Epigenetic regulation is known to shape the genetic landscape of organisms at varying intensities. This study examined the influence of DNA methylation on two cryptic species (B and Q) of B. tabaci 32,33. These two cryptic species are of particular interest due to their differences in biological traits, behavior, xenobiotics’ detoxification (including insecticides used in agricultural landscape), and virus transmission abilities 28,29. Both B and Q cryptic species of B. tabaci exist in the Southeastern United States and have previously been shown to be efficient transmitters of the monopartite ssDNA virus –TYLCV 20,40. Closely related species can exhibit distinct DNA methylation patterns, such as the observed differences between species within genus Triportheus (Characiformes fishes) 60. However, intraspecific differences, such as those in the B. tabaci cryptic species complex, have not been widely investigated.
Previous studies have shown that insects exhibit significantly lower levels of DNA methylation compared with mammals, and the distribution of DNA methylation across the genome also is variable 61. Low genomic DNA methylation in B. tabaci was expected based on other previous studies 14,62. Genes frequently subjected to methylation tend be more highly conserved across invertebrate species, indicating slower evolutionary rates and stronger selective constraint 63. Hypermethylated regions tend to be found within proximity to more highly expressed genes such as housekeeping genes 64–68. In this study, promoter and gene body methylation levels were compared between B and Q cryptic species. The methylation levels were observed to have a similar pattern of bimodality within both cryptic species. However, this bimodality of the promoter region is not common among invertebrates 69, suggesting that B. tabaci might exhibit unique patterns of promoter methylation. It also is worth acknowledging that promoter regions’ annotations are dependent on completeness and accuracy of their respective gene body annotations.
Low methylation levels also were observed across most TE superfamilies. However, copia retrotransposons, one of the largest TE superfamilies in eukaryotes 70,71, were found to be targeted more by DNA methylation than other TE superfamilies. Sequence homology between copia retrotransposons and the promoter regions of heat shock proteins (HSPs) may indicate that this class of TEs could contribute towards whitefly's response to biotic stressors such as pathogens 72,73. HSPs, broadly, are involved in homeostasis and immune response to phytoviruses in several insects including B. tabaci 74. While there was a low number of copia retrotransposons found within the genome assemblies of both cryptic species, these TEs also have been observed to be preferentially methylated and accumulate in regions of high CpG density in other arthropod species 47,48. Another major TE superfamily found in eukaryotes, known as gypsy retrotransposons 70,71, were also found to be likely targets of DNA methylation compared with other TE superfamilies in B. tabaci. Gypsy retrotransposons activation has been found to be involved in Drosophila development and immune responses 75.
The relationship between DNA methylation and gene expression can vary among different insect species and can be context dependent 1. Previously reported transcript-derived expression differences in response to virus acquisition in B and Q cryptic species of B. tabaci 41 was compared to the methylation events. The aim was to uncover key methylation patterns affecting virus acquisition and subsequent inoculation in both B. tabaci cryptic species. Gene expression levels could potentially be influenced by DNA methylation; however, differential promoter methylation was not found to be associated with gene expression differences in either cryptic species in the current study.
Between the two cryptic species, two pairs of orthologs were identified where members from B and Q each exhibited DMRs, whereas 55 and 56 genes exhibited DMRs in only B or Q cryptic species, respectively. The first pair of orthologs with DMRs were: Bta03757 (Brinker) in B cryptic species and BTA018959.1 (unnamed protein product) in Q cryptic species. The second pair that had genes with DMRs and were also found to be DEGs viz., Bta06672 (Threonine–tRNA ligase) in B cryptic species and BTA026677.3 (39S ribosomal protein L39, mitochondrial) in Q cryptic species. Threonine–tRNA ligase, which plays an important role in protein biosynthesis, has been previously identified as a candidate for pest control via RNA interference (RNAi) in three rice planthopper species 76. Differential methylation may be weakly related to the presence of alternative splicing events in B. tabaci. The integration of gene expression analysis with the identification of specific protein clusters associated with GO terms provided valuable insights into B. tabaci-virus interactions. Protein clusters or gene families composed of similar genes and enriched in GO terms such as “transposition”, “DNA-mediated”, “regulation of protein tyrosine kinase activitytranslationprotein localization to nuclear pore”, “ubiquitin-dependent ERAD pathway”, and “spliceosomal snRNP assembly” are of particular interest mainly because of their relevance to phytovirus acquisition and retention.
The low number of genes with DMRs were expected by random chance, suggesting a lack of conservation of site-specific DNA methylation. As this work delves into the epigenetic regulation of gene expression in B. tabaci and its modulation by virus acquisition, it contributes to a broader understanding of insect-vector interactions and the molecular basis of vector-borne pathogen dynamics. This study provides an overview of baseline DNA methylation levels in B. tabaci B and Q cryptic species, as well as shows that non-propagative viruses may have minimal, but differential, impact on the global or targeted methylation patterns of their insect vector. It also may be of interest to look at temporal differences in DNA methylation following acquisition of other persistently transmitted viruses in the two cryptic species. Beyond DNA methylation, the study of other forms of epigenetic alterations in B. tabaci, such as histone modifications and non-coding RNAs (ncRNAs), may further elucidate the intricate epigenetic regulation underlying their response to virus acquisition.