Experimental demonstrations of epigenetic regulations tied to immune priming within a single generation are sparse (17). DNA and RNA methylation was analyzed in the immune-primed mealworm beetle Tenebrio molitor after a second infection with the bacterium Micrococcus lysodeikticus. Immune priming in male and female beetles against M. lysodeikticus infection was associated with reduced RNA methylation (5mC) compared to the control (18) ( Figure 1 ). Simultaneously, RNA methylation was analyzed in immune-primed larvae of T. molitor after exposure to the entomopathogenic fungus Metarhizium. Like adult beetles, immune-primed larvae also displayed different RNA methylation levels compared to the control. The degree of RNA methylation was only restricted within a single generation, as experiments designed to measure its intergenerational inheritance showed no significant difference between the priming group and the control group. However, attempts to analyze the regulatory role of DNA methylation within a single generation of immune-primed beetles proved unsuccessful (5, 18). Nevertheless, this does not eliminate the prospect that alternative epigenetic alterations, like m6A or histone acetylation, might also govern immune priming within the same generation, alongside 5mC RNA methylation.

Histone acetylation has been shown to sustain enduring alterations in transcriptional regulation in immune-primed insects, such as mosquitoes (19). Plasmodium infection induces a long-lasting priming response that enhances antiplasmodial immunity in Anopheles gambiae, the primary vector of malaria in Africa (19). Plasmodium midgut invasion allows prostaglandin E2 (PGE2)-dependent release of hemocyte differentiation factor (HDF) by midgut epithelial cells to enhance the circulation of granulocytes and oenocytes in immune-primed mosquitoes. A double peroxidase (DBLOX) enzyme is essential for HDF synthesis and DBLOX silencing completely abolished the priming response to infection (19). Besides, immune priming is mediated by HATs, which are known to catalyze the transfer of an acetyl group to histones, promoting gene expression. Different HATs expressed in A. gambiae were evaluated for their role in maintaining immune priming following Plasmodium berghei infection. Regulation of immune priming by HATs was only evident for the HAT Tip60, as its gene silencing in A. gambie abolished immune priming ( Figure 2 ). This finding indicates that Tip60 is required for the synthesis of the hemocyte differentiation factor (HDF) that increases oenocyte numbers in immune-primed mosquitoes (19).

Certain epigenetic marks in insects may evade the reprogramming process that usually eliminates all epigenetic data during gametogenesis, enabling these markers to persist through meiosis and be inherited by offspring (20). It raises the possibility that epigenetic changes occurring in immune-primed insects within a generation can be passed on to germ cells and subsequently transmitted to offspring, thereby influencing immune priming of the next generation. Inheritance of epigenetic changes has been experimentally demonstrated to be associated with immune priming at the intergenerational level (parental to offsprings/F1). Feeding Manduca sexta with the entomopathogenic bacterium Serratia entomophila or non-pathogenic E. coli delayed transformation from larvae to pupae (21). Induction of immune response upon exposure to pathogenic and non-pathogenic bacteria in the infected host was associated with shifts in both DNA methylation and histone acetylation. Maternal intergenerational immune priming was mediated by the translocation of bacterial structures from the gut lumen to the eggs, resulting in microbe-specific transcriptional reprogramming of gene expression in larvae of the F1 generation. For example, significant downregulation of HDAC4 and HAT chameau was observed only in the female offspring of parents infected with S. entomophila in the F1 generation (21) ( Figure 2 ). On the other hand, the third-instar F1 larvae displayed sex-specific differences in the expression profiles of immunity-related genes and DNA methylation. Exposure to E. coli in the parental generation induced sex-specific differences in the expression of DNMT1 and DNMT2 in F1 larvae. The DNA methylation level, however, was reduced in the F1 generation male and female offspring of both parents in both treatment groups ( Figure 1 ).

DNMTs may also interfere with the paternal transfer of immunity to the F1 offspring. In the red flour beetle Tribolium castaneum, Dnmt2 expression in the testes was higher than in the wholebody of male beetles of the parental generation (22). RNAi knockdown of the Dnmt2 gene in fathers had systemic consequences, leading to a slowdown in the developmental pace of offspring larvae and an increased mortality rate among adult offspring when exposed to Bt infection. However, Dnmt2 silencing in the paternal generation did not affect Dnmt2 expression in the offspring generation. Also, expression of selected innate immunity genes (hsp83, nimB, and PGRP) was unaffected in the paternal and offspring generations due to Dnmt2 knockdown (22). Despite the limitations in the mechanistic understanding, this finding highlights the involvement of Dnmt2 in paternal effects, wherein its knockdown increases the susceptibility of beetle offspring to pathogens.

Recently, the persistence of pathogen-induced epigenetic alterations in immune-primed insects beyond a single generation has been investigated in G. mellonella. The larvae were consistently exposed to either Bt or M. robertsii across multiple generations, resulting in the emergence of a first generation with heightened resistance to infection (resistant line) compared to susceptible larvae not subjected to selection (susceptible line) (23, 24). Notably, these resistant larvae exhibited various intra-species immuno-physiological adaptations, such as alterations in gut microbiota, melanization, circulating hemocytes, cuticle thickness, and differential expression of immune genes compared to the susceptible line. This phenomenon sparked deeper inquiries into the involvement of DNA methylation and histone acetylation in the evolutionary process underlying G. mellonella’s resistance to Bt and M. robertsii (23, 24). The investigations unveiled distinct alterations in DNA methylation and histone acetylation patterns across tissues of both resistant (selected line) and susceptible (non-selected line) larvae. Particularly noteworthy were tissue-specific changes observed in the midgut, cuticle, and fat body regions. Bt or M. robertsii-resistant larvae showcased heightened levels of histone H3 acetylation in specific tissues: the midgut exhibited increased acetylation in the case of resistance to Bt, while the cuticle displayed elevated acetylation levels in response to resistance against M. robertsii, as opposed to susceptible larvae ( Figure 2 ). Likewise, there was a tissue-specific elevation in 5-methylcytosine levels in uninfected resistant larvae compared to uninfected susceptible larvae ( Figure 1 ). Increased expression of selected histone acetylation and DNA methylation related genes were upregulated in uninfected resistant larvae compared to uninfected susceptible larvae. However, the expression levels of these genes were reversed upon infection of the resistant and susceptible lines with either Bt or M. robertsii (23, 24).