BackgroundThe function of cytosine (DNA) methylation in insects remains inconclusive due to a lack of mutant and/or genetic studies.ResultsHere, we provide evidence for the functional role of the maintenance DNA methyltransferase 1 (Dnmt1) in an insect using experimental manipulation. Through RNA interference (RNAi), we successfully posttranscriptionally knocked down Dnmt1 in ovarian tissue of the hemipteran Oncopeltus fasciatus (the large milkweed bug). Individuals depleted for Dnmt1, and subsequently DNA methylation, failed to reproduce. Eggs were inviable and declined in number, and nuclei structure of follicular epithelium was aberrant. Erasure of DNA methylation from gene or transposon element bodies did not reveal a direct causal link to steady-state mRNA levels in somatic cells. These results reveal an important function of Dnmt1 seemingly not contingent on directly controlling gene expression.ConclusionsThis study provides direct experimental evidence for a functional role of Dnmt1 in egg production and embryo viability and uncovers a trivial role, if any, for DNA methylation in control of gene expression in O. fasciatus.Electronic supplementary materialThe online version of this article (10.1186/s13072-018-0246-5) contains supplementary material, which is available to authorized users.

DNA methylation is an important chromatin modification that can stably alter gene expression in cells and maintain genome integrity in plants and vertebrates. The function of DNA methylation outside of these well-studied systems, however, is unclear. Insects, in particular, represent an understudied group. Variation in the level of DNA methylation and gains and losses in the maintenance methyltransferase, DNMT1, across the insect tree of life suggests that there is much we don't understand about DMNT1 function and evolution. One constant across the studies examining patterns of Dnmt1 expression in insects is that expression is consistently high in reproductive tissues compared to somatic tissue. The explanation for this has been that DNMT1 is required in tissues that have high levels of cell division. Our previous study found that downregulation of Dnmt1 expression in the milkweed bug Oncopeltus fasciatus results in the expected reduction of DNA methylation, no global changes in gene expression reflecting changes in DNA methylation, and the loss of the ability to produce viable oocytes. Here, we show that females treated with ds-Dnmt1 RNA during larval development have a more extreme phenotype; they lack oocytes entirely but develop a normal somatic ovary. Our results indicate a specific role for DNMT1 in the formation of gametes and are consistent with data from other systems, including Tribolium castaneum, a species does not have DNA methylation. We propose that DNMT1 has multiple functional roles in addition to methylating DNA, which explains its complex patterns of evolution.

9DNA methylation is an important chromatin modification that can stably alter gene expression in 10 cells and maintain genome integrity in plants and vertebrates. The function of DNA methylation 11 outside of these well-studied systems, however, is unclear. Insects, in particular, represent an 12 understudied group. Variation in the level of DNA methylation and gains and losses in the 13 maintenance methyltransferase, DNMT1, across the insect tree of life suggests that there is much we 14don't understand about DMNT1 function and evolution. One constant across the studies examining 15 patterns of Dnmt1 expression in insects is that expression is consistently high in reproductive tissues 16 compared to somatic tissue. The explanation for this has been that DNMT1 is required in tissues that 17have high levels of cell division. Our previous study found that downregulation of Dnmt1 expression 18 in the milkweed bug Oncopeltus fasciatus results in the expected reduction of DNA methylation, no 19global changes in gene expression reflecting changes in DNA methylation, and the loss of the ability 20 to produce viable oocytes. Here, we show that females treated with ds-Dnmt1 RNA during larval 21 development have a more extreme phenotype; they lack oocytes entirely but develop a normal 22 somatic ovary. Our results indicate a specific role for DNMT1 in the formation of gametes and are 23 consistent with data from other systems, including Tribolium castaneum, a species does not have 24 DNA methylation. We propose that DNMT1 has multiple functional roles in addition to methylating 25 DNA, which explains its complex patterns of evolution, and suggests that previous inferences of 26 causation from associations are premature. 27

The function of cytosine (DNA) methylation in insects remains unknown. 9Dnmt1 led to reduced egg viability, fecundity, and aberrant follicular epithelium, and thus 3 0 failure to produce a successive generation. Despite finding levels of methylated CG 3 1 (mCG) within coding regions reduced by 83.55%, we found no evidence for DNA 3 2 methylation directly affecting transcription. Our results suggest Dnmt1 plays an 3 3 important role in reproduction in O. fasciatus that is mediated by a gene-regulatory 3 4 independent function of DNA methylation. Oncopeltus fasciatus represents a fruitful 3 5 model species for functional studies of DNA methylation, and continuation of studies in 3 6 this system will unravel the insect epigenome and its functional consequences. 7To assess the function of Dnmt1, double-stranded RNA (dsRNA) targeting Dnmt1

Oncopeltus fasciatus males fed the ancestral diet of milkweed seeds prioritize reproduction over lifespan as evidenced by higher rates of fertility and shorter lifespans than males from the same population fed the adapted diet of sunflower seeds. We examined the proximate mechanisms by which milkweed‐fed males maintained late‐life fertility. We tested the hypothesis that older milkweed‐fed males maintained fertility by producing more, higher quality sperm. Our results, that older males have more sperm, but their sperm do not have higher viability, are in general agreement with other recent studies on how nutrition affects male fertility in insects. We further examined the mechanisms by which sperm are produced by examining the progression of spermatogonial cells through the cell cycle during the transit amplification divisions. We demonstrated that diet affects the likelihood of a spermatocyst being in the S‐phase or M‐phase of the cell cycle. Given work in model systems, these results have implications for subtle effects on sperm quality either through replication stress or epigenetic markers. Thus, viability may not be the best marker for sperm quality and more work is called for on the mechanisms by which the germline and the production of sperm mediate the cost of reproduction.