole genome bisulfite sequencing data are available at GenBank/NCBI under accession number GSE182212.

RNA-sequencing data are available at GenBank / NCBI under accession number PRJNA780766. All other data are available as Source Data files as part of this publication. Source data are

provided with this paper. CODE AVAILABILITY All custom code used for analyses is deposited in the following GitHub repository:

https://github.com/schmitzlab/Methylome-of-clonal-ant_Obir-v5.4.git. REFERENCES * Allis, C. D. et al. _Epigenetics_ 2nd edn (Cold Spring Harbor Laboratory Press, 2015). * Law, J. A. &

Jacobsen, S. E. Establishing, maintaining and modifying DNA methylation patterns in plants and animals. _Nat. Rev. Genet._ 11, 204–220 (2010). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Feng, S. et al. Conservation and divergence of methylation patterning in plants and animals. _Proc. Natl. Acad. Sci. U.S.A._ 107, 8689–8694 (2010). Article  ADS  CAS  PubMed 

PubMed Central  Google Scholar  * Bewick, A. J., Vogel, K. J., Moore, A. J. & Schmitz, R. J. Evolution of DNA methylation across insects. _Mol. Biol. Evol._ 34, 654-665 (2017). * Bewick,

A. J. et al. On the origin and evolutionary consequences of gene body DNA methylation. _Proc. Natl. Acad. Sci. U.S.A._ 113, 9111–9116 (2016). Article  ADS  CAS  PubMed  PubMed Central 

Google Scholar  * Yan, H. et al. DNA methylation in social insects: how epigenetics can control behavior and longevity. _Annu. Rev. Entomol._ 60, 435–452 (2015). Article  CAS  PubMed  Google

Scholar  * Moore, L. D., Le, T. & Fan, G. DNA methylation and its basic function. _Neuropsychopharmacology_ 38, 23–38 (2013). Article  CAS  PubMed  Google Scholar  * Razin, A. &

Riggs, A. DNA methylation and gene function. _Science_ 210, 604–610 (1980). Article  ADS  CAS  PubMed  Google Scholar  * Smith, Z. D. & Meissner, A. DNA methylation: roles in mammalian

development. _Nat. Rev. Genet._ 14, 204–220 (2013). Article  CAS  PubMed  Google Scholar  * Edwards, J. R., Yarychkivska, O., Boulard, M. & Bestor, T. H. DNA methylation and DNA

methyltransferases. _Epigenetics Chromatin_ 10, 23 (2017). Article  PubMed  PubMed Central  Google Scholar  * Schmitz, R. J., Lewis, Z. A. & Goll, M. G. DNA methylation: shared and

divergent features across eukaryotes. _Trends Genet._ 35, 818–827 (2019). Article  CAS  PubMed  PubMed Central  Google Scholar  * Bonasio, R. et al. Genome-wide and caste-specific DNA

methylomes of the ants _Camponotus floridanus_ and _Harpegnathos saltator_. _Curr. Biol._ 22, 1755–1764 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Libbrecht, R., Oxley,

P. R., Keller, L. & Kronauer, D. J. C. Robust DNA methylation in the clonal raider ant brain. _Curr. Biol._ 26, 391–395 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  *

Wang, Y. et al. Functional CpG methylation system in a social insect. _Science_ 314, 645–647 (2006). Article  ADS  CAS  PubMed  Google Scholar  * Schaefer, M. & Lyko, F. DNA methylation

with a sting: an active DNA methylation system in the honeybee. _Bioessays_ 29, 208–211 (2007). Article  CAS  PubMed  Google Scholar  * Bonasio, R. The expanding epigenetic landscape of

non-model organisms. _J. Exp. Biol._ 218, 114–122 (2015). Article  PubMed  PubMed Central  Google Scholar  * Drewell, R. A., Lo, N., Oxley, P. R. & Oldroyd, B. P. Kin conflict in insect

societies: a new epigenetic perspective. _Trends Ecol. Evol._ 27, 367–373 (2012). Article  PubMed  Google Scholar  * Glastad, K. M., Hunt, B. G., Yi, S. V. & Goodisman, M. A. D. DNA

methylation in insects: on the brink of the epigenomic era. _Insect Mol. Biol._ 20, 553–565 (2011). Article  CAS  PubMed  Google Scholar  * Glastad, K. M., Chau, L. M. & Goodisman, M. A.

D. in _Advances in Insect Physiology_ Vol. 48, (eds Zayed, A. & Kent, C. F.) 227–269 (Elsevier, 2015). * Li-Byarlay, H. The function of DNA methylation marks in social insects. _Front.

Ecol. Evol._ 4, (2016). * Lyko, F. & Maleszka, R. Insects as innovative models for functional studies of DNA methylation. _Trends Genet._ 27, 127–131 (2011). Article  CAS  PubMed  Google

Scholar  * Maleszka, R. Epigenetic code and insect behavioural plasticity. _Curr. Opin. Insect Sci._ 15, 45–52 (2016). Article  PubMed  Google Scholar  * Patalano, S., Hore, T. A., Reik, W.

& Sumner, S. Shifting behaviour: epigenetic reprogramming in eusocial insects. _Curr. Opin. Cell Biol._ 24, 367–373 (2012). Article  CAS  PubMed  Google Scholar  * Weiner, S. A. &

Toth, A. L. Epigenetics in social insects: a new direction for understanding the evolution of castes. _Genet. Res. Int._ 2012, 609810 (2012). PubMed  PubMed Central  Google Scholar  * Welch,

M. & Lister, R. Epigenomics and the control of fate, form and function in social insects. _Curr. Opin. Insect Sci._ 1, 31–38 (2014). Article  PubMed  Google Scholar  * Yan, H. et al.

Eusocial insects as emerging models for behavioural epigenetics. _Nat. Rev. Genet._ 15, 677–688 (2014). Article  CAS  PubMed  Google Scholar  * Maleszka, R. Epigenetic integration of

environmental and genomic signals in honey bees: the critical interplay of nutritional, brain and reproductive networks. _Epigenetics_ 3, 188–192 (2008). Article  PubMed  Google Scholar  *

Vaiserman, A. Developmental epigenetic programming of caste-specific differences in social insects: an impact on longevity. _Curr. Aging Sci._ 7, 176–186 (2014). Article  Google Scholar  *

Patalano, S. et al. Molecular signatures of plastic phenotypes in two eusocial insect species with simple societies. _Proc. Natl. Acad. Sci. U.S.A._ 112, 13970–13975 (2015). Article  ADS 

CAS  PubMed  PubMed Central  Google Scholar  * Lattorff, H. M. G. & Moritz, R. F. A. Genetic underpinnings of division of labor in the honeybee (_Apis mellifera_). _Trends Genet._ 29,

641–648 (2013). Article  CAS  PubMed  Google Scholar  * Opachaloemphan, C., Yan, H., Leibholz, A., Desplan, C. & Reinberg, D. Recent advances in behavioral (epi)genetics in eusocial

insects. _Annu. Rev. Genet._ 52, 489–510 (2018). Article  CAS  PubMed  PubMed Central  Google Scholar  * Alaux, C. et al. Honey bee aggression supports a link between gene regulation and

behavioral evolution. _Proc. Natl. Acad. Sci. U.S.A._ 106, 15400–15405 (2009). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar  * Lonsdale, Z. et al. Allele specific expression and

methylation in the bumblebee, _Bombus terrestris_. _PeerJ_ 5, e3798 (2017). Article  PubMed  PubMed Central  Google Scholar  * Elango, N., Hunt, B. G., Goodisman, M. A. D. & Yi, S. V.

DNA methylation is widespread and associated with differential gene expression in castes of the honeybee, _Apis mellifera_. _Proc. Natl. Acad. Sci. U.S.A._ 106, 11206–11211 (2009). Article 

ADS  CAS  PubMed  PubMed Central  Google Scholar  * Lyko, F. et al. The honey bee epigenomes: differential methylation of brain DNA in queens and workers. _PLoS Biol._ 8, e1000506 (2010).

Article  PubMed  PubMed Central  Google Scholar  * Alvarado, S., Rajakumar, R., Abouheif, E. & Szyf, M. Epigenetic variation in the _Egfr_ gene generates quantitative variation in a

complex trait in ants. _Nat. Commun._ 6, 6513 (2015). Article  ADS  CAS  PubMed  Google Scholar  * Kucharski, R., Maleszka, J., Foret, S. & Maleszka, R. Nutritional control of

reproductive status in honeybees via DNA methylation. _Science_ 319, 1827–1830 (2008). Article  ADS  CAS  PubMed  Google Scholar  * Li-Byarlay, H. et al. RNA interference knockdown of _DNA

methyl-transferase 3_ affects gene alternative splicing in the honey bee. _Proc. Natl. Acad. Sci. U.S.A._ 110, 12750–12755 (2013). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

* Herb, B. R. et al. Reversible switching between epigenetic states in honeybee behavioral subcastes. _Nat. Neurosci._ 15, 1371–1373 (2012). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Hunt, B. G., Glastad, K. M., Yi, S. V. & Goodisman, M. A. D. Patterning and regulatory associations of DNA methylation are mirrored by histone modifications in insects.

_Genome Biol. Evol._ 5, 591–598 (2013). Article  PubMed  PubMed Central  Google Scholar  * Hunt, B. G., Glastad, K. M., Yi, S. V. & Goodisman, M. A. D. The function of intragenic DNA

methylation: insights from insect epigenomes. _Integr. Comp. Biol._ 53, 319–328 (2013). Article  CAS  PubMed  Google Scholar  * Sarda, S., Zeng, J., Hunt, B. G. & Yi, S. V. The evolution

of invertebrate gene body methylation. _Mol. Biol. Evol._ 29, 1907–1916 (2012). Article  CAS  PubMed  Google Scholar  * Kronauer, D. J. C., Pierce, N. E. & Keller, L. Asexual

reproduction in introduced and native populations of the ant _Cerapachys biroi_. _Mol. Ecol._ 21, 5221–5235 (2012). Article  PubMed  Google Scholar  * Oxley, P. R. et al. The genome of the

clonal raider ant _Cerapachys biroi_. _Curr. Biol._ 24, 451–458 (2014). Article  CAS  PubMed  PubMed Central  Google Scholar  * Trible, W. et al. _orco_ mutagenesis causes loss of antennal

lobe glomeruli and impaired social behavior in ants. _Cell_ 170, 727–735.e10 (2017). Article  CAS  PubMed  PubMed Central  Google Scholar  * Ravary, F. & Jaisson, P. Absence of

individual sterility in thelytokous colonies of the ant _Cerapachys biroi_ Forel (Formicidae, Cerapachyinae). _Insectes Soc._ 51, 67–73 (2004). Article  Google Scholar  * Teseo, S., Châline,

N., Jaisson, P. & Kronauer, D. J. C. Epistasis between adults and larvae underlies caste fate and fitness in a clonal ant. _Nat. Commun._ 5, 3363 (2014). Article  ADS  PubMed  Google

Scholar  * Arsala, D., Wu, X., Yi, S. V. & Lynch, J. A. Dnmt1a is essential for gene body methylation and the regulation of the zygotic genome in a wasp. _PLoS Genet._ 18, e1010181

(2022). Article  CAS  PubMed  PubMed Central  Google Scholar  * Bewick, A. J. et al. _Dnmt1_ is essential for egg production and embryo viability in the large milkweed bug, _Oncopeltus

fasciatus_. _Epigenetics Chromatin_ 12, 6 (2019). Article  PubMed  PubMed Central  Google Scholar  * Schulz, N. K. E. et al. _Dnmt1_ has an essential function despite the absence of CpG DNA

methylation in the red flour beetle _Tribolium castaneum_. _Sci. Rep._ 8, 16462 (2018). Article  ADS  PubMed  PubMed Central  Google Scholar  * Ventós-Alfonso, A., Ylla, G., Montañes, J.-C.

& Belles, X. DNMT1 promotes genome methylation and early embryo development in cockroaches. _iScience_ 23, 101778 (2020). Article  ADS  PubMed  PubMed Central  Google Scholar  *

Washington, J. T. et al. The essential role of Dnmt1 in gametogenesis in the large milkweed bug _Oncopeltus fasciatus_. _eLife_ 10, e62202 (2021). Article  CAS  PubMed  PubMed Central 

Google Scholar  * Damelin, M. & Bestor, T. H. Biological functions of DNA methyltransferase 1 require its methyltransferase activity. _Mol. Cell. Biol._ 27, 3891–3899 (2007). Article 

CAS  PubMed  PubMed Central  Google Scholar  * Zhang, Q. et al. CRISPR-Cas9 gene editing causes alternative splicing of the targeting mRNA. _Biochem. Biophys. Res. Commun._ 528, 54–61

(2020). Article  CAS  PubMed  PubMed Central  Google Scholar  * Smits, A. H. et al. Biological plasticity rescues target activity in CRISPR knock outs. _Nat. Methods_ 16, 1087–1093 (2019).

Article  CAS  PubMed  Google Scholar  * Li, E. & Zhang, Y. DNA methylation in mammals. _Cold Spring Harb. Perspect. Biol._ 6, a019133–a019133 (2014). Article  PubMed  PubMed Central 

Google Scholar  * Brown, K. D. & Robertson, K. D. DNMT1 knockout delivers a strong blow to genome stability and cell viability. _Nat. Genet._ 39, 289–290 (2007). Article  CAS  PubMed 

Google Scholar  * Unterberger, A., Andrews, S. D., Weaver, I. C. G. & Szyf, M. DNA methyltransferase 1 knockdown activates a replication stress checkpoint. _Mol. Cell. Biol._ 26,

7575–7586 (2006). Article  CAS  PubMed  PubMed Central  Google Scholar  * Li, E., Bestor, T. H. & Jaenisch, R. Targeted mutation of the DNA methyltransferase gene results in embryonic

lethality. _Cell_ 69, 915–926 (1992). Article  CAS  PubMed  Google Scholar  * Okano, M., Bell, D. W., Haber, D. A. & Li, E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de

novo methylation and mammalian development. _Cell_ 99, 247–257 (1999). Article  CAS  PubMed  Google Scholar  * Dearden, P. Germ cell development in the honeybee (_Apis mellifera_); _Vasa_

and _Nanos_ expression. _BMC Dev. Biol._ 6, 6 (2006). Article  PubMed  PubMed Central  Google Scholar  * Kay, S., Skowronski, D. & Hunt, B. G. Developmental DNA methyltransferase

expression in the fire ant _Solenopsis invicta_. _Insect Sci._ 25, 57–65 (2018). Article  CAS  PubMed  Google Scholar  * Amukamara, A. U. et al. More than DNA methylation: does pleiotropy

drive the complex pattern of evolution of _Dnmt1_? _Front. Ecol. Evol._ 8, 4 (2020). Article  Google Scholar  * Dunican, D. S., Ruzov, A., Hackett, J. A. & Meehan, R. R. xDnmt1 regulates

transcriptional silencing in pre-MBT _Xenopus_ embryos independently of its catalytic function. _Development_ 135, 1295–1302 (2008). Article  CAS  PubMed  Google Scholar  * Bhattacharyya,

M., De, S. & Chakrabarti, S. Origin and evolution of DNA methyltransferases (DNMT) along the tree of life: a multi-genome survey. https://doi.org/10.1101/2020.04.09.033167 (2020). *

Katoh, K., Rozewicki, J. & Yamada, K. D. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. _Brief. Bioinform._ 20, 1160–1166 (2019).

Article  CAS  PubMed  Google Scholar  * Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. _Nat. Methods_ 9, 676–682 (2012). Article  CAS  PubMed  Google

Scholar  * Fetter-Pruneda, I. et al. An oxytocin/vasopressin-related neuropeptide modulates social foraging behavior in the clonal raider ant. _PLoS Biol._ 19, e3001305 (2021). Article  CAS

  PubMed  PubMed Central  Google Scholar  * Urich, M. A., Nery, J. R., Lister, R., Schmitz, R. J. & Ecker, J. R. MethylC-seq library preparation for base-resolution whole-genome

bisulfite sequencing. _Nat. Protoc._ 10, 475–483 (2015). Article  CAS  PubMed  PubMed Central  Google Scholar  * Schultz, M. D. et al. Human body epigenome maps reveal noncanonical DNA

methylation variation. _Nature_ 523, 212–216 (2015). Article  ADS  CAS  PubMed  PubMed Central  Google Scholar  * McKenzie, S. K. & Kronauer, D. J. C. The genomic architecture and

molecular evolution of ant odorant receptors. _Genome Res._ 28, 1757–1765 (2018). Article  CAS  PubMed  PubMed Central  Google Scholar  * Li, H. et al. The sequence alignment/map format and

SAMtools. _Bioinformatics_ 25, 2078–2079 (2009). Article  PubMed  PubMed Central  Google Scholar  * Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina

sequence data. _Bioinformatics_ 30, 2114–2120 (2014). Article  CAS  PubMed  PubMed Central  Google Scholar  * Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. _Bioinformatics_ 29,

15–21 (2013). Article  CAS  PubMed  Google Scholar  * Robinson, J. T. et al. Integrative genomics viewer. _Nat. Biotechnol._ 29, 24–26 (2011). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Anders, S., Pyl, P. T. & Huber, W. HTSeq—a Python framework to work with high-throughput sequencing data. _Bioinformatics_ 31, 166–169 (2015). Article  CAS  PubMed  Google

Scholar  * Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. _Genome Biol._ 15, 550 (2014). Article  PubMed  PubMed

Central  Google Scholar  Download references ACKNOWLEDGEMENTS The authors thank Waring Trible and Taylor Hart for advice on egg injections and the CRISPR/Cas9 protocol, and Rebecca Timson

and Vikram Chandra for advice on immunohistochemistry and bioinformatics analyses, respectively. RNA library preparation and sequencing was conducted at the Rockefeller University Genomics

Resource Center. Confocal microscopy was conducted at the Rockefeller University Bio-Imaging Resource Center. Research reported in this publication was supported by the National Institute of

General Medical Sciences of the National Institutes of Health under award no. R35GM127007 to D.J.C.K. The content is solely the responsibility of the authors and does not necessarily

represent the official views of the National Institutes of Health. This work was also supported by a Faculty Scholar Award from the Howard Hughes Medical Institute to D.J.C.K. I.I. was

supported by a Medical Scientist Training Program grant from the National Institute of General Medical Sciences of the National Institutes of Health under award no. T32GM007739 to the Weill

Cornell/Rockefeller/Sloan Kettering Tri-Institutional MD-PhD Program. H.J. and R.J.S. were supported by the National Science Foundation under award no. MCB-1856143. D.J.C.K. is an

investigator of the Howard Hughes Medical Institute. This is Clonal Raider Ant Project paper #21. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Laboratory of Social Evolution and Behavior,

The Rockefeller University, New York, NY, USA Iryna Ivasyk, Leonora Olivos-Cisneros, Stephany Valdés-Rodríguez, Marie Droual & Daniel J. C. Kronauer * Howard Hughes Medical Institute,

New York, NY, USA Stephany Valdés-Rodríguez & Daniel J. C. Kronauer * Department of Genetics, University of Georgia, Athens, GA, USA Hosung Jang & Robert J. Schmitz Authors * Iryna

Ivasyk View author publications You can also search for this author inPubMed Google Scholar * Leonora Olivos-Cisneros View author publications You can also search for this author inPubMed 

Google Scholar * Stephany Valdés-Rodríguez View author publications You can also search for this author inPubMed Google Scholar * Marie Droual View author publications You can also search

for this author inPubMed Google Scholar * Hosung Jang View author publications You can also search for this author inPubMed Google Scholar * Robert J. Schmitz View author publications You

can also search for this author inPubMed Google Scholar * Daniel J. C. Kronauer View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS I.I. and

D.J.C.K. conceived and designed the research. I.I., L.O.C., S.V.R., and M.D. performed CRISPR experiments. I.I. and L.O.C. performed histology and microscopy experiments. H.J. and R.J.S.

performed whole genome bisulfite sequencing and data analysis. I.I. performed all other experiments. I.I. and D.J.C.K. wrote the paper, with feedback from L.O.C., H.J., and R.J.S. All

authors approved the final version. D.J.C.K. supervised the project. CORRESPONDING AUTHORS Correspondence to Iryna Ivasyk or Daniel J. C. Kronauer. ETHICS DECLARATIONS COMPETING INTERESTS

The authors declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Communications_ thanks the anonymous reviewers for their contribution to the peer review of this work.

ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION REPORTING SUMMARY SOURCE DATA SOURCE DATA RIGHTS AND PERMISSIONS OPEN ACCESS This article is licensed under a Creative Commons Attribution 4.0 International

License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source,

provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons

license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by

statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit

http://creativecommons.org/licenses/by/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Ivasyk, I., Olivos-Cisneros, L., Valdés-Rodríguez, S. _et al._ DNMT1 mutant ants

develop normally but have disrupted oogenesis. _Nat Commun_ 14, 2201 (2023). https://doi.org/10.1038/s41467-023-37945-4 Download citation * Received: 24 November 2021 * Accepted: 06 April

2023 * Published: 18 April 2023 * DOI: https://doi.org/10.1038/s41467-023-37945-4 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable

link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative