Infected fruit flies get sick in ways that human patients would recognize; bacterial infections in Drosophila
induce changes in feeding, metabolism and circadian rhythm, and conversely changes in these pathways influence susceptibility to infection 
. Many responses affect survival during infection, but this work remains splintered as the field primarily focuses on individual mechanisms in isolation, offering glimpses of the whole picture. Here we use mutations in two genes, ets21c and wntD, to examine their effect on immune responses and survival during infections with two bacteria, Listeria monocytogenes
and Streptococcus pneumoniae
. Both genes affect multiple arms of the immune system and we wanted to understand how immunity offers protection against pathogens with different lifestyles in Drosophila
. We chose these two microbes because they produced dissimilar phenotypes in previous Drosophila
immunity assays 
. Together these mutants and microbes demonstrate how there can be no perfect immune response, as there are responses that are beneficial during one infection and actively detrimental during another.
immune response can be divided into categories based on the speed at which they act following pathogenic challenge. The fast-acting immune responses, which respond within seconds to minutes, are phagocytosis and melanization 
. Hemocytes are phagocytic cells in the fly and they are concentrated in adherent groups on the dorsal side of the abdomen and the anterior abdominal segment of the heart in adult flies. Inhibition of phagocytosis increases susceptibility to a number of bacteria 
. Insects produce melanin from tyrosine using the enzyme phenoloxidase, which is activated by an immune triggered proteolytic cascade. This process is hypothesized to produce reactive oxygen species, which can harm the host in addition to harming the pathogen, and to physically encapsulate the invaders 
. In Drosophila
, some bacterial pathogens (L. monocytogenes
, Salmonella typhimurium
, and Staphylococcus aureus
) induce visible melanization, and flies defective in the melanization activation pathway are less resistant to these infections 
. Though these relatively quick responses presumably remain active through the whole infection there is at least one response that takes several hours to reach full force. This slow response is the induction of anti-microbial peptides which peak in transcript expression six to 24 hours post infection 
. We do not know when actual antimicrobial activity peaks as this is seldom assayed directly, but presumably this takes even longer than the increase of transcripts.
While it may be simplest to examine the effect of immune components individually, in order to effectively control immunity clinically we need a better understanding of the full immune network; each response doesn't exist in a vacuum. Knowing which immune responses strongly associate with a positive outcome for a given pathogen and which physiological systems are impacted by infection will allow doctors to more effectively treat disease. Patients normally do not have a single pathway or gene responsible for their entire pathology, and we need to develop the tools to deal with these levels of complexity.
To probe changes in the immune response, we turned to two pathogens that previously exhibited opposing phenotypes: Listeria monocytogenes
and Streptococcus pneumoniae
. When injected into the hemocoel, L. monocytogenes
causes lethal infections in Drosophila melanogaster
at doses as low as ten bacteria, and death from infection occurs on the order of one week. L. monocytogenes
lives both intracellular and extracellular in the fly and causes robust disseminated melanization 
. S. pneumoniae
can also cause lethal infections; however, there are sub lethal doses, which prime the fly to become resistant upon subsequent challenges 
. S. pneumoniae
infection kills flies rapidly, within two to four days, and flies surviving past four days have likely cleared the pathogen. S. pneumoniae
is an extracellular pathogen and bead inhibition of phagocytosis increases susceptibility to infection 
. In contrast to L. monocytogenes
, flies deficient in melanization are more resistant to S. pneumoniae
infection, although the mechanism is unknown 
Ets21c (CG2914), a putative transcription factor characterized by its DNA binding ets-domain, was previously implicated in Drosophila
immunity. Ayres et al. found that et21c mutants died more rapidly during L. monocytogenes
infection with similar bacterial loads compared to wild-type, but were no different from wild type flies when challenged with S. typhimurium
or S. aureus
. Studies of immune signaling in Drosophila
S2 cells and hemocyte cell lines used ets21c transcript as a read out of the early immune response and showed that ets21c induction depends on the imd pathway and one of its transcription factors, basket 
WntD (CG8458) is a negative regulator of dorsal signaling in Drosophila
, and wntD mutants are more susceptible to L. monocytogenes
infection than wild type flies 
. Previous work measured the signaling and transcriptional effects of wntD on antimicrobial peptides 
; however, it remains unknown, whether wntD impacts bacterial load during L. monocytogenes
infection and how it affects melanization and phagocytosis.
The effect of a given bacterial load has previously been used to categorize genes as either impacting tolerance or resistance 
. Resistance genes and mechanisms directly impact how well the bacteria grow or are killed, while tolerance genes and mechanisms affect the host's ability to deal with the effect of infection (e.g. energy strain, accumulated damage). While both of these mechanisms are functionally distinct, they way they impact bacterial load cannot be as easily separated and there is a full spectrum of phenotypes possible, from genes that do not impact bacterial load at all to genes that increase bacterial load by hundred-fold in just a day. Determining where in this spectrum our mutants fall helps inform the possible responsible mechanisms.
In this paper, we show that ets21c and wntD mutants are both more susceptible to L. monocytogenes and more resistant to S. pneumoniae, but differ in their ability to control L. monocytogenes bacterial loads. At the levels of specific immune responses, these mutants share an increase in phagocytic activity and a shift in anti-microbial peptide induction, but differ in their melanization capabilities. By examining these differences, we establish the relative contributions of the immune pathways to these outcomes - survival during L. monocytogenes infection depends on multiple factors: melanization and phagocytic ability while phagocytic ability alone predicts survival to S. pneumoniae infection.