Dif1 dl1 double mutants are small and sluggish and only 3.4% survive to adult. Unchallenged Dif1 dl1 double mutant larvae contain many bacteria and yeast in their haemolymph (as many as 105 microbes per animal), while no microbes are observed in wild-type haemolymph. In microbe-free conditions, in the presence of antibiotics, approximately 33% of Dif1 dl1 double mutants survive to adult stages, thats a 10-fold increase compared to normal conditions. Dif1 dl1 double mutants are able to mount a humoral immune response, but this does not prevent constitutive infection of these animals.
Dif1 dl1/+ + double heterozygotes exhibit approximately 5000 blood cells per υl of haemolymph, the same as in wild-type flies. In contrast, Dif1 dl1 double homozygotes exhibit a greatly reduced number of haemocytes, approximately 500 per υl - 10-fold fewer than in wild-type. Approximately 15% of Dif1 dl1 haemocytes undergo apoptosis, compared with only 2.3% in wild-type.
The few blood cells present in Dif1 dl1 mutants exhibit an abnormal morphology. In contrast to wild-type haemocytes, Dif1 dl1 blood cells are enlarged, containing intact intracellular bacteria that are not localised to vacuoles and have not been digested. When Dif1 dl1 larvae are grown on minimal medium in the presence of antibiotics, the number of haemocytes increases 3-fold, to approximately 1400 cells per υl, and the haemocytes are of normal morphology.
Expression of dlScer\UAS.cMa in circulating haemocytes, lymph glands, and epidermis, but not in the fat body, under the control of Scer\GAL4e33C, restores normal haemocyte numbers in Dif1 dl1 double mutants. The rescued blood cells are indistinguishable in morphology from wild-type uninfected haemocytes. In addition, the rescued animals have do not have any microbes in their haemolymph and 66% survive to adult stages.
Expression of dlScer\UAS.cMa in circulating haemocytes, but not in other immune-responsive tissues, under the control of Scer\GAL4He.PZ, results in an absence of microbes from the haemolymph, with approximately 45% of dlScer\UAS.cMa expressing Dif1 dl1 double mutants surviving till adulthood.
Expression of dlScer\UAS.cMa in circulating haemocytes and lymph gland cells, under the control of Scer\GAL4srp.Hemo, increases blood cell number to approximately 90% of wild-type in Dif1 dl1 double mutants and enables approximately 50% survival till adulthood. The majority of these larvae have no microbes in their haemolymph.
The melanotic tumour penetrance and circulating hemocyte levels are partially reduced in lwr4-3 Df(2L)TW119/lwr5 Dif1 double mutants, compared to lwr4-3/lwr5 single mutants. However, the amount of lamellocytes is not reduced.
Expression of BacA\p35GMR.PH in the haemocytes, under the control of Scer\GAL4He.PZ in Dif1 dl1 animals results in the number of blood cells per υl of haemolymph increasing 4-fold, to approximately 2200 cells per υl, whereas the blood cell number remains constant in Dif1 dl1 heterozygotes.