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.

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.

Males have the ability to compete for fertilizations through both precopulatory and postcopulatory intrasexual competition. Precopulatory competition has selected for large weapons and other adaptations to maximize access to females and mating opportunities, while postcopulatory competition has resulted in ejaculate adaptations to maximize fertilization success. Negative associations between these strategies support the hypothesis that there is a trade‐off between success at pre‐ and postcopulatory mating success. Recently, this trade‐off has been demonstrated with experimental manipulation. Males of the leaf‐footed cactus bug Narnia femorata use hind limbs as the primary weapon in male–male competition. However, males can drop a hind limb to avoid entrapment. When this autotomy occurs during development, they invest instead in large testes. While evolutionary outcomes of the trade‐offs between pre‐ and postcopulatory strategies have been identified, less work has been done to identify proximate mechanisms by which the trade‐off might occur, perhaps because the systems in which the trade‐offs have been investigated are not ones that have the molecular tools required for exploring mechanism. Here, we applied knowledge from a related model species for which we have developmental knowledge and molecular tools, the milkweed bug Oncopeltus fasciatus , to investigate the proximate mechanism by which autotomized N . femorata males developed larger testes. Autotomized males had evidence of a higher rate of transit amplification divisions in the spermatogonia, which would result more spermatocytes and thus in greater sperm numbers. Identification of mechanisms underlying a trade‐off can help our understanding of the direction and constraints on evolutionary trajectories and thus the evolutionary potential under multiple forms of selection.

Males have the ability to compete for fertilizations through both pre-copulatory and post-copulatory intrasexual competition. Pre-copulatory competition has selected for large weapons and other adaptations to maximize access to females and mating opportunities while post-copulatory competition has resulted in ejaculate adaptations to maximize fertilization success. Negative associations between these strategies support the hypothesis that there is a trade-off between success at pre- and post-copulatory mating success. Recently, this trade-off has been demonstrated with experimental manipulation. Male leaf-footed cactus bugs, Narnia femorata, that lose a weapon by autotomy during development invest instead in large testes. While evolutionary outcomes of the trade-offs between pre- and post-copulatory strategies have been identified, less work has been done to identify proximate mechanisms by which the trade-off might occur, perhaps because the systems in which the trade-offs have been investigated are not ones that have the molecular tools required for exploring mechanism. Here we applied knowledge from a related model species for which we have developmental knowledge and molecular tools, the milkweed bug Oncopeltus fasciatus, to investigate the proximate mechanism by which autotomized N. femorata males developed larger testes. Autotomized males had evidence of a higher rate of transit amplification divisions in the spermatogonia, which would result in greater sperm numbers. Identification of mechanisms underlying a trade-off can help our understanding of the direction and constraints on evolutionary trajectories and thus the evolutionary potential under multiple forms of selection.

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