Mutant adults show reduced survival compared to wild type after severe wounding with a clean needle (survival after mild wounding is not significantly different).
PPO1Bc/PPO1Bc adult flies have a mild but significant decrease in survival after inoculation with either Providencia rettgeri or Enterococcus faecalis bacteria.
Wandering third instar heterozygous and homozygous larvae show spontaneous melanisation of crystal cells.
Homozygous larvae and adults do not show melanisation at the wound site in response to physical wounding, in contrast to wild type.
Wandering third instar PPO1Bc/PPO1Δ larvae show spontaneous melanisation of crystal cells.
Wandering third instar PPO1Bc/PPO1Δ larvae do not show melanisation at the wound site in response to physical wounding, in contrast to wild type.
Bc1 mutant flies subjected to severe wounding exhibit a reduced survival rate compared to controls. A similar reduction in survival is seen when flies are reared in germ-free conditions, indicating that the observed mortality is not due to infection. The survival rate is improved when flies are exposed to a non-lethal concentration of a pro-oxidant (by ingesting paraquat).
When Bc1 mutant flies are wounded during the adult stages, melanization is severely impaired around the wound site.
Mutant larvae show reduced survival compared to wild-type controls after wounding.
Mutant flies show reduced survival compared to wild-type controls after natural infection with B. bassiana or after challenge with a needle contaminated with either A. tumefaciens or E. faecalis.
The Bc1 mutant causes spontaneous melanisation of the crystal cells.
Spontaneous melanisation of the hemolymph (assayed by dissecting larvae and exposing them to air) is much lower in Bc1 larvae than in wild type.
Bc1 homozygotes have reduced resistance to natural infection with B.bassiana compared to wild-type. Melanization in response to pricking of Bc1 homozygous larvae with a needle is weaker than in wild-type.
Heterozygotes show a dominant melanized crystal cell phenotype, but show a wild-type like survival rate in response to physical wounding, 24 hours after wounding about 20% of animals die. In contrast 91% of homozygous animals have died by 24 hours post wounding, 50% mortality is seen by 6 hours. The wound clots differently in mutant flies; Unlike wild-type there is no evidence of melanin formation in the wounded integument. Furthermore, the two edges of the wound are found apart on mutant flies. This failure to keep the edge of the wound in close proximity leads to death by bleeding.
Blackened crystal cells are not visible in Bc1/+ larvae raised at 29oC though are visible at 27oC.
Crystal cells undergo blackening before completing differentiation in mutants. In homozygous larvae, the circulating black crystal cells are mostly phagocytosed by plasmatocytes; therefore the black inclusions correspond to whole cells in phagosomes and not to blackened crystals within crystal cells. When larvae are raised at 25[o]C, crystal cells are more abundant than at lower temperatures, they agglomerate to form black masses that are encapsulated by lamellocytes and subsist in adults. Isolated black cells are also found within adult phagocytes. Second instar mutant lymph glands contain some black cells in the first lobes together with secretory cells. The posterior lobes are larger than wild type but contain no lamellocytes, although this cell type is already present in the hemolymph (1-5% of circulating cells). During the third instar, all lymph glands appear oversized and contain many lamellocytes. At this stage the abundant circulating lamellocytes encapsulate groups of melanised cells.
Crystal cells show premature melanization of crystalline inclusions.
There is not a significant difference in the survival rate between larvae pricked with a sterile needle or a needle coated with Gram-positive or Gram-negative bacteria, however, infection with fungal spores induces over 60% lethality in 48 hours in homozygous larvae.
Homozygous and heterozygous larvae have circulating melanised crystals. At 21oC larvae have a high frequency of melanotic tumours and a higher them normal haemocyte count.
No haemolymph prophenoloxidase. Eggs of the parasitoid strain L.boulardi are encapsulated by host haemocytes but the cellular capsules do not melanize and harden.
Bc1 causes paracrystalline inclusions in hemocytes of larvae. Heat-induction blackens the crystal cells in the Bc1 hemolymph and lymph glands.
Homozygous and heterozygous third instar larvae have black cells beneath the integument. Many of these pigmented cells appear to sedentary. A few of the black cells are generally found in the anterior pair of the lymph glands, while others are scattered as individual cells throughout the hemocoel. Most of the black cells in the hemocoel are trapped between the intersegmental muscles and the body wall, in the spaces between the alary muscles of the heart and near the ostia of the heart. Numerous black cells are also found in the caudal hemocoel. Some of the black cells near the caudal ostia can be seen to enter the heart, flow anteriorly and then return to the posterior hemocoel via the lateral sinuses in living larvae.
Several lines of evidence suggest that the he black cells seen in the mutant are a mutant form of crystal cells.
Hemolymph from third instar mutant larvae contain black cells, plasmatocytes, podocytes and lamellocytes, but no normal crystal cells (wild-type larvae at this stage contain primarily plasmatocytes and crystal cells). The lamellocytes in the mutant hemolymph samples can be seen adhering to the surfaces of the black cells.
The lymph glands of mutant larvae do not contain the large paracrystalline structures that characterise the crystal cells of wild-type larval lymph glands, but contain melanised cells instead, which may be round or irregular in shape. The surfaces of the melanised cells also show variation, some being smooth and some having surface extensions that are either folded membranes of filamentous projections in section. The internal structures of the black cells, including the nuclei, nucleoli, chromatin and mitochondria are well preserved. The black cells in the mutant larval lymph glands are invariably encapsulated by one or two other nonpigmented cells.
Late homozygous embryos and newly emerged homozygous larvae have black cells. Pigmented cells appear in about the eleventh hour of development in the mutant embryos. Pigmented cells are seen in the vicinity of the first pair of lymph glands while others are scattered along the furrows that form between the folds of the gut in the mutant embryos. When the mutant larvae hatch, these cells are dispersed and there is no indication that the lymph glands disintegrate.
Black cells do not appear until late in the first larval instar in heterozygotes and crystal cells are present in the hemolymph until this time. When the black cells begin to appear in the heterozygous larvae some of them contain distinct paracrystalline inclusions.
Homozygous larval extracts have no detectable phenol oxidase activity, and heterozygous larval extracts show reduced phenol oxidase activity compared to wild type.
Heterozygotes have numerous pigmented cells under the cuticle of the head, thorax and abdomen. This phenotype is very striking in larvae and pupae. In larvae, most of the black cells appear to be attached to the larval integument, but some are floating freely in the body cavity.
Quadrate crystalline bodies in crystal cells of hemolymph replaced by amorphous melanotic mass. Black cells appear in 11-hour embryos in Bc1/Bc1 and in late first-instar larvae in Bc1/+. Crystal cells replaced by black cells during first instar in Bc1/+. Numerousblack cells visible through the integument of larvae, pupae and of head, thorax and abdomen in adults. Bc1/Bc1 larvae have no phenol oxidase activity and larval hemolymph fails to darken upon exposure to air; Bc1/+ intermediate between +/+ and Bc1/Bc1 in these respects.