[Cellular immune system of surgical maggots Lucilia sericata (Diptera, Calliphoridae)].

In the hemolymph of surgical maggots Lucilia sericata seven types of hemocytes were revealed. These are prohemocytes, stable and unstable hyaline cells, thrombocytoids, spindle cells, larval plasmatocytes and plasmatocytes I-IV, which represent sequential stages of one cell line differentiation. In contrast to Calliphora hyaline cells, this type of hemocytes in cropemptying larvae of Lucilia is elongated or vermiform in shape. Hyaline cells may be transformed to both prothrombocytoids and unstable prophenoloxydase-producing cells. Appearance and differentiation of each hemocyte type is rigidly linked with a definite stage of development. In cellular defense the main role play juvenile plasmatocytes, plasmatocytes II and III and trombocytoides. Juvenile plasmatocytes are the most active ones. After charcoal particles injection they were instantly surrounded by the thick envelope of adhered alien particles and form uniform morules aggregations or conglomerates together with thrombocytoidal agglutinates. Plasmatocytes II and III during the early stages of differentiation may be involved in adhesion and phagocytosis of alien particles and during the last stages in the engulfing of apoptose desintegrated tissues. Thus the cellular defense reaction is assisted by 4 hemocyte types--prophenoloxydase-unstable hyaline cells, thrombocytoids, juvenile plasmatocytes and plasmatocytes I-IV.

[Cellular defense system of some synanthropic dipterans inhabitant of bacterially aggressive environment].

The hemocytic count and defense reaction within 4 families of higher Diptera: Tabanidae, Syrphidae, Muscidae and Sarcophagidae, whose larvae inhabit bacterially aggressive environment, were investigated. The least hemocytes types (3) were revealed in Tabanidae and Syrphidae larvae--prohemocytes, plasmatocytes and prophenoloxydase-containing unstable hyaline cells (oenocytoids). In Sarcophaga crassipalpis and Musca domestica stable hyaline cells and thrombocytoids or podocytoid-like cells can be added to this set. At the time of pupariation in Sarcophaga, new generation of prohemocytes is segregated into the hemolymph, which form small round or spindle-shaped hyaline cells. So, the number of plasmatocyte types in Sarcophaga increase to six. Typical to Calliphoridae juvenile plasmatocytes in the members of investigated families are absent. Among the one hemocyte type morphology also can vary, especially in unstable prophenoloxydase hyaline cells. In Drosophila there are crystal cells containing in the cytoplasm paracrystalloidal inclusions. In Calliphoridae there are big hyaline cells with homogenous cytoplasm producing circumferential bubbles. Both in Sarcophaga and Tabanidae they contain in their cytoplasm big globules. However in Sarcophaga they rapidly disintegrate, while in Tabanidae are maintained unchanged during hours. In Muscidae and Syrphidae prophenoloxydase extrusion occurs very early and these cells obtain pycnotic nuclei and very liquid cytoplasm with strings of granules. Thrombocytoids in Musca larvae are represented by big flattened anucleated irregular cytoplasm and "naked" nuclei and cytoplasmic fragments often with fan-like projections. Plasmatocytes in all species studied are the cells with pronounced phylopodies. In larvae they contain cytoplasmic catabolic inclusions and in pupa--ragments of apoptotic tissues. Clearance of hemolymph from alien particles in Sarcophagidae and Muscidae occur by thrombocytoides, while in Tabanidae by plasmatocyte nodulation. A differing case is Syrphidae whe-e charcoal injection produce depletion of hemolymph both from particles and all types of hemocytes. So the specimen of different higher Diptera families can use different schemes of cellular defense reaction.

[Response of Calliphora vicina larval hemocytes to abiotic and biotic foreign particles injection].

Human erythrocytes injection into the body cavity of Calliphora vicina postfeeding larvae results to their fast binding by thrombocytoidal fragments with agglutinates formation. There were almost none sites of lysis and degradation of erythrocytes in agglutinates even after shape modification and strands generation. Exceptions are zones of agglutinates with juvenile hemocytes, where destruction of erythrocytes is seen. The sequential injection of erythrocytes and charcoal particles leads to charcoal adhesion at first to agglutinates periphery and later to more deep stratum of cytoplasm between the erythrocytes. Under such conditions agglutinate formation period is accompanied with morphology variations which do not influence the intensity of agglutinating reaction. Juvenile plasmatocytes phagocytized the charcoal particles regardless of their concentration and duration of previous contact with erythrocytes. When mixture of abiotic and biotic particles was injected into post feeding larvae, crythrocytes and charcoal generate independent aggregations in the range of separate agglutinates. At the same time plasmatocytes form nodules consisting of temporary cell aggregations covered with cores of non phagocytized charcoal particles. These data testified that presumably lectin receptors responsible for foreign biotic and abiotic particles recognition are very near but not identical for different types of hemocytes. They may be specifical (for plasmatocytes) or integrated to different parts of cellular membrane (in thrombocytoids).

[Functions of Calliphora vomitotia larval hemocytes in recognition and elimination from hemolymph human erythrocytes and charcoal particles].

Investigation of Calliphora vomitotia hemocyte defense reaction to human erythrocytes shows that erythrocytes are recognized mainly by thrombocytoids. Adhesion to plasmatocytes and subsequent phagocytosis also takes place, but in a less degree. Agglutination of erythrocytes by thrombocytoids is increased after feeding cessation and remains at a high level during all the period of crop emptying. Entrapped erythrocytes not later than after five to eight minutes show signs of destruction and disintegrate into fragments. Later structureless masses can arise. The results of secondary injections of charcoal particles reveal that both thrombocytoidal agglutinates and plasmatocytes can engulf additional abiotic invaders even after filling by erythrocytes. Meanwhile despite the changing morphology of agglutinates their capability to adhere new batches of aliens remains unchanged at enough high level. These agglutinates and plasmatocytes phagocytized the charcoal particles independently of previous erythrocyte exposition. Injection of charcoal and erythrocytes mixture leads to appearance of agglutinates with erythrocytes and charcoal mixed together. We guess that foreign receptors obtain wide spectrum of affinity to all kinds of invaders.

[Functional morphology of blowfly Calliphora vicina hemocytes].

In the hemolymph of Calliphora seven types of hemocytes were revealed. These are prohemocytes, which are the stem cells, stable and unstable hyaline cells, thrombocytoids, spindle cells, juvenile plasmatocytes and plasmatocytes I-IV, which represent sequential stages of one cell line differentiation were registered. The margin between them is completion of the crop emptying and beginning of wandering stage. In the feeding and crop emptying larvae take place rising of hyaline cells, thrombocytoids and hyaline cells amount with parallel growth of their defense function. The second wave of hemogenesis occur in the end of crop emptying period. It is accompanied by burst of plasmatocyte I production with their subsequent differentiation to plasmatocytes II-IV. Production of stable hyaline cells and respectively prothrombocytoids may be regulated not only by hormonal background but also by inorganic or organic particles invaded into the hemocel. Three types of hemocytes are involved in loosing of hemolymph from alien particles, notably thrombocytoids, juvenile plasmatocytes and plasmatocytes I and II. Thrombocytoids are responsible for parasitic eggs encapsulation. In addition they can phagocytize tiny organic and inorganic particles. Juvenile plasmatocytes respond to alien invasion almost as quickly as thrombocytoids at the onset of invasion. Plasmatocytes I and II start phagocytosis more slowly, hours post invasion, frequently accumulating the particles previously catched by thrombocytoids. Plasmatocytes I can absorb foreign particles and group in morules and can also surround filled thrombocytoids forming distinctive capsules. Both morules and capsules are temporary structures and disintegrate some hours lately. It is supposed the existence of three levels of immune defence: the fast response reaction of thrombocytoids and juvenile plasmatocytes and slow cellular reactions of plasmatocytes I. They are prerequisites for more extensive humoral response.

[Agglutination and phagocytosis of foreign abiotic particles by bluebottle Calliphora vicina haemocytes in vivo. II. Influence of the previous septic immune induction on haemocytic activity].

The rate of Calliphora vicina haemocytic defense reaction to foreign particles injection depends on the larval age and on the previous bacterial immunization. Immunization of crop-empting larvae induces an evident increase in particles phagocytosis by juvenile plasmatocytes in 24 h after injection. Both the hemogram and the pattern of cellular defense reaction change significantly after crop-empting. Immunized larvae start intensive adhesion of foreign particles to plasmatocytes surface and formation of great aggregations of plasmatocytes (morules) no longer than in 34 min after injection. The period of particle-haemocyte adhesion is short-termed and no more than after 30 min cell aggregates dissociate and adhered charcoal particles pass to thrombocydoidal agglutinates. Unimmunized control larvae of the same age have shown no adhesion and morules formation. In immunized wadering and diapausing larvae, formation of capsules consisting of central thrombocydoidal agglutinate filled with alien particles and adherent plasmatocytes I is intensified. In contrast to moru-les, this capsule formation is not accompanied by charcoal particles adhesion to plasmatocytes. Immunization of mature larvae of C. vicina shown no prominent influence on both the rate of phagocytosis and the hyaline cells differentiation. It might be supposed that the receptors system is complex and the immunization both the mechanisms of foreigners recognition (adhesion, morulation and incapsulation) and the far more lately occurring phagocytosis.

[Effect of immunization on the in vivo immunocytes reaction to foreign particles in the larvae of the flesh fly Calliphora vomitoria].

Bacterial immunization of Calliphora vomitoria larvae induces hemocytic defense reaction in response to abiotic foreign particles injections. This reaction depends on the larval age and, consequently, the immunocytes composition. The juvenile plasmatocytes which are abundantly present in the end feeding and crop emptying larvae are initially very active and their reaction rate does not depend on the immunization. The plasmatocytes I appear after crop emptying. Immunization has a positive effect on their differentiation rate and, correspondently, the rapid progress in the defense response. The proportion of competent plasmatocytes in immunized larvae increases. The stable hyaline cells formation also depends on immunization, although elements of this response vary at different age steps. immunization of the end feeding and crop emptying larvae induces an increase in the percentage of hyaline cells, but alien particles injection does not provide additional stimulating effect. After crop emptying, immunization of wandering larvae ceases to exert an obvious direct influence, but significantly increases the sensitivity to charcoal injection. The emergence of foreign particles in the hemolymph of immunized wandering larvae causes a rapid increase in the number of hyaline cells due to prohemocytes and undifferentiated plasmatocytes differentiation.

[The foreign particles injection induces stable hyaline cells differentiation in the hemolymph of the blowfly Calliphora vicina larvae].

The stable hyaline cells (thrombocytoids precursors) are prevailing haemocytes type in young larvae of Calliphora vicina. Their concentration decreased significantly during the crop emptying and became completely absent in wandering larvae. However, the injection of foreign particles into the haemocoel induced evident increase in the number of stable hyaline cells by means of transformation from prohaemocytes within 24 h after the treatment. Maximum of hyaline cells concentration is achieved on the 2-3 day when the part of them starts to transform into prothrombocytoids. Injection of both abiotic (charcoal) and biotic (human erythrocytes) foreign particles exerts an identical effect. Puncture of the body wall, bacterial immunization and injection of saline did not induce hyaline cells appearance. In crop emptying larvae, the stable hyaline cells originate within the clusters of undifferentiated steam cells, i. e. prohaemocytes. After the completion of crop emptying in wandering and diapausing larvae, preliminary dedifferentiation of very young plasmatocytes may be also observed. It is suggested that specification of the stable hyaline cells is induced by thrombocytoids after engulfing of the injected foreign particles and forming of their agglutinates.

[Differentiation of the stable hyaline cells in response to foreign particles injections into the larvae of blowfly calliphora vomitoria].

Injection of foreign particles (charcoal and human erythrocytes) into the larvae of Calliphora vomitoria provokes the complex immune response including their phagocytosis, nodulation and encapsulation by plasmatocytes and thrombocytoids. Precursors of thrombocytoids and analogs of Drosophila lamellocytes are very frequent during the periods of feeding and crop emptying, but fully disappear in wandering larvae. Injection of charcoal or erythrocytes into crop emptying larvae leads also to a dramatic increase in the number of stable hyaline cells, precursors of thrombocytoids. The hyaline cells differentiate from prohaemocytes and, quite possibly, from young weakly-specialized plasmatocytes in a day after injection. Later they are transformed to prothrombocytoids and thrombocytoids. The number of hyaline cells and young plasmatocytes in the crop emptying larvae of C. vomitoria is far greater than that in the same age larvae of C. vicina. Presumably it accounts for significantly increasing rate of stable hyaline cells differentiation in the injected larvae of C. vomitoria. Their part after injection of charcoal particles or erythrocytes may reach 40-50 % of the main haemocyte number compared to 20-25% in C. vicina. After completion of the crop emptying, the rate of hyaline cells differentiation in response to the foreign particles injection is evidently reduced but remains to be distinctly visible. Injections of saline also stimulate the differentiation of the stable hyaline cells from prohaemocytes but elevation of their amount is more weak and gradual. The bacterial immunization and needle prick show no effect. The treatments, inducing the rising of hyaline cells differentiation, lead also to pupariation delay. This correlation suggests involvement of the endocrine system into this process.

[Agglutination and phagocytosis of foreign abiotic particles by hemocytes of the blowely, Calliphora vicina in vivo. I. Dynamics of hemocyte activity during larval development].

Three types of Calliphora larval hemocytes have been revealed to be involved in phagocytosis of abiotic foreign particles: thrombocytoids, larval plasmatocytes and plasmatocytes I. Thrombocytoids are the quickest to respond to the appearance of invaders. The onset of test particle entrapment by thrombocytoid cytoplasmic fragments was observed, depending on the larval age within 0.5-5.0 min after injection. Separated fragments were fused, forming strands or roundish agglutinates. Phagocytosis of carbon, carmine or Indian ink particles by larval plasmatocytes occurs far more lately, and no earlier than 20-30 min after injection. Plasmatocytes I are capable of foreign particles adhesion on their surface, with a subsequent morule formation, and of engulfing these particles. These two events start in different time periods: adhesion occurs in 5-10 min, while phagocytosis is observed in 1--3 h. The rate of test particle entrapment and stability of agglutinales clearly depends on the larval age. The most pronounced reaction of hemocytes to foreign particles may be observed by the end of feeding and crop emptying. The second, somewhat less expressed rise of activity occurs in mature larvae not long before the onset of pupariation. Diapause induction is accompanied by reducing activities of both plasmatocytes and thrompocytoids. The importance of different hemocyte types in cellular immune reaction of Calliphora vicina larvae, and co-ordination between plasmatocytes and thrombocytoids are discussed.

[Hemocytes of the blowfly Calliphora vicina and their dynamics during larval development and metamorphosis]. / Gemotsity miasnoi mukhi Calliphora vicina i ikh dinamika pri razvitii lichinok i induktsii metamorfoza

On the basis of in vitro observation of live cells and examination of stained slides of larval and prepupal Calliphora vicina hemolymph, seven types of hemocytes have been detected: prohemocytes, stable and unstable hyaline cells, thrombocytoids, spindle cells, larval plasmatocytes, and plasmatocytes I-IV, a. The last representing sequential stages of one cell line differentiation. Prohemocytes are basic cells, from which other forms of hemocytes derive outside the hemopoietic tissue, i.e. in free hemolymph. At the last larval instar, three waves of hemopoiesis occur. Either wave tends to increase the general number of cells and to change the quality of hemocyte population. The first wave occurs at the close of larva feeding and is accompanied by increase in the number of hyaline hemocytes, thrombocytoids and larval plasmatocytes. The second wave of hemopoiesis occurs after the larva's crop emptying. In this period the main increase of hemocyte population occurs at the expense of prohemocytes and plasmatocytes I. The most significant (five-fold) explosion of the population of free hemocytes takes place at the onset of pupariation and correlates with the rise of ecdysone titer. At the first stage of this peak, the amount of plasmatocytes I sharply increases. Further on these are rapidly differentiated into plasmatocytes II and III. After the puparium formation, hemocytes are reduced in number. Plasmatocytes III phagocytose fragments of destroyed larval tissues, pass to the stage of plasmatocytes IV (macrophages), and partially settle on tissues.