Epigenetic Control of Temperature-Dependent Female Reproductive Life History Trade-Offs in Seed Beetles, Callosobruchus maculatus

Many species are threatened by climate change and must rapidly respond to survive changing environments. Epigenetic modifications, such as DNA methylation, can facilitate plastic responses by regulating gene expression in response to environmental cues. Understanding epigenetic responses is therefore essential for predicting species’ ability to rapidly adapt in the context of global environmental change. Here, we investigated the functional significance of DNA methylation on temperature-dependent life history in seed beetles, Callosobruchus maculatus. We assessed changes in DNA methyltransferase (Dnmt1 and Dnmt2) expression levels under ambient conditions and thermal stress, and reproductive performance following artificially-induced epimutation via 3-aminobenzamide (3AB) and Zebularine (Zeb), at a range of ambient and warmer temperatures over two generations. We found that Dnmt1 and Dnmt2 were greatly expressed in females, throughout the body, and exhibited temperature-dependence; in contrast, Dnmt expression was minimal in males. Epimutation led to shifts in female reproductive life history trade-off allocation, and differentially altered thermal optima of fecundity and offspring viability. This study revealed the optimal allocation strategy among these fitness components is temperature-dependent, and trade-offs become increasingly difficult to resolve epigenetically under more extreme warming. Results suggest that epigenetic mechanisms are strongly implicated in, and perhaps limiting of, invertebrate life history responses to temperature change. Further investigation will reveal targeted DNA methylation patterns and specific loci associated with temperature-dependent life history trade-offs in seed beetles and other invertebrates.

in response to novel thermal regimes [39], which has also been recently experimentally 133 demonstrated [40]. Additionally, preliminary studies assessing C. maculatus thermal that optimal trade-off strategies among these traits may be temperature-dependent. 138 Similarly, Berger et al. [41] found a temperature-dependent trade-off between longevity 139 and reproductive effort in thermally stressed C. maculatus associated with improved 140 germline repair of stress-induced DNA mutations -which may have strong implications 141 on adaptive abilities to climate change. These earlier results suggest that temperature-142 dependent, plastic shifts in life history trade-offs may facilitate adaptive responses to 143 climate warming in our study organism. 144 To test our prediction that DNA methylation influences temperature-dependent life 145 history trait expression and optimal life history trade-off allocations, first we examined 146 sex and anatomical differences in Dnmt gene expression to test for sex-specific 147 differences in expression of enzymes that initiate DNA methylation, and also whether 148 these represent whole organism differences or anatomical region specificity. We then Callosobruchus maculatus are an invasive pest that have evolved to complete its entire 160 developmental cycle within stored, dried leguminous agricultural products [39]. Female  female C. maculatus. The putative de novo methylation gene, Dnmt3, was not studied 178 as C. maculatus does not appear to express Dnmt3 [43]. Although Dnmt2 is not directly 179 involved in DNA methylation, we were nevertheless interested in examining sex and 180 temperature effects on tRNA methylation, to inform future studies of its role in insects.

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Genes that are not epigenetic enzyme encoding genes were also examined, to create 182 an overall picture of how epigenetic genes may be responding similarly or differently 7 183 to other genes with divergent functions in thermal stress (heat shock proteins 70 184 (Hsp70) and 90 (Hsp90); [44]) and life history (vitellogenin (Vtg); [45] annealing of primers at their annealing temperatures for 30 seconds (S4 Table) and 3)  in an oven at 60°C to return moisture levels similar to that of normal dried beans.

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To determine whether DNA methylation influences life history trait allocation in 260 response to warming, C. maculatus beetles were exposed to one of three treatments: averaged within F 1 families) and F 2 adult lifespan (also averaged within F 1 families). 274 We also investigated temperature and epimutation treatment effects on whole-genome 275 methylation using methylation-sensitive ELISAs (Study in Supporting Information).      with no effect of Zeb at these temperatures (Table 1; Figure 3B).    insects as an epigenetic response to temperature change [59][60][61] which could explain their ability to rapidly adapt to novel host species and climates as 539 they spread.

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Our preliminary analyses of how whole-genome methylation is affected by temperature 541 and treatment revealed suggestive trends but did not yield conclusive results likely due The results of this study provide novel findings that DNA methylation plays a functional 554 role in regulating C. maculatus female reproductive life history strategies and how they, 555 and their optima, vary with temperature, reflected by offspring thermal performance.

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The results suggest that the observed temperature dependence of Dnmt expression in 557 adult females, rather than being simply a thermodynamic property of the molecule, Dunan for their guidance and support throughout the study.