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).

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.

Generation of specific immune memory by bacterial exposure in the major malaria vector Anopheles arabiensis (Diptera: Culicidae).

There is a growing body of evidence that invertebrates can generate improved secondary responses after a primary challenge. This immunological memory can be primed by a range of pathogens, including bacteria. The generation of immunological memory has been demonstrated in mosquitoes, with the memory primed by a range of initial stimuli. This study aimed to examine whether insecticide resistance affects the capacity to generate immunological memory. The primary hypothesis was tested by examining the capacity of genetically related laboratory-reared Anopheles arabiensis strains that differ by insecticide resistant phenotype to generate immunological memory. The competing hypothesis tested was that the bacterial virulence was the key determinant in generating immunological memory. Immune memory was generated in F1 females but not males. Immunological memory was demonstrated in both laboratory strains, but the efficacy differed by the insecticide resistant phenotype of the strain. An initial oral challenge provided by a blood meal resulted generated better memory than an oral challenge by sugar. The efficacy of memory generation between the two bacterial strains differed between the two mosquito strains. Regardless of the challenge, the two strains differed in their capacity to generate memory. This study therefore demonstrated that insecticide resistant phenotype affected the capacity of the two strains to generate immunological memory. Although this study needs to be replicated with wild mosquitoes, it does suggest that a potential role for insecticide resistance in the functioning of the immune system and memory generation of An. arabiensis.

Marked Effects of Larval Salt Exposure on the Life History and Gut Microbiota of the Malaria Vector Anopheles merus (Diptera: Culicidae).

Anopheles merus can breed in a range of saltwater concentrations. The consequences of this ability on the life history of adult An. merus are poorly understood. This study examined the effects of exposure to 0, 2.1875, 4.375, 8.75, and 17.5 g/L of sodium chloride on An. merus. The effects on larval development, adult longevity, fertility, and fecundity, as well as deltamethrin tolerance were examined. The effect of larval salt exposure on the expression of defensin-1 in adults was examined by quantitative Real-Time PCR. Finally, the effect of the larval salt concentration on microbial dynamics was assessed by 16S Next Generation Sequencing. High concentrations of saltwater increased larval development time and number of eggs laid, as well as deltamethrin tolerance. Larval exposure to salt also reduced the expression of defensin-1. The exposure also had a significant effect on microbial diversity in larvae and adults. The diversity of larvae decreased once adults emerged. Salt-tolerant bacterial genera predominated in larvae but were absent in adults. High salt concentrations resulted in greater abundance of Plasmodium-protective genera in adults. Although this study was conducted on a laboratory strain of An. merus, these data suggest that osmoregulation has a significant effect on the life history of the species with potential epidemiological consequences.