A Regulatory Gene for Phenoloxidase Activity in Drosophila melanogaster

A recessive lethal mutationl(2)hemocausing the occurrence of melanotic tumors in homozygous Drosophilalarvae was found. The study of phenoloxidase (PO) activity revealed that the number of hemocytes with PO activity in homozygous larvae was significantly reduced (0.4 ± 0.24%), compared to wild-type larvae (6.3 ± 0.5%). On injury followed by injection with bacterial cells, the formation of melanotic thrombus did not occurred and hemocytes with PO activity were not recorded in homozygotes of line P103. Suppression of the activity of PO isozymes A₁and A₃was detected by means of electrophoretic analysis of homozygotes. According to gene mapping data, the localization of this mutation did not match any structural gene for known PO forms and is therefore related to a regulatory gene controlling the activity of the immune system ofDrosophila.     

Drosophila Immunity: Analysis of Larval Hemocytes by P-Element-Mediated Enhancer Trap

Our aim was to identify new genes involved in the cellular aspects of defense mechanisms of Drosophila, as well as in melanotic tumor formation processes that are linked to blood cell disregulation. We have screened 1341 enhancer detector fly lines for expression of the lacZ reporter gene in larval hemocytes at the end of the third instar. We have selected 21 lines in which we observed a reproducible lacZ expression in blood cells. These lines were classified according to the subsets of hemocytes in which lacZ was expressed, and we identified five lines that can be used as lamellocyte markers. Three lines were selected for further analysis. The first exhibited strong lacZ expression in all lamellocytes. The second expressed lacZ in plasmatocytes and lamellocytes, and exhibited a melanotic tumor phenotype in larvae homozygous for the insertion. A third line showed a striking insertion-linked phenotype of melanized lymph glands (the hematopoietic organ), which resulted in the total absence of circulating hemocytes in the mutant larvae. We anticipate that this mutation, which we named domino, will prove a useful tool in the analysis of the role of hemocytes during the various aspects of immune response and melanotic tumor formation.     

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

Summary 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.     

Sensitivity of larvae of Drosophila melanogaster mutant mus(2)201G1 to various analgesics

Sensitivity of larvae from mutagen-sensitive mus(2)201G1 mutant of Drosophila melanogaster to analgin, amidopyrine and antipyrine (4-64 mkmol/ml of medium) was studied. Relative frequency of flies homozygous for this mutation after combined development of homozygous and heterozygous flies in the medium with the analgetic was used as a criterion for larval sensitivity. It is shown that sensitivity of homozygotes was significantly higher than that of heterozygotes to all three analgetics. Relative sensitivity of homozygotes increased with the elevation of drugs concentration. The data obtained demonstrate the mutagenicity of the analgetics tested for Drosophila.     

Prophenoloxidase binds to the surface of hemocytes and is involved in hemocyte melanization in Manduca sexta

In insects, melanotic encapsulation is an important innate immune response against large pathogens or parasites, and phenoloxidase (PO) is a key enzyme in this process. Activation of prophenoloxidase (proPO) to PO is mediated by a serine proteinase cascade. PO has a tendency to adhere to foreign surfaces including hemocyte surfaces. In this study, we showed that in the naïve larvae of the tobacco hornworm Manduca sexta, hemolymph proPO bound to the surface of granulocytes and spherule cells but not to oenocytoids, and about 10% hemocytes had proPO on their surfaces. When larvae were injected with water (injury) or microsphere beads (immune-challenge), hemolymph proPO was activated, and the number of hemocytes with surface proPO/PO increased at 12 h post-injection, but dropped to the normal level at 24 h. Hemocyte surface proPO can be activated in vitro, leading to melanization of these hemocytes. The number of melanized hemocytes from the larvae injected with water or microsphere beads significantly increased. We also showed that neither hemocytes nor cell-free plasma alone triggered melanization of immulectin-2-coated agarose beads in vitro. However, agarose beads were effectively melanized by isolated hemocytes in the presence of cell-free plasma. Our results suggest that activation of hemocyte surface proPO may initiate melanization, leading to the systemic melanization of hemocyte capsules.     

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.     

Hemocyte reactions and early cellular changes during melanotic tumor formation in Drosophila melanogaster

In Drosophila melanogaster tu bw larvae melanotic tumors form as a result of a cell-mediated immune response involving the encapsulation and melanization by hemocytes of portions of the caudal adipose tissue. The tissue-specific encapsulation response is not due to the disintegration of the basement membrane surrounding the adipose tissue as is reported to be the case in other melanotic mutants. Prior to encapsulation large numbers of hemocytes appear in the circulation and begin to differentiate into flattened cells termed lamellocytes. This transformation occurs at a time when changes are noted within the adipose cells. The localized accumulation of blood cells near intact basement membrane suggests that abnormally developing adipose cells acquire altered molecular surfaces or release substances to which the hemocytes respond. The initial reaction of the hemocytes with the adipose tissue is cell lysis, and this is rapidly followed by the aggregation of numerous additional blood cells which eventually cohere to one another to form a multilayered capsule. What little evidence there is of disintegration of the basement membrane occurs only after hemocytes have lysed at the surface, and other blood cells begin to invade the adipose tissue. Melanization occurs first in the intercellular spaces along the plasma membranes of the lysed cells, and progresses from the innermost layers toward the periphery of the encapsulating cells. Since the changes observed in the hemocytes and adipose cells are precocious, occurring to a lesser degree later in normal development, the initial effect of the genetic mutation in tu bw larvae may be an endocrine dysfunction which causes an asynchronous and abnormal development of the caudal adipose tissue and/or the hematopoietic organs.     

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.     

高流动性蛋白基因过量表达对果蝇发育的影响

HmgD基因编码果蝇高流动性蛋白（High mobility group proteins, HMG）的同源物,它可以参与染色质的组装。目前关于HMGD蛋白在果蝇胚胎发育过程中的作用尚无定论。我们采用UAS-Gal4系统,通过功能获得性突变的方法研究了HmgD基因过量表达对果蝇发育的影响。结果表明：HmgD过量表达对果蝇胚胎期发育的影响较弱,而对后期幼虫的发育具有很大的影响;HmgD过量表达的果蝇胚胎死亡率增高,但这种影响不是很大,因为一部分胚胎仍然能够发育至成体;但是当HmgD在广泛表达的Gal4驱动子（ActGal4）的控制下过量表达时导致子代大量死亡,特别是用4个拷贝的转基因果蝇进行杂交时,后代中的突变型在三龄幼虫末期全部死亡;部分突变型幼虫体内长有黑色素瘤,其血淋巴中的血细胞数量极显著地高于野生型。RT-PCR分析表明,突变幼虫中与血细胞增殖有关的Ras-MAPK途径和Toll途径被异常激活。这些结果显示：HmgD过量表达可能引起染色质结构疏松,激活了特定的转录因子,从而引发了三龄幼虫期异常的转录调控,并导致幼虫死亡。     

Evidence for an elevated frequency of in vivo somatic cell mutations in ataxia telangiectasia.

Somatic cell mutation frequency in vivo was measured in individuals with high cancer risk who were from ataxia telangiectasia (A-T) families. The assay for somatic mutation measures the frequency of variant erythrocytes which are progeny of erythroid precursor cells with mutations that result in a loss of gene expression at the polymorphic glycophorin A (GPA) locus. Samples from 14 of 15 A-T homozygotes showed high frequencies of GPA gene expression-loss variant cells with normal expression of only one of the two alleles at the GPA locus (i.e., GPA hemizygous variant cells). The mean elevation of the frequency of hemizygous variant cells over those in normal controls and unaffected family members was 7-14-fold. A-T homozygotes also showed an increase in the frequency of cells in which one allele at the GPA locus had lost expression and in which the remaining allele was expressed at a homozygous level (i.e., GPA homozygous variant cells). Family members who are obligate A-T heterozygotes did not appear to have a significantly elevated frequency of GPA hemizygous or homozygous variant cells. These indications of elevated in vivo frequencies of variant erythrocytes in A-T homozygotes support a causal link between susceptibility to somatic mutation and susceptibility to cancer.     

Effect of gamma irradiation on the hemocyte-mediated immune response of Aedes aegypti against microfilariae

The effect of gamma irradiation on the melanotic encapsulation response of Aedes aegypti black eye Liverpool strain against inoculated Dirofilaria immitis microfilariae (mff) was assessed at 1, 2, 3, and 6 days postinoculation (PI). Mosquitoes received 6000 rad from a 137Cs source (Shepard Mark I irradiator) at 3 days postemergence and were inoculated with 15-20 mff 24 hr later. These mosquitoes were compared to nonirradiated controls that also were inoculated with 15-20 mff at 3 days postemergence. The immune response was significantly reduced in irradiated mosquitoes as compared with controls at all days PI. Although the response was significantly inhibited compared with controls, irradiated mosquitoes were still capable of eliciting a response against 69% of recovered mff at 6 days PI. External gamma irradiation did not significantly affect the proliferation of hemocytes associated with the melanotic encapsulation response of A. aegypti. The number of circulating hemocytes increased in irradiated mosquitoes in response to inoculated mff in a manner similar to nonirradiated, inoculated controls. Hemocyte monophenol oxidase activity, however, was significantly reduced in gamma-irradiated mosquitoes at 12 hr PI as compared with controls. The reduced immunological capacity of irradiated mosquitoes might be related to an interference with gene activity required for the synthesis or activation of enzymes that are directly or indirectly involved in the biochemical processes associated with the production of melanotic substances that sequester mff.     

Activation or detoxification of mutagenic and carcinogenic compounds in transgenic Drosophila expressing human glutathione S-transferase.

Sensitivity of transgenic Drosophila melanogaster with expression of a human gene encoding the glutathione S-transferase alpha subunit (GSTA1-1) to 1,2:5,6-dibenzanthracene (DBA) and 1,2-dichloroethane (DCE) was investigated in the somatic mutation and recombination test (SMART). We performed the same assay in control transgenic flies expressing the bacterial lacZ gene. Three types of transgenic Drosophila strains carrying GSTA1-1 were used: two transgenic strains homozygous for the second chromosome with a single-copy transgene insertion and one strain with two transgene insertions. Larvae carrying the lacZ gene were significantly more sensitive to genotoxic effects of DBA than those carrying three copies of the GSTA1-1 gene. The larvae with lacZ expression showed significantly lower sensitivity to DCE compared with those expressing GSTA1-1. Finally, a pretreatment with buthionine-sulphoximine (BSO) in experiment with DCE significantly decreased the frequency of mutation events in larvae with three GSTA1-1 copies in comparison with others.     

Developmental modulation of immunity: changes within the feeding period of the fifth larval stage in the defence reactions of Manduca sexta to infection by Photorhabdus

In insect pathogen interactions, host developmental stage is among several factors that influence the induction of immune responses. Here, we show that the effectiveness of immune reactions to a pathogen can vary markedly within a single larval stage. Pre-wandering fifth-stage (day 5) larvae of the model lepidopteran insect Manduca sexta succumb faster to infection by the insect pathogenic bacterium Photorhabdus luminescens than newly ecdysed fifth-stage (day 0) caterpillars. The decrease in insect survival of the older larvae is associated with a reduction in both humoral and cellular defence reactions compared to less developed larvae. We present evidence that older fifth-stage larvae are less able to over-transcribe microbial pattern recognition protein and antibacterial effector genes in the fat body and hemocytes. Additionally, older larvae show reduced levels of phenoloxidase (PO) activity in the cell-free hemolymph plasma as well as a dramatic decrease in the number of circulating hemocytes, reduced ability to phagocytose bacteria and fewer melanotic nodules in the infected tissues. The decline in overall immune function of older fifth-stage larvae is reflected by higher bacterial growth in the hemolymph and increased colonization of Photorhabdus on the basal surface of the insect gut. We suggest that developmentally programmed variation in immune competence may have important implications for studies of ecological immunity.     

Characteristics of the endocrine system in Drosophila melanogaster 1 (2)gl mutants, differing in time of death]

A comparative study of the state of the endocrine system and one of the larval organs, the salivary gland, has been carried out in larvae homozygous for the 1(2)gl gene. They differ in time of death (death at the third larval instar--larval allele, and at the prepupal state--prepupal allele). It is shown that homozygotes for the larval and prepupal allele have underdeveloped prothoracal glands. Corpora allata in homozygotes for the larval allele does not differ from the norm. Corpora allata in homozygotes for the prepupal allele is decreased proportionally to the decrease of prothoracal glands. A decrease of gland size is due to a decrease of the volume of cell but not to their number; this decrease is accompanied by the decrease of their relative DNA content. Salivary glands in homozygotes are reduced and comprise 80% of the normal size in homozygotes for the prepupal allele and 50% for homozygotes for the larval allele. Polyteny level in the salivary gland nuclei is much decreased as compared with the normal level. DNA level is more reduced in larvae homozygous for the larval allele.     

Identification of Immune System and Response Genes, and Novel Mutations Causing Melanotic Tumor Formation in Drosophila Melanogaster

We are using Drosophila as a model system for analysis of immunity and tumor formation and have conducted two types of screens using enhancer detector strains to find genes related to these processes; genes expressed in the immune system (type A; hemocytes, lymph glands and fat body) and genes increased in expression by bacterial infection (type B). For type A, tissue-specific reporter gene activity was determined. For type B, a variation of enhancer detection was devised in which β-galactosidase is assayed spectrophotometrically with and without bacterial infection. Because of immune system involvement in melanotic tumor formation, a third type was hypothesized to be found among types A and B: genes that, when mutated, have a melanotic tumor phenotype. Enhancer detector strains (2800) were screened for type A, 900 for B, and 11 retained for further analysis. Complementation tests, cytological mapping, P-element mobilization, and determination of lethal phase and mutant phenotype have identified six novel genes, Dorothy, wizard, toto, viking, Thor and dappled, and one previously identified gene, Collagen IV. All are associated with reporter gene expression in at least one immune system tissue. Thor has increased expression upon infection. Mutations of wizard and dappled have a melanotic tumor phenotype.