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.
Dif1 dl1 animals, in which BacA\p35GMR.PH is expressed in the haemocytes, under the control of Scer\GAL4He.PZ still have microbes in the haemolymph, even though the microbial load is 100-fold lower than in Dif1 dl1 larvae. After injection with E.coli, nearly all of the BacA\p35GMR.PH expressing Dif1 dl1 larvae become flaccid and die with in a few hours. At 5 hours post infection, the few surviving animals exhibit up to 1000 times more bacteria per animal than wild-type larvae. Bacteria are engulfed by the BacA\p35GMR.PH-rescued haemocytes but are not digested, subsequently, many of the haemocytes rupture.
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)J4/lwr5 dl1 double mutants compared to lwr4-3/lwr5 single mutants. However, the amount of lamellocytes is not reduced.
In lwr4-3/lwr5; dl1/Df(2L)J4, total numbers of hemocytes are reduced compared to lwr4-3/lwr5 single mutants. Plasmatocyte levels are reduced by 50% compared to single mutants, while lamellocyte levels are not significantly reduced.
95% of twi1/+; dl1/+ animals raised at 18oC survive to adulthood whereas only 0.6% of those raised at 25oC do. This temperature sensitive lethality is not significantly affected by the presence or absence of scM6/+ or sc+t.XSE.
Embryos carrying foghs.PM show a flattened stretched appearance around their entire circumference and disorganisation of the ordered arrangement of cells.
A partial restoration of cuticle pattern elements along the dorso-ventral axis is seen in embryos derived from dl1 females if the females also carry Difbcd.PS. The degree of ventralisation seen in these rescued embryos is increased if the females are also heterozygous for cact7. The gastrulation defects of embryos derived from dl1 females is partially rescued if the females also carry Difbcd.PS; anterior movement of the posterior midgut invagination is seen along the dorsal side of the embryo, but the ventral furrow does not form.
The degree of lethality of embryos derived from dl1/+ females at 29[o]C is not significantly altered if the females are expressing of one copy of Zzzz\Pvank1Scer\UAS.T:Zzzz\FLAG under the control of Scer\GAL4mat.αTub67C.T:Hsim\VP16. However, if the females are expressing two copies of Zzzz\Pvank1Scer\UAS.T:Zzzz\FLAG, the fraction of embryos failing to hatch is increased, and the degree of dorsalisation of the unhatched embryos is more severe.
The degree of lethality of embryos derived from dl1/+ females at 29[o]C is not significantly altered if the females are expressing of one copy of Zzzz\I2vank3Scer\UAS.T:Zzzz\FLAG under the control of Scer\GAL4mat.αTub67C.T:Hsim\VP16. However, if the females are expressing two copies of Zzzz\I2vank3Scer\UAS.T:Zzzz\FLAG, the fraction of embryos failing to hatch is increased, and the degree of dorsalisation of the unhatched embryos is more severe.
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.