Abstract
Although invasive generalist insect herbivores can migrate and rapidly adapt to a broad range of suitable host plants, they can face significant difficulties when accidentally migrating to novel and marginally-suitable hosts. What happens, at both the genome-wide regulatory and transcriptional levels, if these marginally-suitable hosts must be used for multiple generations before migration to a suitable host can take place, largely remains unknown. Moreover, if adaptation to the new marginally-suitable hosts and transcriptomic changes do occur, how are they regulated? Does it involve changes in epigenetic patterning and processing (such as alterations in DNA methylation) and/or sequence changes in cis- or trans-regulatory elements in the genome (such as enhancers and promoters)? Using as a model the invasive and generalist whitefly species, Bemisia tabaci, we established a multi-generational experimental system that allowed us to explore the evolved differences between two populations that were subjected to cotton (a suitable host) and habanero-pepper (a marginally-suitable host) on which the initial survival is 5%. First, we conducted reciprocal host performance assays under both optimal (30°C) and mildly stressful (24°C) temperature conditions to assess the adaptive changes in the two populations. We found that the habanero-adapted population exhibited a substantial increase in survival on habanero pepper (exceeding 60%) but did not reach the survival rates observed on the original cotton host (80–90%). Furthermore, this population displayed a trade-off, exhibiting reduced performance on cotton relative to the non-adapted population, and an antagonistic interaction with temperature stress at 24°C. Transcriptomic profiling revealed that adaptation to habanero pepper was associated with two major molecular changes: (1) an upregulation of genes involved in cuticle structural component synthesis and (2) a downregulation of genes encoding cysteine-type peptidases, particularly cathepsin B proteins, which are implicated in the activation of plant defense responses. Next, we analyzed the evolution of 5-methylcytosine (5mC) DNA methylation and single nucleotide polymorphisms (SNPs) in promoter regions after 34 generations. Our findings revealed only slight divergence in 5mC methylation levels between populations reared on suitable versus marginally-suitable hosts. These methylation differences were not directly correlated with changes in gene expression levels. Instead, we observed a strong association between 5mC methylation patterns, gene function, and expression variability. Housekeeping genes exhibited hypermethylation and lower expression variability, whereas environmentally responsive genes were hypomethylated and displayed higher expression variability. Additionally, SNP analysis in promoter regions suggested selection for specific alleles that may contribute to adaptive processes. These findings indicate that both epigenetic modifications and genetic variation in regulatory elements play a role in the multi-generational adaptation of invasive generalist herbivores to marginally-suitable hosts. In conclusion, our study provides novel insights into the interplay between epigenetic regulation and genetic selection during host adaptation in invasive insect herbivores. The observed shifts in gene expression, 5mC methylation, and promoter-region polymorphisms highlight the complex mechanisms underlying long-term adaptation to suboptimal host plants. These results contribute to a broader understanding of how invasive generalists persist and evolve in novel environments, with implications for pest management and ecological resilience.