show Abstracthide AbstractThe role of mitochondria in both adaptive and innate immune responses is increasingly recognized, but the role of natural mitochondrial DNA (mtDNA) variation as an immunomodulatory factor has received less attention. One reason for this is the difficulty of separating the effect of mtDNA from that of the nuclear genome. By utilizing the fruit fly Drosophila melanogaster, a powerful model system, we created cytoplasmic hybrids, aka. cybrid lines, where unique mtDNAs (mitotypes) were introgressed into a controlled isogenic nuclear background. We harnessed a panel of cybrid lines to study the effect of mtDNA variation on humoral and cell-mediated innate immune responses. Mitotypes exhibited heterogeneity in infection outcomes upon bacterial, viral and parasitoid infections. One mitotype of note, mtKSA2 appeared to be more immunocompetent when compared to other mitotypes. We performed transcriptomic profiling of uninfected flies and flies infected with bacteria or a virus and compared them to a different mitotype in the same nuclear background to find the mechanistic basis of the immunocompetence. We found that mtKSA2 caused an upregulation of TCA and OXPHOS related genes and a downregulation of a multiple genes encoding for antimicrobial peptides in uninfected flies. Upon infection, mtKSA2 flies produced unique transcriptomes that were separated by infection type and duration. When we examined immune cells (hemocytes) in mtKSA2 larvae, we noted an increase in hemocyte numbers. These hemocytes were activated in the absence of infection, increased their production of ROS, and showed evidence of increased encapsulation efficiency upon parasitoid wasp infection. Overall, our results show that mtDNA variation acts as an immunomodulatory factor in both humoral and cell-mediated innate immunity and that specific mitotypes provide enhanced protection against infections. Overall design: 1-4 days old, mated females and males were infected with Staphylococcus aureus (PIG1) or Providencia rettgeri (DSM4542) by pricking the flies with a tungsten needle dipped in bacterial solution (OD600=0.1). Samples were collected 8 and 20 hours after infecting the flies. Systemic infection with DCV was introduced to 2-5 days old, mated females and males by pricking as described with the bacterial infections. DCV inoculum of 10^8 viral particles was used. Samples were collected 3 days after the infection with DCV. As controls, uninfected flies of similar age were collected.