Comparison of the effect of bacterial stimulation on the global epigenetic landscape and transcription of immune genes in primarily zoophilic members of the Anopheles gambiae complex (Diptera: Culicidae)

Abstract

Members of the Anopheles gambiae complex vary in their vector competence, and this is often attributed to behavioural differences. Similarly, there are differences in transmission capabilities of the zoophilic members of this complex despite exhibiting similar behaviours. Therefore, behavioural differences alone cannot fully explain vector competence variation within members of the An. gambiae complex. The immune system of mosquitoes plays a key role in determining susceptibility to parasite infection and consequently transmission capacity. This study aimed to examine variations in the immune response of An. arabiensis, An. merus and An. quadriannulatus, a major, minor, and non-vector respectively. The global epigenetic landscape was characterised and the expression of Defensin-1 and Gambicin was assessed in response to Gram-positive (Streptococcus pyogenes) and Gram-negative (Escherichia coli) bacterial infections. The effect of insecticide resistance in An. arabiensis on these aspects was also assessed. The immune system was stimulated by a blood-borne bacterial supplementation. The 5mC, 5hmC, m6A methylation levels and Histone Acetyl Transferase activity were assessed with commercial ELISA kits. The transcript levels of Defensin-1 and Gambicin were assessed by quantitative Real-Time Polymerase Chain Reaction. Species-specific differences in 5mC and m6A methylation existed both constitutively as well as post immune stimulation. The epigenetic patterns observed in the laboratory strains were largely conserved in F1 offspring of wild-caught adults. The methylation patterns in the major vector typically differed from that of the minor/non-vectors. The differences between insecticide susceptible and resistant An. arabiensis were more reflected in the expression of Defensin-1 and Gambicin. The expression of these peptides differed in the strains only after bacterial stimulation. Anopheles merus and An. quadriannulatus expressed significantly higher levels of antimicrobial peptides, both constitutively and after immune stimulation. These findings suggest molecular variations in the immune response of members of the An. gambiae complex.

Keywords: Anopheles arabiensis; Antimicrobial peptides; Epigenetics; Histones; Insecticide resistance; Methylation.

Graphical abstract

Fig. 1

Global changes in DNA methylation levels in laboratory strains and F1 An. arabiensis in response to immune stimulation administered via a blood meal. Calorimetric analysis of DNA methylation levels compared to an internal positive control determined by Enzyme Linked Immunosorbent Assay (ELISA) (A) Changes in 5-methylcysteine (5mC) levels. Data is presented as relative % methylation. (B) Changes in 5-hydroxymethyl cysteine (5hmC) levels. Data is presented as relative to a 0.55 % methylated DNA control. SENN–insecticide susceptible An. arabiensis, SENN DDT–insecticide resistant An. arabiensis, MAFUS–An. merus, SANGWE–An. quadriannalatus and F1–Progeny of wild An. arabiensis. Asterisks (*) indicate a significant difference from the control of the same strain. Significant differences between strains within the same treatment are indicated by capital letters.

Fig. 2

Global changes in N6-methyladenosine m6A mRNA methylation levels in laboratory strains and F1 An. arabiensis in response to immune stimulation administered via a blood meal. Calorimetric analysis of m6A RNA methylation levels compared to an internal positive control determined by Enzyme Linked Immunosorbent Assay (ELISA). Asterisks (*) indicate a significant difference from the control of the same strain. Significant differences between strains within the same treatment are indicated by capital letters.

Fig. 3

Global changes in Histone Acetyl Transferase (HAT) activity in laboratory strains in response to immune stimulation administered via a sugar-meal. Calorimetric analysis of HAT activity compared to an internal positive control. Asterisks (*) indicate a significant difference from the control of the same strain. Significant differences between strains within the same treatment are indicated by capital letters.

Fig. 4

Relative normalized mRNA transcript levels of basal Defensin-1 and Gambicin in laboratory strains. (A) Relative Defensin-1 (Def) expression levels in untreated adults. (B) Relative Gambicin (Gamb) expression levels in untreated adults. (C) Relative Defensin-1 (Def) expression levels after Gram-positive exposure. (D) Relative Gambicin (Gamb) expression levels after Gram-positive exposure. (E) Relative Defensin-1 (Def) expression levels after Gram-negative exposure. (F) Relative Gambicin (Gamb) expression levels after Gram-negative exposure. Significant differences are indicated by different lower-case letter.

Fig. 5

Relative normalized mRNA transcript levels of basal Gram-negative binding protein-1 (GNBP-1) and post immune stimulation in laboratory strains. (A) Relative expression levels in untreated adults. (B) Relative expression levels post Gram-positive bacterial challenge. (C) Relative expression levels post Gram-negative. Significant differences are indicated by different lower-case letter.