Given the importance of DNA methylation in protection of the genome against transposable elements and transcriptional regulation in other taxonomic groups, the diversity in both levels and patterns of DNA methylation in the insects raises questions about its function and evolution. We show that the maintenance DNA methyltransferase, DNMT1, affects meiosis and is essential to fertility in milkweed bugs, Oncopeltus fasciatus, while DNA methylation is not required in somatic cells. Our results support the hypothesis that Dnmt1 is required for the transition of germ cells to gametes in O. fasciatus and that this function is conserved in male and female gametogenesis. They further suggest that DNMT1 has a function independent of DNA methylation in germ cells. Our results raise the question of how a gene so critical in fitness across multiple insect species can have diverged widely across the insect tree of life.

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

While DNA methylation is an important chromatin modification in many groups of organisms, the function of DNA methylation within the insects is unclear. The taxonomic distribution of DNA methyltransferase genes in insects is highly variable, as is the presence of methylated genomes. In the large milkweed bug, Oncopeltus fasciatus, we have shown the maintenance methyltransferase Dnmt1 is required for oocyte production but this appears to be unrelated to methylation given that demethylating somatic cells causes no loss of somatic cell function. One hypothesis is that Dnmt1 is affecting meiosis. Here we used RNAi to downregulate Dnmt1 in males at two stages where meiosis is occurring; during testis development and in adults replenishing sperm stores following sperm depletion. We found that downregulation of Dnmt1 in stages where meiosis is required resulted in the greatest disruption to spermatogenesis. Our results support the hypothesis that Dnmt1 is required for the transition of germ cells to gametes in O. fasciatus and that this function is conserved in male and female gametogenesis. In addition, the role of Dnmt1 was specific to the germ cells. Downregulation of Dnmt1 across all tissues resulted in a germline-specific phenotype. These results suggest that the reduction of methylation has a phenotype restricted to the germ cells. Our results raise the question of how a gene so critical in fitness across multiple insect species can have diverged widely across the insect tree of life.Significance StatementGiven the importance of DNA methylation in protection of the genome against transposable elements and transcriptional regulation in other taxonomic groups, the diversity in both levels and patterns of DNA methylation in the insects raises questions about its function and evolution. We show that the maintenance DNA methyltransferase, DNMT1, affects meiosis and is essential to fertility in milkweed bugs, Oncopeltus fasciatus, while DNA methylation is not required in somatic cells. Our results suggest that DNMT1 has a function independent of DNA methylation in germ cells. The evolutionary lability of a gene with such a fundamental fitness activity suggests that the function Dnmt1 in germ cell development is easily lost or replaced.

T3 and T4 DNA ligase have contrasting performance in ligase-catalysed oligonucleotide polymerisations.