Quantitative analysis of hemolymph monophenol oxidase activity in immune reactive Aedes aegypti

A hemocyte-mediated melanotic encapsulation reaction is elicited in adult Aedes aegypti in response to intrathoracically inoculated microfilariae of Dirofilaria immitis. The activity of monophenol oxidase in cell-free hemolymph collected from uninoculated, microfilariae-inoculated and saline-inoculated control mosquitoes was investigated using a quantitative radiometric assay that measured the amount of tritiated water formed during the hydroxylation of l-[3,5-³H]tyrosine to dopa. Enzyme activity in immune reactive hosts examined 2 days postinoculation was aproximately twice as high (96–206 nmol/min per mg protein) as in uninoculated or saline inoculated insects (34–80 nmol/min per mg protein). The augmented activity of the enzyme coincides in time with the early development of melanotic capsules around the microfilariae. The possible involvement of hemocytes in the activation and/or generation of monophenol oxidase in response to infection, and the metabolism of catecholamines in relation to insect immune responses are discussed.     

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.     

Immune expression in a damselfly is related to time of season, not to fluctuating asymmetry or host size

1. Variation in immune responsiveness within and among species is the subject of the emerging field of ecological immunology. The work reported here showed that individuals of Lestes forcipatus Rambur differ in their likelihood of mounting immune responses, and in the magnitude of those responses, against a generalist ectoparasite, the water mite Arrenurus planus Marshall. 2. Immune responses took the form of melanotic encapsulation of mite feeding tubes, occurred in the few days after host emergence, and resulted in mites dying without engorging. Such immune responses were more probable and stronger for hosts sampled later rather than earlier in the season. Such responses may act as selection affecting seasonal patterns of egg hatching and larval abundance of mites. 3. Contrary to expectation, metrics of host size (wing length) and wing cell fluctuating asymmetry were not related to the likelihood of immune responses. 4. The importance of season on immune expression of insects has not been explored in detail. These results suggest possible trade‐offs in allocation of melanin (or its precursors) to maturation versus immunity, and indicate the need for studies on the synergistic effects of weather and parasitism on host species that use melanotic encapsulation to combat parasites and pathogens.     

Drosophila serpin 27A is a likely target for immune suppression of the blood cell-mediated melanotic encapsulation response

Avirulent strains of the endoparasitoid Leptopilina boulardi succumb to a blood cell-mediated melanotic encapsulation response in host larvae of Drosophila melanogaster. Virulent wasp strains effectively abrogate the cellular response with substances introduced into the host that specifically target and effectively suppress one or more immune signaling pathways, including elements that control phenoloxidase-mediated melanotic encapsulation. The present study implicates involvement of the Drosophila Toll pathway in cellular innate immunity by regulating the serine protease inhibitor Serpin 27A (Spn27A), which normally functions as a negative regulator of phenoloxidase. The introduction of Spn27A into normally highly immune competent D. melanogaster larvae significantly reduced their ability to form melanotic capsules around eggs of L. boulardi. This study confirms the role of Spn27A in the melanization cascade and establishes that this pathway and associated blood cell responses can be activated by parasitization. The activation of phenoloxidase and the site-specific localization of the ensuing melanotic response are such critical components of the blood cell response that Spn27A and the signaling elements mediating its activity are likely to represent prime targets for immune suppression by L. boulardi.     

Genetic basis of encapsulation response in Anopheles gambiae

The phenomenon of encapsulation of invading organisms is widespread in insects. Co-evolution has produced an intricate balance between the immune responses of the host and immune-suppressive (or immune-evading) properties of the parasite. Genome-wide genetic mapping revealed different loci in Anopheline mosquitoes were involved in melanotic encapsulation of different malaria parasites. Certain isolates of human malaria parasites can still suppress or avoid the immune response from refractory mosquitoes. Similar interactions with parasitoids were observed in Drosophila melanogaster. Species-specific encapsulation locus was identified for two parasitoids, respectively, and virulent strain of parasitoid can suppress the immune system of an otherwise resistant fruitfly. It is believed that the encapsulation loci in both mosquitoes and fruitfly may encode gene products that function at the early stages of parasite/parasitoid recognition or immediate signaling events. Future research on membrane receptor molecules and their roles in insect immunity will yield interesting insights into mosquito-parasite interactions.     

Melanotic mutants in Drosophila: pathways and phenotypes

Mutations in >30 genes that regulate different pathways and developmental processes are reported to cause a melanotic phenotype in larvae. The observed melanotic masses were generally linked to the hemocyte-mediated immune response. To investigate whether all black masses are associated with the cellular immune response, we characterized melanotic masses from mutants in 14 genes. We found that the melanotic masses can be subdivided into melanotic nodules engaging the hemocyte-mediated encapsulation and into melanizations that are not encapsulated by hemocytes. With rare exception, the encapsulation is carried out by lamellocytes. Encapsulated nodules are found in the hemocoel or in association with the lymph gland, while melanizations are located in the gut, salivary gland, and tracheae. In cactus mutants we found an additional kind of melanized mass containing various tissues. The development of these tissue agglomerates is dependent on the function of the dorsal gene. Our results show that the phenotype of each mutant not only reflects its connection to a particular genetic pathway but also points to the tissue-specific role of the individual gene.     

The role of surface characteristics in eliciting humoral encapsulation of foreign bodies in Plasmodium-refractory and -susceptible strains of Anopheles gambiae

A refractory strain of the mosquito, Anopheles gambiae, melanotically encapsulates and kills many species of malaria parasites, whereas susceptible strains allow the parasites to develop normally. To study the role of surface characteristics in eliciting this immune response, 27 types of chromatography beads that differed in matrix type, charge, functional group, and functional group density were assayed for degree of melanotic encapsulation in refractory and susceptible mosquitoes. Overall, two glucan-based matrices, Sephadex (dextran) and cellulose, stimulated the strongest responses, regardless of functional group. Substituting matrix hydroxyl groups with functional groups on Sephadex and cellulose beads decreased the level of encapsulation. These results demonstrate that glucans induce melanotic encapsulation in An. gambiae. Beads with agarose, polystyrene, and acrylic matrices, and most methacrylate-based beads elicited little or no melanization; however, epoxide-methacrylate beads were encapsulated, demonstrating that glucans are not essential for eliciting a response. Comparisons between the two strains demonstrated that refractory mosquitoes melanized many bead types to a greater degree than did susceptible mosquitoes. On this basis, we propose that an important difference between the two strains is that one of the enzymes involved in the melanization pathway functions at a higher level in the refractory strain. Finally, of all beads tested, only 85% substituted CM-Sephadex beads were virtually unmelanized in susceptible mosquitoes but highly melanized in the refractory strain; thus, a specific surface microenvironment is necessary to demonstrate this effect.     

Immune response to nylon filaments in two damselfly species that differ in their resistance to ectoparasitic mites

1. Insects commonly resist parasites using melanotic encapsulation. Many studies measuring immune response use the amount of melanin deposited on an artificial object that has been inserted into the animal as a proxy of the amount of resistance that the host is capable of mounting to natural parasites. 2. The relevance of this methodology to immune response in natural insect populations needs further study. Here, we examined two temperate damselfly species to elucidate the relationships among damselfly size, natural resistance to mites, and the immune response mounted by the same damselflies against nylon filaments. 3. The damselfly species that had high rates of melanotic encapsulation of mites in nature deposited more melanin on the nylon inserts than the species with low rates of natural resistance. 4. In females of this species, those that had resisted mites naturally melanised the nylon filament more aggressively than those that did not resist mites. 5. Our results show some support for the use of nylon filaments to assess natural patterns of immune response in these damselflies, but also suggest that caution should be used in interpreting the responses.     

The effects of host age and superparasitism by the parasitoid, Microplitis rufiventris on the cellular and humoral immune response of Spodoptera littoralis larvae

To study the dynamics of stage-dependent immune responses in Spodoptera littoralis (Boisd.) larvae (Lepidoptera: Noctuidae), single and superparasitism experiments were carried out using the parasitoid Microplitis rufiventris Kok. (Braconidae: Hymenoptera). Compared to younger (preferred) host larvae, the older (non-preferred) host larvae displayed a vigorous humoral response that often damaged and destroyed the single wasp egg or larva. Superparasitism and host age altered both the cellular and humoral immune responses. Younger host larvae showed a stronger encapsulation response compared to older host larvae. Moreover encapsulation rates in younger hosts (e.g., second instar) decreased with increasing numbers of parasitoid eggs deposited/larvae. In older larvae, the encapsulation rate was low in fourth, less in fifth and absent in sixth instar hosts. Conversely, the order and magnitude of the cellular immune response in S. littoralis hosts were highest in second instar larvae with the first instar larvae being a little lower. The immune response steadily decreased from the third through to the fifth instar and was least obvious in the sixth instar. In contrast, the general humoral immune response was most pronounced in sixth instar larvae and diminished towards younger stages. The results suggest that both cellular and humoral responses are stage-dependent. Wasp offspring in younger superparasitized host larvae fought for host supremacy with only one wasp surviving, while supernumerary wasp larvae generally survived in older superparasitized larvae, but were unable to complete development. Older instars seem to have a method for immobilizing/killing wasp larvae that is not operating in the younger instars.     

Effects of juvenile hormones and precocenes on the immune response of Spodoptera littoralis larvae to supernumerary larvae of the solitary parasitoid,Microplitis rufiventris Kok

The study represents one of the first direct tests of juvenile hormones (JHs) on the cellular immune response of a lepidopteran insect, Spodoptera littoralis (Boisd.), superparasitized by a solitary parasitoid, Microplitis rufiventris Kok. JHII-treated S. littoralis larvae exhibited a significant reduction in encapsulation response to the supernumerary M. rufiventris larvae at different developmental ages (0-4, 10-14 and 24-26 h) of supernumerary M. rufiventris embryos within 48 or 144 h after parasitization than either JHI-treated or control hosts. The reduction in encapsulation response was significantly greater in hosts which received higher JHII dose (5 &mgr;g) than those treated with lower one (1 &mgr;g).The progression of the encapsulation, culminating in melanization in the capsules occurred more frequently in precocene-treated hosts than either JH-treatment or control ones. Application of the anti-juvenile hormones (PI or PII) caused stronger encapsulation reaction to the injured supernumerary parasitoid larvae than JHI or JHII. In PI-treatments, the reaction was rapid and dramatic by lower (25 &mgr;g) dose treatments than higher ones (70 &mgr;g).The capsules formed around the supernumerary larvae of M. rufiventris showed signs of melanization within 48 h in PI-treated hosts. Melanization was more pronounced in either PI- or PII-treated hosts prior emergence of successful parasitoid larvae. Generally, the injured supernumerary larvae were seen within melanin-rich capsules in PI- and PII-treated hosts. Melanization was not observed in most capsules of JHI- and JHII-treated hosts. Additional physiological effects were observed: (1) in some cases, application of JHI to parasitized S. littoralis larvae inhibited subsequent emergence of its wasp, M. rufiventris, and (2) teratocytes (cells of wasp origin) from PI or PII-treated hosts were smaller in size than those observed in the JH-treated or control hosts.Finally, the dramatic increase in encapsulation responses of precocene-treated S. littoralis larvae of the supernumerary of M. rufiventris and definitely opposite reactions in typical hosts but JHII-treated hosts suggested that the cellular defense reaction may be under inhibitory hormonal control.The results of the study have implications for medically important insects such as mosquitoes and their interactions with parasites, as well as having significance for biocontrol and mass rearing programs using parasitoids.     

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.     

Genetic correlation between melanization and antibacterial immune responses in a natural population of the malaria vector Anopheles gambiae

The immune system of invertebrates can mount different responses, including melanotic encapsulation and several antibacterial defense mechanisms. Variation of the efficacies of these responses is generally considered to be a product of the evolutionary pressure on each response due to infection by parasites. However, potential interactions and trade-offs among the different responses of the immune system could constrain the evolutionary potential of each response. In a natural population of the mosquito Anopheles gambiae, we measured the genetic association between the melanization response and an antibacterial response in two environmental qualities (well-fed and undernourished larvae). In both environments the two immune responses were positively genetically correlated: in full-sib families that were most likely to melanize a bead, injected bacteria were most likely to be cleared. Thus, our data do not support the idea of a trade-off among different outcomes of the invertebrate immune system, but rather that some families are overall immunologically superior to others.     

Effects of the mermithid nematode Ovomermis sinensis on the hemocytes of its host Helicoverpa armigera

Little is known about the mechanism by which mermithid nematodes avoid encapsulation responses of insect hosts. In this study, we investigated the influence of the mermithid nematode Ovomermis sinensis on host Helicoverpa armigera hemocyte number, encapsulation activity, spreading behavior and cytoskeleton. Parasitism by O. sinensis caused a significant increase in the total hemocyte counts (THC) and plasmatocyte numbers of H. armigera. However, in vivo encapsulation assays revealed that hemocyte encapsulation abilities of H. armigera were suppressed by O. sinensis. Moreover, parasitism by O. sinensis changed the spreading behavior and cytoskeletons of the host hemocytes. The results suggested that O. sinensis could actively suppress the hemocyte immune response of its host, possibly by destroying the host hemocyte cytoskeleton. This is the first report of a possible mechanism by which mermithid nematodes suppress encapsulation responses of insect hosts.     

Nitric oxide involvement in Drosophila immunity.

The augmented production of nitric oxide (NO) was observed during the hemocyte-mediated melanotic encapsulation responses of Drosophila melanogaster and D. teissieri. When introduced into the hemocoel of D. melanogaster larvae, NO activated the gene encoding the antimicrobial peptide Diptericin. These observations, together with previous studies documenting the production of superoxide anion (O(*-)(2)) and H(2)O(2) in immune-challenged Drosophila, provide evidence that reactive intermediates of both oxygen (ROI) and nitrogen (RNI) constitute a part of the cytotoxic arsenal employed by Drosophila in defense against both microbial pathogens and eukaryotic parasites. These ROI and RNI appear to represent an evolutionarily conserved innate immune response that is mediated by regulatory proteins that are homologous to those of mammalian species.     

Aedes aegypti: characterization of hemocyte polypeptide synthesis during wound healing and immune response to inoculated microfilariae

Hemocytes from adult, female Aedes aegypti, intrathoracically inoculated with microfilariae (mf) of the nematode Dirofilaria immitis, were compared to saline-inoculated and uninoculated controls using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 125I-labeling, and wheat germ agglutinin (WGA) binding techniques. Activation of wound healing and/or melanotic encapsulation responses by the inoculation of saline or mf into the host hemocoel induced alterations in the hemocyte activity of these mosquitoes. Protein assays of whole hemocyte lysates revealed that hemocytes from saline- and mf-inoculated mosquitoes had higher protein concentrations than uninoculated controls. Many polypeptides were seen within all three hemocytes preparations when stained with silver nitrate, but there was an overall increase in protein synthesis in hemocytes from inoculated mosquitoes. In addition, a 200-kDa polypeptide was uniquely expressed in hemocytes from inoculated mosquitoes. There were several prominent surface proteins labeled with 125I, and several of these increased dramatically in intensity during wound healing and/or a melanotic encapsulation response. Similar results were seen in two-dimensional separations. A set of basic polypeptides comigrated with an acidic polypeptide resulting in a surface protein of approximately 80-90 kDa that increased in inoculated mosquitoes. Hemocytes from inoculated mosquitoes exhibited a group of three acidic polypeptides, whereas hemocytes from uninoculated mosquitoes exhibited only one of these protein fragments. Three surface polypeptides bound 125I-labeled WGA, and binding of WGA to hemocyte surface polypeptides was successfully inhibited by the incubation of cells with the lectin and its competing sugar.     

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.     

Sexually dimorphic effect of mating on the melanotic encapsulation response in the harem‐defending Wellington tree weta,Hemideina crassidens (Orthoptera: Tettigonioidea: Anostostomatidae)

Reproductive activities are generally costly to immune responsiveness because limited resources required by reproduction are diverted away from immunity (and vice versa). Reproduction, however, is not expected to affect the immune response in males and females similarly as mating is expected to negatively affect male immunity more so than female immunity. Here, I test the phenotypic plasticity hypothesis in the Wellington tree weta (Hemideina crassidens), a sexually dimorphic orthopteran insect that is endemic to New Zealand. My laboratory experiment showed that although males had higher rates of melanotic encapsulation than females, contrary to prediction, females were the only sex significantly affected by mating and the effect was positive. In addition to immunity differing between the sexes, immune function can differ intrasexually, particularly when males are polymorphic and different investment strategies are used to maximize fitness. Male H. crassidens exhibit alternative mating strategies that are represented by three different morphotypes. I therefore explored whether the morphs differed in their melanotic encapsulation response and whether mating affected the morphs differently. I found no difference among morphs or an effect of mating on male immune response.     

Lethal(1) aberrant immune response mutations leading to melanotic tumor formation in Drosophila melanogaster

Using P element-mediated mutagenesis we have isolated 20 X-linked lethal mutations, representing at least 14 complementation groups, which exhibit melanotic tumor phenotypes. We present the systematic analysis of this interesting group of lethal mutations that were selected for their visible melanotic or immune response. The lethal and melanotic tumor phenotypes of each lethal(1) aberrant immune response (air) mutation are pleiotropic effects of single genetic lesions. Lethality occurs throughout the larval and early pupal periods of development and larval development is extended in some air mutants. The air mutant lethal syndromes include abnormalities associated with the brain, haematopoietic organs, gut, salivary glands, ring glands, and imaginal discs. Additional characterization of the melanotic tumor mutations Tuml and tu(1)Szts have indicated that the melanotic tumor phenotype is similar to that observed in the air mutants. These studies have led to the proposal that two distinct classes of melanotic tumor mutations exist. Class 1 includes mutants in which melanotic tumors result from "autoimmune responses" or the response of an apparently normal immune system to the presence of abnormal target tissues. The Class 2 mutants display obvious defects in the haematopoietic organs or haemocytes, manifested as overgrowth, and the resulting aberrant immune system behavior may contribute to melanotic tumor formation.