Parasitoid-induced cellular immune deficiency in Drosophila.

L. heterotoma females inject VLPs along with their eggs into the hemocoel of the Drosophila larva. The VLPs destroy lamellocytes that are potentially harmful for the parasite eggs, but the other types of host hemocytes retain their functions. The parasitized third instar Drosophila larvae continue to grow and pupariate, showing no outward adverse effects. Minimum disruption to the growth and development of the host is advantageous to the endoparasite egg, since survival of the endoparasite depends on the health of the host to the developmental stage at which the endoparasite begins feeding on host tissues. The target of the VLPs must be microtubule components, because VLP entry into the lamellocyte induces depolymerization and repolymerization of microtubules. Microtubule rearrangement changes the discoidal lamellocyte to a bipolar cell. Concomitant with the modification in cell morphology, lamellocytes lose their surface adhesivity so they cannot adhere to a foreign object or to each other to form a capsule around endoparasite eggs. VLP-affected lamellocytes continue to elongate, lose cytoplasmic contents at the poles as blebs full of microvesicles, and are eventually destroyed. The molecular nature of the L. heterotoma VLP and the mechanisms underlying its specificity for lamellocyte cytoplasm are not known. An interesting consideration is the evolution of a particle whose selectivity for a host hemocyte protects the eggs of the parasitic insect. On the basis of information available at this time, it is clear that the L. heterotoma VLP is a useful tool for studying molecular aspects of microtubules and cytoskeleton.

Lamellocyte differentiation in Drosophila larvae parasitized by Leptopilina.

The presence of Leptopilina heterotoma or Leptopilina boulardi eggs in the hemocoel of a Drosophila melanogaster larva induces the differentiation of lamellocytes, the blood cells that encapsulate foreign objects. L. boulardi eggs are encapsulated by the newly differentiated lamellocytes, but L. heterotoma eggs are not. The induced lamellocytes in host larvae with L. heterotoma eggs undergo the same destructive morphological changes as reported previously for lamellocytes present in melanotic tumor mutant larvae at the time of parasitization. Thus, the virus-like particles produced by the L. heterotoma female to protect its eggs from encapsulation do not block the differentiation of lamellocytes, but rather destroy lamellocytes whenever they are present in the hemocoel.

Effects of lamellolysin from a parasitoid wasp on Drosophila blood cells in vitro.

Female parasitoid Leptopilina heterotoma inject a factor, lamellolysin, along with their eggs into the host hemocoel to destroy selectively host hemocytes that encapsulate foreign objects. In parasitized Drosophila melanogaster larvae, these hemocytes (lamellocytes) change from discoidal cells to bipolar cells that no longer adhere to each other to form capsules. To study the effects of lamellolysin on Drosophila lamellocytes in vitro, a giant strain of D. melanogaster was constructed to yield hemolymph with an abundance of lamellocytes. The effect of lamellolysin on the adhesivity of lamellocytes in vitro was demonstrated when the cells were gently rotated in the culture medium. Under these conditions, the bipolar shape of the affected lamellocytes resembled that of lamellocytes in parasitized hosts. When lamellocytes were exposed to lamellolysin in stationary culture medium, the elongation of the bipolar cells continued until they became threadlike. Lamellocytes fragmented in both stationary and rotating culture medium in the presence of lamellolysin, although loss of cellular material was more pronounced in the latter. This study demonstrates that lamellolysin acts directly and destructively on lamellocytes.

Parasitoid virus-like particles destroy Drosophila cellular immunity.

Parasitoid wasps must avoid the destructive effects of the host's cellular defense system in order to exploit the host hemocoel as a suitable environment for their survival. To protect their eggs from encapsulation by Drosophila melanogaster blood cells, Leptopilina heterotoma females inject a factor that selectively destroys lamellocytes, the type of Drosophila blood cell involved in recognition and encapsulation of large foreign objects. Other types of host blood cells, including the phagocytic plasmatocytes, remain functional. This report demonstrates that the destructive factor for lamellocytes is a virus-like particle (VLP) stored in the reservoir of an accessory gland associated with the female wasp reproductive system. We show that VLPs enter Drosophila blood cells in vitro. VLPs are found free in the cytoplasm of lamellocytes but are confined to phagocytic vesicles of plasmatocytes. As lamellocytes are susceptible to the VLP infection and plasmatocytes are not, we conclude that the mode of VLP entry and its disposition in the cytoplasm determine the fate of the infected host blood cell.

Leptopilina heterotoma and L. boulardi: strategies to avoid cellular defense responses of Drosophila melanogaster.

Eggs of three strains of the cynipid parasitoid Leptopilina heterotoma and a Tunisian strain (G317) of L. boulardi are not encapsulated by hemocytes of Drosophila melanogaster hosts, but the eggs of a Congolese strain (L104) of L. boulardi are encapsulated. To determine the reason for the difference in host response against the parasitoid eggs, lamellocytes (hemocytes that encapsulate foreign objects and form capsules around endogenous tissues in melanotic tumor mutants) were examined in host larvae parasitized by the five Leptopilina strains. Parasitization by the three L. heterotoma strains affected the morphology of host lamellocytes and suppressed endogenous melanotic capsule formation in melanotic tumor hosts. L104 did not alter the morphology of host lamellocytes nor block tumor formation in melanotic tumor mutant hosts. The morphology of some lamellocytes was affected by G317 parasitization but host lamellocytes were still capable of forming melanotic tumors and encapsulating dead supernumerary parasitoid larvae. Therefore, the eggs of strains affecting lamellocyte morphology are protected from encapsulation by the host's blood cells. L. heterotoma eggs float freely in the host hemocoel but L. boulardi eggs are attached to host tissue surfaces. Lamellocytes cannot infiltrate the attachment site so the capsule around the L104 egg remains incomplete. The wasp larva uses this gap in the capsule as an escape hatch for emergence.

Microtubule inhibitors block the morphological changes induced in Drosophila blood cells by a parasitoid wasp factor.

The shape change of Drosophila melanogaster blood cells (lamellocytes) from discoidal to bipolar that is caused by a factor from the female parasitoid Leptopilina heterotoma is blocked by the tubulin inhibitors vinblastine and vincristine in vitro. The actin inhibitor, cytochalasin B, causes arborization of Drosophila lamellocytes and acts synergistically with the wasp factor to alter lamellocyte morphology. Lamellocyte aborization induced by cytochalasin B is blocked by simultaneous treatment with vinblastine. These observations indicate that the changes in lamellocyte shape induced by both the wasp factor and cytochalasin B require microtubule assembly.

Regulation of synthesis of larval serum proteins after transplantation of larval fat body into adult Drosophila melanogaster.

The larval serum proteins, LSP1 and LSP2, of Drosophila melanogaster are synthesized by the fat body during the third instar. We examined the potential for LSP synthesis by fat body implants in adult flies. Fat body from third instar donors will continue to synthesize LSPs in both males and females. Implants from late second instar larvae will start synthesizing LSP1 and LSP2 in females but only LSP1 in males, suggesting that regulation of these proteins is not the same and that the physiological milieu in the two sexes differs. The newly synthesized LSPs are secreted into the hemolymph for approximately 48 hr when secretion stops but synthesis continues. This sequence follows the pattern for LSP secretion in situ. Fat body from mid second instar larvae is variable in its ability to synthesize LSPs. LSPs are not detected in implants from first instar larvae despite there being a high level of protein synthesis in the implant and considerable growth of the fat body cells. We conclude that there is a critical stage of differentiation during the latter half of the second instar when the fat body becomes independent of the larval milieu and can synthesize LSPs in the adult.

Genetics of a Drosophila phenoloxidase.

An electrophoretic mobility variant of phenoloxidase in a lz stock of Drosophila melanogaster was identified as the A3 component of the phenoloxidase complex by using two different activators to study enzyme activity-natural activator isolated from pupae and 50% 2-propanol. The structural gene for the A3 proenzyme, Dox-3, was not associated with lz on the X chromosome; it mapped to the right of rdo (53.1) and left of M(2)m in the second linkage group. The lz locus affects the differentiation of the crystal cell, the type of hemocyte that carries prophenoloxidase(s) in paracrystalline form. Alleles of lz lacking paracrystalline inclusions in their hemocytes do not have phenoloxidase activity whereas alleles with paracrystalline inclusions have enzyme activity. The presence of proenzyme in the paracrystalline inclusions was demonstrated by in situ activation with natural activator or propanol followed by incubation in buffered dopa.

Selective destruction of a host blood cell type by a parasitoid wasp.

Foreign objects that enter the hemocoel of Drosophila melanogaster larvae are encapsulated by one type of blood cell, the lamellocyte, yet eggs of the parasitoid wasp Leptopilina heterotoma remain unencapsulated in D. melanogaster larval hosts that have many lamellocytes. Here we demonstrate that shortly after a female wasp oviposits in the hemocoel the lamellocytes undergo morphological changes and lose their adhesiveness. These affected blood cells are eventually destroyed as the parasitoid egg continues its development. The factor responsible for lamellocyte destruction, lamellolysin, is contained in an accessory gland of the female reproductive system and is injected along with the egg into the host hemocoel. Lamellolysin does not alter the morphology or the defense functions of the other types of blood cells in the host.

Basement membrane polarizes lectin binding sites of Drosophila larval fat body cells.

Vertebrate epithelial cells in monolayers are asymmetrical in that their apical and basal membranes differ in morphology and function. That this cell polarity depends on the presence of tight junctions can be demonstrated by labelling one surface of a cell monolayer in culture with fluorescent lectins and lipid probes, and subsequently observing whether the labels disperse to the opposite cell surfaces. Here we report on a differential distribution of binding sites for the lectin wheat germ agglutinin (WGA) on the cells of Drosophila melanogaster larval fat body, and show that the pattern is correlated with the structural association between the cell surfaces and their overlying basement membrane.

Blood cell surface changes in Drosophila mutants with melanotic tumors.

When wheat germ agglutinin conjugated to fluorescein isothiocyanate is bound to hemocytes from larvae of Drosophila melanogaster, two populations of hemocytes are distinguished. One shows a fluorescent speckled surface (spk+) and the other lacks this characteristic (spk-). In mutant larvae with melanotic tumors and in larval hosts with heterospecific implants, most of the lamellocytes (a hemocyte variant involved in capsule formation and tissue rejection) are spk+, whereas the lamellocytes in nontumorous larvae are spk-. This suggests that spk+ lamellocytes are necessary for encapsulation of aberrant tissues in the mutant larvae and are responsible for rejection of foreign tissue implants.

Drosophila larval fat body surfaces : Changes in transplant compatibility during development.

The hemocytes oftu-Sz ts melanotic tumor larvae ofDrosophila melanogaster encapsulate heterospecific and surface-modified homospecific tissue implants, but do not encapsulate unmodified homospecific implants (R. Rizki and Rizki 1980). In the present study we usedtu-Sz ts hosts to assay changes in larval fat body surfaces during development. Donor fat bodies from various ages of larvae were accepted (remained unencapsulated) intu-Sz ts hosts whereas fat bodies from donors with everted spiracles and all subsequent stages of development that were tested were rejected (encapsulated). Since the demarcation between acceptance and rejection by thetu-Sz ts blood cells did not coincide with the gross morphological changes that appear in the fat body during metamorphosis (dissolution of the basement membrane and dispersal of the freed fat body cells at pupation), we compared acceptable and nonacceptable fat body surfaces by three other methods. Fat body surface ultrastructure was examined, fat bodies were treated with fluorescein isothiocyanate-conjugated lectins, and fat body surfaces were reacted with a monoclonal antibody specific for basement membrane. These approaches did not uncover fat body surface changes associated with eversion of the anterior spiracles, suggesting that recognition of tissue surface heterogeneities by the insect hemocytes exceeds the resolving power of the other three methods. However, the monoclonal antibody fails to bind to the basement membrane ofD. virilis larvae, whose fat body is always rejected intu-Sz ts hosts. This supports our suggestion that the molecular architecture of the basement membrane may be important in eliciting the encapsulation response.

Overlapping deficiencies refine the map position of the sex-linked actin gene of Drosophila melanogaster.

A 3H-labelled actin-specific probe was hybridized to Drosophila melanogaster X chromosomes heterozygous for deficiencies in the 5C region. The results suggest that the sex-linked actin gene resides in the overlap region of Df (1) C149 and Df (1) N73 at 5C3-4.

A mutant affecting the crystal cells inDrosophila melanogaster.

Black cells (Bc, 2-80.6±) mutant larvae ofDrosophila melanogaster have pigmented cells in the hemolymph and lymph glands. In this report we present evidence that these melanized cells are a mutant form of the crystal cells, a type of larval hemocyte with characteristic paracrystalline inclusions.Bc larvae lack crystal cells. Furthermore, the distribution pattern of black cells inBc larvae parallels that of experimentally-blackened crystal cells in normal larvae (phenocopy).InBc/Bc zygotes black cells appear during mid embryonic development but inBc +/Bc zygotes pigmented cells are not found until late in the first larval instar.Crystal cells are present in the heterozygous larvae until this time, and paracrystalline inclusions can be seen in some of the cells undergoing melanization in these larvae.The rate of phenol oxidase activity inBc +/Bc larval cell-free extracts is less than half that ofBc +/Bc +extracts whereas enzyme activity is undetectable inBc/Bc larvae. We propose that theBc +gene product is required for maintaining the integrity of the paracrystalline inclusions; inBc/Bc larvae either the product is absent or nonfunctional so an effective contact between substrate and enzyme results in melanization of the cells.Phenol oxidase itself is either destroyed or consumed in the melanization process accounting for the absence of enzyme activity inBc/Bc larvae. These studies confirm that the crystal cells store phenolic substrates and are the source of the hemolymph phenol oxidase activity in the larva ofD. melanogaster.

Developmental analysis of a temperature-sensitive melanotic tumor mutant inDrosophila melanogaster.

A sex-linked, temperature-sensitive melanotic tumor mutation inDrosophila melanogaster, tu (1) Sz ts, was mapped at 34.3±and localized to bands 10A10-11 of the polytene chromosomes. At 26°Ctu-Sz ts larvae develop melanotic tumors whereas 18°C is non-permissive for tumor formation. Tumorigenesis at 26°C involves the encapsulation of abnormal caudal fat body regions by precociously differentiated hemocytes. Low temperature blocks the development of the abnormal adipose cells and the overlying aberrant tissue surfaces but does not inhibit precocious differentiation of the hemocytes to the lamellocytic form. This phenotypic difference at the two temperatures indicates that lamellocyte encapsulation to form melanotic tumors is directed against abnormal tissue surfaces. On the basis of these observations and an earlier study (Rizki and Rizki 1979) we propose that hereditary melanotic tumors inD. melanogaster are a calss of autoimmune disorders in which affected tissue surfaces arouse the body's cellmediated defense response.

Hemocyte responses to implanted tissues inDrosophila melanogaster larvae.

At 26° C temperature-sensitivetu(1) Sz ts larvae ofDrosophila melanogaster develop melanotic tumors consisting of aberrant caudal adipose tissue encapsulated by precociously differentiated hemocytes (lamellocytes). Whentu-Sz ts larvae are grown at 18° C, lamellocytes are present but the caudal fat body surfaces remain normal and melanotic tumors do not develop (Rizki and Rizki, preceding paper). In this paper we demonstrate that the lamellocytes intu-Sz ts larvae at 18° C encapsulate implants of mechanically-damaged fat bodies and adipose cells devoid of basement membrane, while leaving host fat bodies or implanted fat bodies with intact basement membrane unencapsulated. Therefore, low temperature blocks melanotic tumor formation by normalizing the surfaces of the prospective tumor-forming sites intu-Sz ts.The discriminatory ability oftu-Sz ts lamellocytes was examined by challenging them with undamaged heterospecific tissues. Tissues from sibling species ofD. melanogaster were not encapsulated whereas tissues fromDrosophila species outside theD. melanogaster species subgroup were. Ultrastructural examination of encapsulated heterospecific tissues showed intact basement membrane, so we propose that distinction between "self" and "not self" by lamellocytes depends upon the molecular architecture of the basement membrane. In similar series of experiments usingD. virilis donor tissues inOre-R wild type larval hosts, fat bodies remained unencapsulated and imaginal disks metamorphosed. These studies suggest that continued presence of lamellocytes in the larval host is a prerequisite for encapsulation.

Cell interactions in the differentiation of a melanotic tumor in Drosophila.

The cellular events in the formation of melanotic tumors in the tu-W mutant larva of Drosophila melanogaster are described. The first step is the differentiation of spherical hemocytes to flattened cells, the lamellocyte variants. Subsequently, the surface of the caudal fat body undergoes changes to which the hemocytes respond by forming cellular capsules. The hemocytes utilize two mechanisms in this process: (1) phagocytosis of small particulate materials escaping from the adipose cells, (2) adhesion to form a multilayered wall of lamellocytes. Differentiating hemocytes in the vicinity of the tumor-forming site extrude membrane-bound vesicles that tend to adhere to the hemocyte surfaces. These vesicles are trapped between the lamellocytes as they pile in layers to form the capsule wall. It is suggested that the vesicles play a role in lamellocyte-to-lamellocyte adhesion during the initial stages of hemocyte aggregation at the tumor-forming site.

Modification of Drosophila cell surfaces by concanavalin A.

The effect of Con A on the surface morphology of cultured cells of Drosophila melanogaster growing on coverglasses was examined by scanning electron microscopy. With low lectin concentrations (5--10 microgram/ml) surface filaments disappeared and the cells flattened and spread against the glass surface. Cytoplasmic fusion bridges were observed in areas where cells made contact. Concentrations of Con A ranging between 50--500 microgram/ml caused cell shrinkage and surface distortions without cell flattening and filament loss. These morphologic effects were not apparent if Con A binding sites were blocked by preincubation with alpha-methyl-D-mannopyranoside before application to the cell cultures. However, once the Con A-mediated changes were in effect, the cells failed to show recovery when they were returned to growth medium and a majority of the cells on the coverglasses degenerated. Presumably the cells whose morphology appears unaffected by Con A treatment are the survivors that repopulate cultures returned to growth medium.