Genetics, and not diet, drives DNA methylation in the water strider Microvelia longipes

Phenotypic plasticity, the ability of a single genotype to produce alternative phenotypes in response to environmental cues, is a key driver of evolutionary change. In the water strider Microvelia longipes, males display remarkable continuous variation in hindleg length, a sexually selected trait used as a weapon in male male contests for access to females. To determine whether DNA methylation mediates this environmentally induced phenotypic variation, we used three inbred lines of M. longipes showing differences in mean hindleg length, body size, and allometric coefficients. Whole-genome bisulfite sequencing (WGBS) on adult males and females from all lines determined that about 12% of the 12,684,876 CpG sites found in the genome were methylated. This global level of DNA methylation is among the highest reported in insects. DNA methylation was predominantly concentrated within or near gene bodies (77% of methylated CpGs), consistent with patterns observed in other insects. Unsupervised clustering and principal component analyses revealed that methylation patterns differed significantly between genetic lines but showed minimal differences between sexes, indicating a strong genetic influence. Most surprisingly, nutritional treatment followed by leg-specific WGBS failed to identify any change in DNA methylation despite nutrition having a pronounced effect on leg length. These results show that in M. longipes, DNA methylation patterns are largely stable across nutritional treatments and primarily determined by genetic background. This challenges the common assumption that DNA methylation universally mediates environmentally induced phenotypic plasticity and suggests that other epigenetic mechanisms, such as histone modifications or non-coding RNAs, may play a more direct role in regulating continuous plastic traits. Our study underscores the complexity of epigenetic regulation and highlights the need for broader investigation of molecular pathways to fully understand the molecular basis of phenotypic variation in natural populations.