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.     

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.     

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

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.     

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).     

Cold resistance in all life stages of two blowfly species (Diptera, Calliphoridae)

1. Freezing was lethal in the eggs, larvae, pupae and adults of the blowflies Calliphora vicina R-D. and Calliphora vomitoria (L.), but varying degrees of supercooling were found. 2. Cold resistance (as determined by their supercooling capacity) was greatest in eggs (to c. -25 degrees C), and moderate in other life stages (range -7 to -13 degrees C), which was evident from the distributions of their supercooling points. 3. Water contents of all life stages varied from 63% to 77% of fresh weight; pupae of C. vomitoria contain the smallest proportion of water. No relationship between the amount of body water and supercooling ability was detected. 4. Glucose was the major compound in all life stages of C. vicina, with a maximum concentration of 8.22 micrograms mg-1 fresh weight in the adults. In addition, five other potential cryoprotectants occurred in concentrations greater than 0.1% of fresh weight. No correlation between solute concentration and supercooling was observed. 5. Both C. vicina and C. vomitoria have sufficient cold hardiness to avoid lethal freezing in sheltered habitats in southern Britain, which may render diapause unnecessary in both species.     

Effects of the parasitization of a braconid, Apanteles, on the blood of its host, Pieris

Parasitization of a braconid wasp, Apanteles glomeratus, of larvae of a common cabbage butterfly, Pieris rapae crucivora, caused changes in differential haemocyte count (DHC), total haemocyte count (THC), and encapsulative capacity against dead eggs of Apanteles in the fourth and fifth instar host larvae.However, no correlation could be found between the number of Apanteles eggs deposited and THC of the middle fourth instar host larvae or between the number of parasitoid larvae and specific gravity of the haemolymph from the late fifth instar host larvae.From the changes in DHC and in THC of both non-parasitized and parasitized Pieris larvae, an increase in the number of plasmatocytes of non-parasitized Pieris larvae in the early fourth instar period was supposed to be due to transformation of prohaemocytes into plasmatocytes, and a low population of plasmatocytes of parasitized larvae in the comparable period was assumed to be due to a suppression of transformation of prohaemocytes by some factor released from the parasitoid eggs.Failure of the parasitized fourth instar Pieris larvae to encapsulate injected dead eggs of Apanteles indicated that the parasitoid embryos were, in some way, actively inhibiting the encapsulation reactions of the host.The increase in THC of the parasitized fifth instar larvae could not be ascribed to a decrease in the volume of host haemolymph. Rather it could be interpreted by a suppression of adhesive capacity of haemocytes in the host haemocoel to tissue surfaces.Reduced encapsulative capacity of the parasitized fifth instar larvae might be attributed either to a depression of the adhesive activity of plasmatocytes resulting from a depletion of energy source for haemocytes in the host haemolymph by parasitization, or from an active suppression of adhesiveness of the plasmatocytes by secretions from ‘giant cells’ (teratocytes) originated from the parasitoid.     

Comparative study of hemocytes and associated cells of some medically important dipterans

The aim of this work is to study, characterize, and compare different morphological types of hemocytes of Glossina austeni, G. morsitans, Calliphora erythrocephala, Stomoxys calcitrans, Lucilia sericata, Aedes aegypti, and Culex quinquefasciatus. This information is intended to provide a basis for future studies of the cellular defense mechanisms of these dipterans. Seven morphological types of hemocytes were identified by phase-contrast optics: prohemocytes, plasmatocytes, thrombocytoids, granulocytes, adipohemocytes, oenocytoids, and spindle cells of various sizes. Adipohemocytes are difficult to distinguish from both fat body cells and granulocytes. All seven cell types are not present in every species. For example, thrombocytoids and spindle cells were not found in A. aegypti or C. quinquefasciatus, and oenocytoids were observed only in A. aegypti, C. quinquefasciatus, and in C. erythrocephala. In addition to the hemocytes, fat body cells and nephrocytes are also freely present in the hemolymph of some species but may have gained access to the blood during the bleeding process. With electron microscopy and with thick plastic sections of G. austeni hemolymph, only nephrocytes, plasmatocytes, granulocytes, and spindle cells were identified with any certainty, and the large spindle cells are morphologically very different from those found in the other dipterans. They are rigid cells supported by microtubules running throughout their entire length. These hemocytes are present in large numbers only in newly emerged flies, they are absent in larvae and young pupae, and are rare in old adults. Their disappearance from the hemolymph of newly emerged Glossina appears to be a result of phagocytosis by the plasmatocytes.     

Development rates of some pre-adult stages in blowflies with reference to low temperatures

Abstract. Duration of some pre-adult stages of Calliphora vicina Robineau-Desvoidy, C.alpina Zett., C.vomitoria Linn., Phormia terraenovae R.-D. and Lucilia sericata Meig. were determined at 3.5–26^oC, in the context of use of these insects in forensic estimates of post-mortem intervals. Egg development in C. vicina occurs down to at least 3.5^oC, larval growth to 4^oC and puparial formation, development and perfect adult emergence to 5^oC. Growth curves by successive weighing of individual larvae showed specific differences in rates of development of the above species at constant temperatures. Alternating temperatures for 12 h periods (such as 10^oC and 20^oC) to simulate diurnal changes, usually, but not at all temperature pairs tested, caused acceleration of growth relative to the corresponding constant temperature (15^oC in the above case), in C.vomitoria, P.terraenovae and L.sericata , but usually caused retardation of larval growth in C.vicina. In this species, larvae as single individuals on beef liver in small tubes grew on average at the same rates as those (50–100 larvae) growing communally. All species showed variation in larval growth between individuals, more marked in C.vicina , and slower-growing larvae showed increased unexplained mortality. Because of this and to avoid complication by fast-growing larvae from later laid egg batches on a carcass, growth curves given are those of mean weights of the heaviest 25% (upper quartile) of groups of 40–90 feeding larvae. Variation in growth rates, and acceleration or retardation of growth in different species under alternating temperatures, thus affect suggested procedures in obtaining estimates of post-mortem intervals using these insects.     

Hemocytes of Calliphora vicina larvae. I. Histological analysis

The rational classification of blood cells of a blowfly Calliphora vicina larva has been worked out basing on the studies of hemocyte morphology and their dynamics while ageing. Eight morphological cell types can be found during the third age in larva's hemolymph. Hemocytes of type I (prohemocytes) are undifferentiated cells, and, probably, serve as cambial elements of hemocytes of other types. Hemocytes of types II (thrombocytoids), III (cell platelets), IV, and V (early- and late post-thrombocytoids) possibly correspond to consecutive stages of a certain differentiation pattern. Hemocytes of types VI (filopodocytes), VII (crystal cells), and VIII (histolysocytes) posses a number of specific features and represent three independent cell lines. The existence of four independent cell lines in C. vicina hemolymph supports the polygenetic conception of hematopoiesis in insects. We compared our classification with other existing classifications of C. vicina larva blood cells, and proposed a new nomenclature of these cells. The existence of cells with crystal inclusions (type VII hemocytes) in C. vicina larva hemolymph has been shown for the first time. Data on two generations of blood cells during larval development of C. vicina were obtained. Hemocytes of types II, VI and VII belong to the first generation. These cells disappear completely at the stage of empty-poropped larva, when hemocytes of the second generation i.e. those of types III and VIII, appear in the hemolymph.     

Changes in haemolymph constituents of American bollworm, Helicoverpa armigera (Hübner), infected with nucleopolyhedrovirus

Six types of haemocytes viz., prohaemocytes, plasmatocytes (round, fusiform, vermiform and spindle shaped), granular cells, spherule cells, oenocytoids and adipohaemocytes were found in the haemolymph of larvae of American bollworm H. armigera. The total and differential haemocyte counts (THC and DHC) in H. armigera haemolymph were affected by nucleopolyhedrovirus (NPV) treatment. There was a general decrease in THC in response to NPV treatment in both young and old larvae. However the decrease was more apparent in 5 and 8 day old larvae than in 10 day old larvae. The differential haemocytes showed less of granular cells and more of spherule cells and prohaemocytes in the old larvae. Plasmatocytes and granular cells in 10 day old larvae initially phagocytosed polyhedra; however, disintegrated after 3 to 4 hr. The haemolymph of NPV treated larvae melanized slowly particularly in old larvae. Phenoloxidase (PO) activity decreased positively with granular cells and oenocytoids in 10 day old treated larvae. Cellular fraction had high level of PO activity, which was transferred to plasma in response to NPV infection in the older larvae. The role of NPV pathogenesis vis-à-vis immunity in insect is discussed.     

Selective depletion of the plasmatocytes in Galleria mellonella following injection of bacteria

Injection of a suspension of a bacterial pathogen, Bacillus cereus, into larvae of the wax moth, Galleria mellonella, resulted in the disappearance of plasmatocytes from the haemolymph. This depletion effect was dose dependent, and occurred within 5 min of injection of the bacteria. Similar effects, though of lesser intensity, followed injection of a number of other species of both pathogenic and nonpathogenic bacteria. This rapid and specific reaction may play a part in the natural response of insects to the injection of foreign bodies.     

Haemopoietic organs and effect of their ablation on total haemocyte count in lemon-butterfly,Papilio demoleus L

The haemopoietic organs in Vth instar larvae of P. demoleus are in the form of thin transparent cellular sheets, closely wrapped around the base of each wing-pad. Three cell types viz; prohaemocytes, plasmatocytes and oenocytoids appear to be derived from these organs and their ablation caused a reduction in cell number which, in turn, revealed that the haemocytes in general are derived both from the haemopoietic organs as well as from the circulating blood cells.     

黑翅痂蝗血细胞形态学的研究

【目的】蝗虫血细胞的研究以及不同血细胞类型间的演化关系,是蝗虫细胞免疫学的依据,也是防蝗治蝗的重要依据。【方法】本研究通过Wright-Giemsa对黑翅痂蝗Bryodema nigroptera Zheng血细胞进行了混合染色,并使用光学显微镜进行了观察。【结果】发现黑翅痂蝗血细胞包括原血胞、浆血胞、粒血胞、类绛色血细胞、囊血胞、脂血胞、蠕形细胞和巨核血细胞8种类型。【结论】在成虫体内,原血胞占细胞总数的1%,浆血胞占32.2%,粒血胞占57.3%,类绛色血细胞占1%,囊血胞占6%,脂血胞占1%,蠕形细胞占1%,巨核细胞占细胞总数的0.5%,粒血胞数量最多,巨核细胞最少。     

Ex vivo observation of insect hemocyte behavior against beads and nematodes in the presence of insect plasma

Recognition of foreign targets by insect hemocytes is a crucial first step for insect immunity against invading multicellular organisms in the hemocoel. To understand the mechanism of recognition, we observed the hemocyte behavior of Galleria mellonella Linnaeus (Lepidoptera: Pyralidae) larvae against beads and the nonparasitic nematode Caenorhabditis elegans (Maupas) (Rhabditida: Rhabditidae) in the presence of plasma ex vivo using time-lapse microscopy. Both granular cells and plasmatocytes adhered to and spread on the surface of beads and nematodes. In addition, the spread plasmatocytes actively moved over the beads and nematodes. These results suggest that not only granular cells but also plasmatocytes can recognize foreign targets in the presence of insect plasma and that spread plasmatocytes can actively search for foreign targets. Hemocyte adhesion to beads and nematodes ex vivo was similar to that of the in vivo 1?h after injection. A divalent cation chelator inhibited the spreading and adhesion of plasmatocytes ex vivo, but it did not affect the adhesion of granular cells. The present method enables the analysis of acute hemocyte response against foreign targets in the presence of plasma.

     

Ontogenetic study of three Calliphoridae of forensic importance through cuticular hydrocarbon analysis

Abstract.  The ontogeny of the cuticular hydrocarbons of three dipterans of importance to forensic entomology, Calliphora vomitoria (Linné), Calliphora vicina (Robineau-Desvoidy) and Protophormia terraenovae (Robineau-Desvoidy) (Diptera: Calliphoridae), was explored using gas chromatography analysis. The stages examined ranged from eggs to 8-day-old adults. Cuticular hydrocarbon profiles revealed that odd linear alkanes dominate in all three species. Short-chain carbon compounds in larvae and post-feeding larvae were seen to evolve to long-chain carbon compounds in pupae and adults. Discriminant analysis of cuticular hydrocarbon profiles showed a clear differentiation among the different stages (larvae, post-feeding larvae, pupae and adults) and within stages, according to the age of individuals. This study concluded that the postmortem interval can be determined from the composition of cuticular waxes in Calliphoridae in forensic situations.     

Impact of insecticides on the haemogram of Rhynocoris kumarii Ambrose and Livingstone (Hem., Reduviidae)

Abstract:  The haemogram of Rhynocoris kumarii Ambrose and Livingstone comprises prohaemocytes, plasmatocytes, granular haemocytes, cystocytes and oenocytoids. The impact of five insecticides, viz. monocrotophos, dimethoate, methylparathion, quinalphos and endosulfan on the total haemocyte count (THC) and differential haemocyte counts (DHC) was studied. All of the insecticides except endosulfan initially reduced both prohaemocytes and plasmatocytes, increased the granular haemocytes, altered the percentage of cystocytes and oenocytoids and increased the total haemocyte count (THC). On the contrary, endosulfan initially increased the prohaemocytes and plasmatocytes, decreased the granular haemocytes and also the THC. The highest impact on the DHC and THC was caused by methylparathion and monocrotophos and the least impact by endosulfan. Hence, endosulfan is considered as the safest insecticide followed by dimethoate and quinalphos among these five insecticides to use with R. kumarii .     

Separation and behavior in vitro of hemocytes from the moth,Pseudoplusia includens

Hemocytes collected from larvae of Pseudoplusia includens (Lepidoptera: Noctuidae) were separated by centrifugation on Percoll cushions. The procedure resulted in 95% purity of plasmatocytes and greater than 99% purity of granular and spherule cells. Medium supplemented with chicken serum enhanced cell viability and promoted spreading of plasmatocytes. Cell-free plasma and medium preconditioned by plasmatocytes or granular cells stabilized cells in vitro and also accelerated spreading of plasmatocytes relative to medium supplemented with chicken serum. Oenocytoids were the only morphotype that exhibited endogenous phenoloxidase activity, while granular cells and plasmatocytes were the only cells that endocytosed fluorescent beads in vitro. Granular cells and plasmatocytes ingested fluorescently labelled beads, both in mixed populations of hemocytes and after separation. Plasmatocytes were the only morphotype that encapsulated large foreign targets in vitro following separation. Separated granular cells attached and spread on the surface of foreign targets but never formed a multilayered capsule.     

Different defense strategies of Dendrolimus pini, Galleria mellonella, and Calliphora vicina against fungal infection

The resistance of Galleria mellonella, Dendrolimus pini, and Calliphora vicina larvae against infection by the enthomopathogen Conidiobolus coronatus was shown to vary among the studied species. Exposure of both G. mellonella and D. pini larvae to the fungus resulted in rapid insect death, while all the C. vicina larvae remained unharmed. Microscopic studies revealed diverse responses of the three species to the fungal pathogen: (1) the body cavities of D. pini larvae were completely overgrown by fungal hyphae, with no signs of hemocyte response, (2) infected G. mellonella larvae formed melanotic capsules surrounding the fungal pathogen, and (3) the conidia of C. coronatus did not germinate on the cuticle of C. vicina larvae. The in vitro study on the degradation of the insect cuticle by proteases secreted by C. coronatus revealed that the G. mellonella cuticle degraded at the highest rate. The antiproteolytic capacities of insect hemolymph against fungal proteases correlated well with the insects' susceptibility to fungal infection. The antiproteolytic capacities of insect hemolymph against fungal proteases correlated well with the insects' susceptibility to fungal infection. Of all the tested species, only plasmatocytes exhibited phagocytic potential. Exposure to the fungal pathogen resulted in elevated phagocytic activity, found to be the highest in the infected G. mellonella. The incubation of insect hemolymph with fungal conidia and hyphae revealed diverse reactions of hemocytes of the studied insect species. The encapsulation potential of D. pini hemocytes was low. Hemocytes of G. mellonella showed a high ability to attach and encapsulate fungal structures. Incubation of C. vicina hemolymph with C. coronatus did not result in any hemocytic response. Phenoloxidase (PO) activity was found to be highest in D. pini hemolymph, moderate in G. mellonella, and lowest in the hemolymph of C. vicina. Fungal infection resulted in a significant decrease of PO activity in G. mellonela larvae, while that in the larvae of D. pini remained unchanged. PO activity in C. vicina exposed to fungus slightly increased. The lysozyme-like activity increased in the plasma of all three insect species after contact with the fungal pathogen. Anti E. coli activity was detected neither in control nor in infected D. pini larvae. No detectable anti E. coli activity was found in the control larvae of G. mellonella; however, its exposure to C. coronatus resulted in an increase in the activity to detectable level. In the case of C. vicina exposure to the fungus, the anti E. coli activity was significantly higher than in control larvae. The defense mechanisms of D. pini (species of economic importance in Europe) are presented for the first time.     

东亚飞蝗血细胞形态学研究

通过Wright's染色和光学显微镜对不同地区东亚飞蝗的血细胞进行了观察,发现东亚飞蝗Locusta migratoria manilensis(Meyen)的血细胞包括原血胞、浆血胞、粒血胞和类绛血胞4种类型.原血胞的细胞核为红色,而其它3种血细胞的细胞核均被Wright's染液染成红色和蓝色2种类型.故认为浆血胞是...