Functional Significance of the Crystal Cells in the Larva of Drosophila melanogaster

Cytoplasmic crystalline inclusions are found in some larval haemocytes of Drosophila melanogaster. Blackening can be experimentally induced in these cells, and previously it was suggested that either the substrate or enzyme for the tyrosine-tyrosinase system leading to melanin production in Drosophila larvae is found in these inclusions in the crystal cells. The present report is an attempt to further localize the enzyme and substrate. Larvae have been fed on food containing α-C¹⁴-tyrosine and autoradiographs of the blood cells taken from these larvae subsequently prepared. The C¹⁴ activity in the crystal cells is restricted to the crystal inclusions in the cells and is significantly higher than that found in the other type of haemocytes, the plasmatocytes. When samples of the blood cells are incubated in DOPA solution, the extra-crystalline cytoplasm becomes blackened while the crystals themselves remain colorless. These observations are consistent with the hypothesis that the substrate is localized in the crystal inclusions whereas enzyme is found in the surrounding cytoplasm. An in vivo structural isolation would serve to separate enzyme and substrate rather than an inhibition by dehydrogenases as postulated by previous authors. In vitro examination with the phase microscope has shown that the crystal cells rupture easily and the crystals dissolve in the haemolymph. Therefore any treatment which tends to disrupt the structural integrity of the cell will allow the enzyme and substrate to come together. Humoral factors preceding metamorphosis might account for the in vivo release of the enzymatic reaction by initially altering the permeability of the cell.     

A mutant affecting the crystal cells inDrosophila melanogaster

Summary 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.     

Zinc induces caspase-dependent mitochondrial pathway of the programmed cell death in haemocytes of Drosophila melanogaster

Zinc is an essential trace element in cells. However, its high level in cytoplasm promotes activation of stress signaling pathways and may lead to cell death. In the present study we used Drosophila melanogaster blood cells (haemocytes), obtained from the third instar larvae, to study the effects of high concentrations of Zn(2+) on programmed cell death (PCD). We analyzed the activity of caspases, the level of caspase inhibitor protein DIAP1 and metallothioneins, as well as calcium concentrations and activity of mitochondria in haemocytes exposed to 0.35 and 1.7 mM concentrations of Zn. The obtained results showed that rapid increase of [Zn(2+)]( i ) in the cytoplasm up-regulates metallothionein MtnB but not MtnA gene expression in cells treated with Zn(2+) in both concentrations. Excess of Zn(2+) also induced activation of the initiator caspase Dronc, associated with the mitochondrial pathway of PCD, and the effector caspase DrICE. In turn, the activity of receptor-regulated Dredd caspase was not changed. The level of DIAP1 decreased significantly in haemocytes in the presence of high Zn(2+) concentration in comparison to untreated cells. Moreover, mitochondrial membrane potential was significantly decreased after exposure to Zn ions. These results indicate that high concentration of Zn(2+) in the cytoplasm of haemocytes induces PCD via a mitochondrial pathway and that caspases play a pivotal role in this process.     

Acid phosphatase activity in the larval salivary glands of developing Drosophila melanogaster.

Both the biochemical profile and the optical and fine structural localization of acid phosphatase activity in the larval salivary glands of developing Drosophila melanogaster is described. Biochemically, acid phosphatase shows peak activity in the glands of feeding larvae, followed by a marked decline. Directly preceding the onset of cell histolysis however, enzyme activity increases 1.5 fold and is maintained at this level. Histochemically, acid phosphatase activity initially appears as discrete point or lysosomal sources. As development proceeds, an intense and diffuse form of enzyme is seen, accompanying an extremely vacuolated cytoplasm. Ultrastructurally, the enzyme is located in lysosomes, Golgi elements, multivesicular bodies and both within, and on the extracisternal surface of the rough endoplasmic reticulum. This extracisternal or cytosolic form appears directly preceding cell lysis and eventually shows a comprehensive cellular distribution. Large numbers of acid phosphatase positive haemocytes are attached to the basal glandular surface at all developmental stages. In morphologically intact gland cells, discrete extracisternal enzyme activity appears associated with local areas of degradation.     

Haemocyte changes in D. Melanogaster in response to long gland components of the parasitoid wasp Leptopilina boulardi: a Rho-GAP protein as an important factor

The hymenopteran wasp Leptopilina boulardi (Figitidae) is a larval solitary parasitoid of Drosophila larvae of the melanogaster sub-group. The factors used by parasitoid females to prevent encapsulation of their eggs by the host are localized in the female long gland and reservoir. We report here the physiological effects of these factors on host haemocytes using in vivo injection experiments. The total number of haemocytes, the number of plasmatocytes and the number of crystal cells were not modified by injection of long gland extracts. In contrast, long gland extracts either from virulent or avirulent strains had a significant effect on the lamellocyte number. Compared to the Ringer control, the avirulent long gland products induced an increase of the lamellocyte number while virulent extracts induced a drastic decrease together with an alteration of the morphology of these cells. Interestingly, changes in the lamellocyte morphology were also observed following injection of the P4 protein, a major component of L. boulardi female long glands that displays a strong immune suppressive effect on Drosophila larvae. The implication of the P4 protein in suppressing the host cellular immunity is discussed in correlation with its predicted molecular function as a Rho-GAP protein.     

Ultrastructure of the haemocytes of Tetrodontophora bielanensis Waga (Collembola)

Five types of haemocytes: prohaemocytes, plasmatocytes, granular haemocytes, spherule cells and phagocytes, have been distinguished on the basis of ultrastructural studies. Prohaemocytes are ovoid cells with a simple structural organization. Plasmatocytes are larger; their cytoplasm contains well-developed rough endoplasmic reticulum, numerous mitochondria and free ribosomes. Granular haemocytes are the most numerous of the blood cells, characterized by the presence of electron-dense granules. The cytoplasm of spherule cells contains many spherules made up of filamentous material of medium electron density. Rough endoplasmic reticulum, free ribosomes and mitochondria are also found in the cytoplasm. Phagocytes are the largest haemocytes. Their cytoplasm contains an abundance of lysosomes and myelin structures. In addition to haemocytes, cells intermediate between plasmatocytes and granular haemocytes have been observed, which indicates that the granular haemocytes are derived from plasmatocytes.     

In vivo nanoscale analysis of the dynamic synergistic interaction of Bacillus thuringiensis Cry11Aa and Cyt1Aa toxins in Aedes aegypti

The insecticidal Cry11Aa and Cyt1Aa proteins are produced by Bacillus thuringiensis as crystal inclusions. They work synergistically inducing high toxicity against mosquito larvae. It was proposed that these crystal inclusions are rapidly solubilized and activated in the gut lumen, followed by pore formation in midgut cells killing the larvae. In addition, Cyt1Aa functions as a Cry11Aa binding receptor, inducing Cry11Aa oligomerization and membrane insertion. Here, we used fluorescent labeled crystals, protoxins or activated toxins for in vivo localization at nano-scale resolution. We show that after larvae were fed solubilized proteins, these proteins were not accumulated inside the gut and larvae were not killed. In contrast, if larvae were fed soluble non-toxic mutant proteins, these proteins were found inside the gut bound to gut-microvilli. Only feeding with crystal inclusions resulted in high larval mortality, suggesting that they have a role for an optimal intoxication process. At the macroscopic level, Cry11Aa completely degraded the gastric caeca structure and, in the presence of Cyt1Aa, this effect was observed at lower toxin-concentrations and at shorter periods. The labeled Cry11Aa crystal protein, after midgut processing, binds to the gastric caeca and posterior midgut regions, and also to anterior and medium regions where it is internalized in ordered “net like” structures, leading finally to cell break down. During synergism both Cry11Aa and Cyt1Aa toxins showed a dynamic layered array at the surface of apical microvilli, where Cry11Aa is localized in the lower layer closer to the cell cytoplasm, and Cyt1Aa is layered over Cry11Aa. This array depends on the pore formation activity of Cry11Aa, since the non-toxic mutant Cry11Aa-E97A, which is unable to oligomerize, inverted this array. Internalization of Cry11Aa was also observed during synergism. These data indicate that the mechanism of action of Cry11Aa is more complex than previously anticipated, and may involve additional steps besides pore-formation activity.     

Effect of the insect pathogenic bacterium Photorhabdus on insect phagocytes

Photorhabdus are insect pathogenic bacteria that replicate within the insect haemocoel following release from their entomopathogenic nematode symbionts. To investigate how they escape the cellular immune response we examined the effects of two strains of Photorhabdus, W14 and K122, on Manduca sexta phagocytes (haemocytes), in vitro and in vivo. Following injection of Esherichia coli into Manduca larvae, these non-pathogenic bacteria are rapidly cleared from the haemolymph and the number of free haemocytes transiently increases. In contrast, following injection of either strain of pathogenic Photorhabdus, the bacteria grow rapidly while the number of haemocytes decreases dramatically. In vitro incubation of haemocytes with either Photorhabdus supernatant reduced haemocyte viability, and the W14 supernatant caused distinct changes in the actin cytoskeleton morphology of different haemocyte cell types. In phagocytosis assays both Photorhabdus strains can inhibit their own phagocytosis whether the bacterial cells are alive or dead. Further, the supernatant of W14 also contains a factor capable of inhibiting the phagocytosis of labelled E. coli. Together these results suggest that Photorhabdus evades the cellular immune response by killing haemocytes and suppressing phagocytosis by mechanisms that differ between strains.     

Proteinaceous crystals in cells of virus infected plants

Crystal-containing organelles in cells of virus infected plants lying at chloroplasts and mitochondria are identical with single membrane-bound microbodies containing crystals of catalase described in healthy plants. Massive complex inclusions caused by turnip mosaic virus very frequently contain the same microbodies with crystal inclusions; that phenomenon may be related to some pathophysiological changes of virus infected plants. Comparable proteinaceous crystals, but not lying within microbodies limited by a membrane, may also be found in cytoplasm of infected cells. These crystals are sometimes surrounded by a substance resembling the microbody matrix. Disintegrated cytoplasm of virus infected cells may also contain the same crystals lying free in “empty spaces”. Cytopathological effects responsible for this phenomenon and possible artifacts as well are discussed.     

Suppression of Drosophila cellular immunity by directed expression of the ExoS toxin GAP domain of Pseudomonas aeruginosa

We show here that transgenic Drosophila can be used to decipher the effect of a bacterial toxin on innate immunity and demonstrate the contribution of blood cells in fly resistance to bacterial infection. ExoS is a Pseudomonas aeruginosa exotoxin directly translocated into the host cell cytoplasm through the type III secretion system found in many Gram-negative bacteria. It contains a N-terminal GTPase activating (GAP) domain that prevents cytoskeleton reorganization by Rho family of GTPases in cell culture. Directed expression of the ExoS GAP domain (ExoSGAP) during fly eye morphogenesis inhibited Rac1-, Cdc42- and Rho-dependent signalling, demonstrating for the first time its activity on RhoGTPases in a whole organism. We further showed that fly resistance to P. aeruginosa infections was altered when ExoSGAP was expressed either ubiquitously or in haemocytes, but not when expressed into the fat body, the major source of NF-(kappa)B-dependent anti-microbial peptide synthesis. Fly sensitivity to infection was also observed with Gram-positive Staphylococcus aureus strain and was associated to a reduced phagocytosis capacity of ExoSGAP-expressing haemocytes. Our results highlight the major contribution of cellular immunity during the first hours after Drosophila infection by P. aeruginosa, an opportunist pathogen affecting patients with pathologies associated to a reduced leukocyte number.     

Basis of the trade-off between parasitoid resistance and larval competitive ability in Drosophila melanogaster

Drosophila melanogaster can be artificially selected for increased resistance against parasitoid wasps that attack the larvae. Lines selected for greater resistance are poorer larval competitors under conditions of resource scarcity. Here we investigated the mechanistic basis of this apparent trade-off. We found that resistant lines have approximately twice the density of haemocytes (blood cells) than that of controls. Haemocytes are involved in encapsulation, the chief cellular immune defence against parasitoids. We have previously shown that resistant lines feed more slowly than controls and hypothesize that limiting resources are being switched from trophic to defensive functions.     

HISTOPATHOLOGICAL AND HISTOCHEMICAL CHANGES IN HONEYBEE LARVAE (APIS MELLIFERA L.) AFTER INFECTION WITH BACILLUS LARVAE,THE CAUSATIVE AGENT OF AMERICAN FOULBROOD DISEASE

Morphological, histochemical and cytochemical changes were examined in honeybee larvae after infection with the bacterium Bacillus larvae. The results indicate cell necrosis in the midgut epithelium accompanied by increasing cell vacuolization and nuclear pyknosis following per os inoculation with B. larvae. Many autolysosomes were positive for acid phosphatase. Non-vacuolar acid phosphatase activity was also found in lysed cell compartments. No such activity was found in regenerative epithelial cells. Degradation of haemocytes, salivary glands and other tissues was also observed. Histochemical analyses after per cutaneous inoculation with B. larvae of three- and five-day-old honeybee larvae show intense non-vacuolar acid phosphatase activity followed by disintegration of infected salivary glands, epithelial cell cytoplasm and haemocytes.     

Iron containing nuclear inclusions in human pigmentary cirrhosis (author's transl)]

In human pigmentary cirrhosis nuclear (pseudo-)inclusions of cytoplasmic material, containing less or more degenerated and therefore faintly stained hemosiderin granules, are to be observed. But sometimes there are also finely fibrillar or granular proteinaceous materials, stainable by the Prussian-blue reaction, lying between the chromatin-strands or occupying the whole nucleus and displacing the chromatin to the nuclear envelope (margination of chromatin). Such uncoloured substances may condense into homogeneous masses and nearly hexagonal (0r related) crystals with a diameter up to 14 micron and a yellow-brownish colour, giving a strongly positive PERL's reaction. In contrast to the preceding stages intranuclear crystals of this kind have been observed in one case only. After destruction of the nuclear envelope and the marginated chromatin the crystals are lying free in the cytoplasm and later on, the cytoplasm being destroyed too, they may be ingested by von Kupffer cells. All the iron containing crystals, to be found in the cytoplasm, derive from former intranuclear inclusions. The intranuclear deposits of iron containing protein are interpreted as ferritin-aggregates. It is supposed that ferritin molecules, built up in the cytoplasm, do enter the nucleus via the pores of the nuclear envelope. Such an event not only signalizes a cytopathologic reaction but in turn may give rise to such additional cytopathologic lesions as cell shrinking and cell death.     

Insect haemocytes: what type of cell is that?

Classification of insect larvae circulating haemocytes is the subject of controversy, and the terminology used to designate each cellular type is often different from one species to another. However, a survey of the literature on insect haemocytes suggests that there are resemblances for most of the cell types and functions, in different insect species. In this review paper, we compare the structure and functions of circulating haemocytes in those insect species that are, by far, the most often used species for insect physiology studies, i.e. lepidopteran species and Drosophila. We show that there is high degree of homology of haemocyte types and suggest possible synonymies in terminology among species from these taxa.     

Notch signaling controls lineage specification during Drosophila larval hematopoiesis

Drosophila larval hemocytes originate from a hematopoietic organ called lymph glands, which are composed of paired lobes located along the dorsal vessel. Two mature blood cell populations are found in the circulating hemolymph: the macrophage-like plasmatocytes, and the crystal cells that contain enzymes of the immune-related melanization process. A third class of cells, called lamellocytes, are normally absent in larvae but differentiate after infection by parasites too large to be phagocytosed. Here we present evidence that the Notch signaling pathway plays an instructive role in the differentiation of crystal cells. Loss-of-function mutations in Notch result in severely decreased crystal cell numbers, whereas overexpression of Notch provokes the differentiation of high numbers of these cells. We demonstrate that, in this process, Serrate, not Delta, is the Notch ligand. In addition, Notch function is necessary for lamellocyte proliferation upon parasitization, although Notch overexpression does not result in lamellocyte production. Finally, Notch does not appear to play a role in the differentiation of the plasmatocyte lineage. This study underlines the existence of parallels in the genetic control of hematopoiesis in Drosophila and in mammals.     

Structural and functional characterisation of the blood cells of the bivalve mollusc,Scrobicularia plana

Light and electron microscopical studies were carried out in order to characterise the blood cells of the bivalve mollusc, Scrobicularia plana. Three types of haemocytes were recognised: eosinophilic granular haemocytes, basophilic granular haemocytes and basophilic agranular haemocytes. The eosinophilic granulocytes were vesicular and contained large granules whereas the basophilic granulocytes were found to contain small granules and glycogen 'lakes'. The basophilic agranular haemocytes were significantly smaller than the granular haemocytes and had a high nucleus to cytoplasm ratio. Functional characterisation of the blood cells identified activity for the lysosomal enzymes: acid phosphatase, beta-glucuronidase, non-specific esterase and arylsulphatase. There was also a weak staining reaction for phenoloxidase and peroxidase activities. Phagocytosis of Gram-positive bacteria was demonstrated by the haemocytes and antibacterial activity was shown by cell-free haemolymph. Assays to determine release of reactive oxygen species from the haemocytes did not detect any reactive oxygen generation.     

Drosophila haematopoiesis

Like in vertebrates, Drosophila haematopoiesis occurs in two waves. It gives rise to three types of haemocytes: plasmatocytes (phagocytosis), crystal cells (melanization) and lamellocytes (encapsulation of parasites). A first population of haemocytes, specified during embryogenesis, gives rise to an invariant number of plasmatocytes and crystal cells. A second population of haemocytes is specified during larval development in a specialized haematopoietic organ, the lymph gland. All three types of haemocytes can be specified in this organ, but lamellocytes only differentiate in response to parasitism. Thus, larval in contrast to embryonic haematopoiesis can be modulated by physiological constraints. Molecular cascades controlling embryonic haematopoiesis are relatively well established and require transactivators such as GATA, FOG and Runx factors, which are also co-opted in mammalian haematopoiesis. Mechanisms involved during larval haematopoiesis are less well understood although a number of chromatin remodelling factors and signalling pathways (JAK/STAT, Toll, Hedgehog, Notch) are required. In healthy larvae a pool of progenitors is maintained within the lymph gland, under the control of a signalling centre which expresses Collier, Serrate, Antennapedia and Hedgehog, and controls haemocyte homeostasis. Its key role in haemocyte homeostasis is reminiscent of interactions described in vertebrates between haematopoietic stem cells and their microenvironment (niche).