Hemocyte load and immune resistance to Asobara tabida are correlated in species of the Drosophila melanogaster subgroup

Larvae from six Drosophila species of the melanogaster subgroup were compared for both the hemolymph concentration of hemocytes and the ability to encapsulate the eggs of the parasitoid Asobara tabida (Hymenoptera; Braconidae). Results showed a high correlation between the parasitized hosts' concentration of circulating hemocytes and their aptitude to form a hemocytic capsule around the parasitic eggs. Two conditions seem to be required for the encapsulation of A. tabida eggs to succeed: one condition, which may relate to the recognition of the parasite by the host defense system, is the occurrence of a primary hemocytic response, which gives rise to the amplification of the hemocyte population; the other condition is the presence in the parasitized hosts of a hemocyte load large enough for the cellular capsule to be completed before the parasitic egg becomes protected by embedment within the host tissues. Since the concentration in hemocytes of the parasitized hosts is partially related to the concentration in hemocytes before parasitization, Drosophila species carrying a high hemocyte load could be better predisposed to resist A. tabida. Results are discussed in regard to the importance of a non-specific, quantitative character, such as the host hemocyte load, for the co-evolutionary immune interactions between A. tabida and its Drosophila hosts.     

Geographical variation in resistance of the parasitoid Asobara tabids against encapsulation by Drosophila melanoqaster larvae: the mechanism explored

Abstract. The braconid parasitoid Asobara tabida Nees attacks larvae of several Drosophila species in fermenting substrates. Northwestern and central European populations of the parasitoid attack mainly D.subobscura Collin. Southern European parasitoids attack mainly D.melanogaster Meigen. Larvae of this last species can defend themselves against parasitoids by encapsulating the parasitoid egg. Parasitoids from southern European populations are better able to resist encapsulation of their eggs than their northwestern and central European conspecifics. The eggs of southern European parasitoids appear to have a 'sticky' egg chorion. As a result of this 'stickiness' the eggs become embedded in host tissue where they are not completely covered by the host's blood cells. This leads to, at most, partial encapsulation of the egg. Parasitoid larvae can escape from partially closed 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.     

COSTS OF COUNTERDEFENSES TO HOST RESISTANCE IN A PARASITOID OF DROSOPHILA

Abstract The ability of a parasitoid to evolve enhanced counterdefenses against host resistance and its possible costs were studied in a Drosophila -parasitoid system. We reared Asobara tabida (Braconidae, Hymenoptera) exclusively on D. melanogaster to impose artificial selection for improved counterdefenses against cellular encapsulation, the main host defense against parasitism. Controls were reared on D. subobscura , the main host of the population of wasps from which the laboratory culture was derived and a species that never encapsulates parasitoids. We observed improved survival and avoidance of encapsulation in all five selection lines compared to their paired control lines, although there was unexpected variation among pairs. Improved survival was associated with parasitoid eggs becoming embedded in host tissue, where they were protected from circulating haemocytes. There were no differences among lines in average adult size, fat content, egg load, or performance on D. subobscura . However, the duration of the egg stage in selection lines was longer than that of control lines, probably because of reduced nutrient and/or oxygen supply when eggs are embedded in host tissue. We suggest that this delay in hatching reduces the probability of parasitoid survival if another parasitoid egg is laid in the same host (superparasitism or multiparasitism) and hence is a cost of enhanced counterdefenses against host resistance.     

Host refractoriness of the tobacco hornworm, Manduca sexta, to the braconid endoparasitoid Cotesia flavipes

Cotesia flavipes (Hymenoptera:Braconidae) is a gregarious endoparasitoid of several pyralid stemborer larvae of economic significance including the sugarcane borer, Diatraea saccharalis. In this study, the ability of this parasitoid to develop in a sphingid host, Manduca sexta, was tested. First, second, third, fourth, and even pharate fifth instar host tobacco hornworm larvae were readily parasitized by the female C. flavipes parasitoids but no wasp larvae hatched from the eggs in this refractory host. Instead, the parasitoid eggs were invariably encapsulated by the host's hemocytes and, ultimately, no parasitoids emerged from tobacco hornworm hosts. The first stages of encapsulation were evident at 2 h post-parasitization of the host M. sexta larvae, when the beginning stages of capsule formation were seen. The developmental fate of the host larvae with encapsulated parasitoids was variable. Most succumbed as abnormally small fifth instars or as post-wandering prepupal animals, while a few developed normally to the pupal stage. Dissection of all the larvae or pupae with encapsulated wasp eggs showed evidence of hemocytic encapsulation and melanization of the C. flavipes eggs. This report describes the association between C. flavipes and M. sexta, which appears to be an excellent model system for studying the physiological processes accompanying wasp egg encapsulation that result in death of the host as well as the parasitoid. Since the parasitoid egg never hatches, the system offers an excellent opportunity to identify and study the effects of parasitoid-injected polydnavirus and venom on host physiology.     

Passive evasion of encapsulation in Macrocentrus cingulum Brischke (Hymenoptera: Braconidae), a polyembryonic parasitoid of Ostrinia furnacalis Guenée (Lepidoptera: Pyralidae)

The hymenopteran Macrocentrus cingulum usually deposits one egg into the larval body cavity of lepidopteran Ostrinia furnacalis, and the egg subsequently splits into several dozens of embryos during its development. How the parasitoid eggs and embryos avoid encapsulation by the host's immune response remains unknown. We compared hemocyte counts, morphologies and behaviors between unparasitized O. furnacalis larvae, and larvae parasitized by M. cingulum. No distinct differences were observed. Sephadex A-25 beads elicited a strong encapsulation response when injected into the parasitized host larvae, which indicates that parasitism by M. cingulum does not affect host's cellular immunity. However, there were significant differences in the host's encapsulation reactions towards injected eggs from different sources. Injected M. cingulum mature eggs excised from the lateral oviducts of the female wasps were not encapsulated, while immature eggs or driselase treated mature ones provoked an encapsulation response within 2 h after injection. Inspection of eggs by transmission electron microscopy revealed that the driselase collapsed the surface fibrous layer of the eggs, indicating that surface fibrous layer may play a role in protecting eggs from host's immune attack.     

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.     

Drosophila hemopoiesis and cellular immunity

In Drosophila melanogaster larvae, three classes of circulating cellular immune surveillance cells (hemocytes) can be identified: plasmatocytes, crystal cells, and lamellocytes. Plasmatocytes are professional phagocytes most similar to the mammalian monocyte/macrophage lineage and make up approximately 95% of circulating hemocytes. The other approximately 5% of circulating hemocytes consists of crystal cells, which secrete components necessary for the melanization of invading organisms, as well as for wound repair. A third cell type known as lamellocytes are rarely seen in healthy larvae and are involved in the encapsulation of invading pathogens. There are no obvious mammalian counterparts for crystal cells or lamellocytes, and there is no equivalent to the lymphoid lineage in insects. In this review, I will discuss what is currently known about Drosophila hemopoiesis and the cellular immune response and where possible compare it to vertebrate mechanisms.     

A NOVEL TYPE OF HEMOCYTES LOCALIZING MELANIZATION WITH HIGH‐SPREADING BEHAVIOR IN MYTHIMNA SEPARATA

Lepidopteran larvae show a cellular response to invading foreign substances that are larger than hemocytes, for example, parasitoid eggs or larvae. This response is called hemocyte encapsulation and is often accompanied by phenoloxidase (PO)‐catalyzed melanization. In the present study, we artificially transplanted endoparasitoid larvae and small glass fragments into the hemocoel of the common armyworm, Mythimna separata. We observed that the host larva showed a cellular response and that, 2–4 h after transplantation, melanin formation was spatially confined to the surface of the encapsulated substances. We further noted that specific morphological hemocytes surrounded by melanin formation became attached to the surface of the foreign substances. We designated these hemocytes hyperspread cells (HSCs) on the basis of their specific characteristics and circumferential spread. We confirmed the occurrence of prophenoloxidase (PPO)/phenoloxidase (PO) on the periphery of the HSCs and in the substance secreted around the HSCs by using anti‐PPO antibody. We were unable to detect PPO‐mRNA in HSCs by using in situ hybridization, although we showed that oenocytoids contained PPO‐mRNA and PPO protein. We used light microscopy and scanning electron microscopy to discriminate five main types of circulating M. separata hemocytes. We observed that HSCs differed from plasmatocytes, but spread out well. Further, during the encapsulation process, HSCs appeared to provide a localized melanization spot on the surface of foreign invaders.     

Parasite suppression of the oxidations of eumelanin precursors in Drosophila melanogaster

In insects, eukaryotic endoparasites encounter a series of innate immune effector responses mediated in large part by circulating blood cells (hemocytes) that rapidly form multilayer capsules around foreign organisms. Critical components of the encapsulation response are chemical and enzyme-catalyzed oxidations involving phenolic and catecholic substrates that lead to synthesis of eumelanin. These responses are initiated immediately upon infection and are very site-specific, provoking no undesirable systemic responses in the host. In this study, we were interested to learn if the principal oxidation pathways leading to the synthesis of eumelanin in larvae of Drosophila melanogaster were targets for inhibition by immune suppressive factors (ISF) derived from a virulent strain of the endoparasitic wasp Leptopilina boulardi. Comparative in vitro assays monitored by sensitive electrochemical detection methods showed that ISF derived from female reproductive tissues significantly diminished the oxidations of the two diphenol eumelanin precursors, dopamine and 5,6-dihydroxyindole (DHI). The oxidations of the monophenol tyrosine, and two other related diphenols, dopa and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), were not significantly inhibited by ISF. The data suggest that melanogenesis represents at least one of the host responses suppressed by L. boulardi ISF, and that the oxidation pathways selectively targeted for inhibition are those synthesizing decarboxylated pigment precursors derived from DHI. These observations, together with previous reports of adverse effects of ISF on the ability of hemocytes to adhere to foreign surfaces, suggest a multifaceted approach by the parasitoid to circumvent the innate immune response of D. melanogaster.     

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.     

Parasitization of Manduca sexta larvae by the parasitoid wasp Cotesia congregata induces an impaired host immune response

During oviposition, the parasitoid wasp Cotesia congregata injects polydnavirus, venom, and parasitoid eggs into larvae of its lepidopteran host, the tobacco hornworm, Manduca sexta. Polydnaviruses (PDVs) suppress the immune system of the host and allow the juvenile parasitoids to develop without being encapsulated by host hemocytes mobilized by the immune system. Previous work identified a gene in the Cotesia rubecula PDV (CrV1) that is responsible for depolymerization of actin in hemocytes of the host Pieris rapae during a narrow temporal window from 4 to 8h post-parasitization. Its expression appears temporally correlated with hemocyte dysfunction. After this time, the hemocytes recover, and encapsulation is then inhibited by other mechanism(s). In contrast, in parasitized tobacco hornworm larvae this type of inactivation in hemocytes of parasitized M. sexta larvae leads to irreversible cellular disruption. We have characterized the temporal pattern of expression of the CrV1-homolog from the C. congregata PDV in host fat body and hemocytes using Northern blots, and localized the protein in host hemocytes with polyclonal antibodies to CrV1 protein produced in P. rapae in response to expression of the CrV1 protein. Host hemocytes stained with FITC-labeled phalloidin, which binds to filamentous actin, were used to observe hemocyte disruption in parasitized and virus-injected hosts and a comparison was made to hemocytes of nonparasitized control larvae. At 24h post-parasitization host hemocytes were significantly altered compared to those of nonparasitized larvae. Hemocytes from newly parasitized hosts displayed blebbing, inhibition of spreading and adhesion, and overall cell disruption. A CrV1-homolog gene product was localized in host hemocytes using polyclonal CrV1 antibodies, suggesting that CrV1-like gene products of C. congregata's bracovirus are responsible for the impaired immune response of the host.     

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.     

Development of the braconid wasp Cotesia flavipes in two Crambids, Diatraea saccharalis and Eoreuma loftini: Evidence of host developmental disruption

Cotesia flavipes is an important gregarious larval endoparasitoid of several crambid stem borers, including Diatraea saccharalis. The suitability of two crambid species, Eoreuma loftini and D. saccharalis, pests of sugarcane and rice in Texas, for C. flavipes development was tested. The effect of parasitization by C. flavipes on encapsulation response was assessed in vivo in both D. saccharalis and E. loftini. The results indicated that the parasitoid developed and emerged successfully in D. saccharalis larvae. Although E. loftini larvae were readily parasitized by C. flavipes parasitoids, no wasp larvae hatched from the eggs in this host because eggs were encapsulated by the host's hemocytes. The developmental fate of the E. loftini larvae with encapsulated parasitoids was variable. Most died as abnormal fifth instars or as post-wandering prepupae, while a few developed normally to the pupal stage. In vivo experiments, there was a significant reduction in the percent of beads encapsulated in parasitized larvae in both hosts. However, the percent of beads showing melanization decreased significantly in parasitized D. saccharalis larvae but did not differ significantly in parasitized or unparasitized E. loftini larvae. Our results showed that D. saccharalis is a suitable host for C. flavipes whereas E. loftini is an unsuitable host. This study indicated that lepidopteran stem borers that are taxonomically, behaviorally, and ecologically very similar can differ in their ability to encapsulate a parasitoid species.     

Effect of the venom of the gregarious parasitoid Apanteles glomeratus on its hemocytic encapsulation by the host, Pieris

When eggs from the lateral oviduct of the gregarious parasitoid Apanteles glomeratus were injected with calyx fluid and venom apparatus material into host larvae, Pieris rapae crucivora, most of the eggs were not encapsulated. Apanteles eggs deposited by the parasitoid from which the venom apparatus was removed were usually encapsulated by the host. These results indicate that the parasitoid venom apparatus material is an important factor in suppressing the encapsulation of 1- or 2-day-old eggs in the host. In order to clearly demonstrate that the venom suppresses egg encapsulation but not the encapsulation of other foreign objects, DEAE-Sephadex A-50 ion-exchange particles stained with 0.001% (w/v) Congo Red solution were injected into hosts together with venom apparatus material. The Sephadex particles were encapsulated by host hemocytes. The results suggest that the venom does not inhibit the encapsulation ability of the host.     

Are insect immune suppressors driving cellular uptake reactions?

Many insect parasitoids that deposit their eggs inside immature stages of other insect species inactivate the cellular host defence to protect the growing embryo from encapsulation. Suppression of encapsulation by polydnavirus-encoded immune-suppressors correlates with specific alterations in hemocytes, mainly cytoskeletal rearrangements and actin-cytoskeleton breakdown. We have previously shown that the Cotesia rubecula polydnavirus gene product CrV1 causes immune suppression when injected into the host hemocoel. CrV1 is taken up by hemocytes although no receptors have been found to bind the protein. Instead CrV1 uptake depends on dimer formation, which is required for interacting with lipophorin, suggesting a CrV1-lipophorin complex internalisation by hemocytes. Since treatment of hemocytes with oligomeric lectins and cytochalasin D can mimic the effects of CrV1, we propose that some dimeric and oligomeric adhesion molecules are able to cross-link receptors on the cell surface and depolymerise actin by leverage-mediated clearance reactions in the hemolymph.     

Effects of lamellolysin from a parasitoid wasp on Drosophila blood cells in vitro

Female parasitoid Leptopilina heterotoma inject a factor, lamellolysin, along with their eggs into the host hemocoel to destroy selectively host hemocytes that encapsulate foreign objects. In parasitized Drosophila melanogaster larvae, these hemocytes (lamellocytes) change from discoidal cells to bipolar cells that no longer adhere to each other to form capsules. To study the effects of lamellolysin on Drosophila lamellocytes in vitro, a giant strain of D. melanogaster was constructed to yield hemolymph with an abundance of lamellocytes. The effect of lamellolysin on the adhesivity of lamellocytes in vitro was demonstrated when the cells were gently rotated in the culture medium. Under these conditions, the bipolar shape of the affected lamellocytes resembled that of lamellocytes in parasitized hosts. When lamellocytes were exposed to lamellolysin in stationary culture medium, the elongation of the bipolar cells continued until they became threadlike. Lamellocytes fragmented in both stationary and rotating culture medium in the presence of lamellolysin, although loss of cellular material was more pronounced in the latter. This study demonstrates that lamellolysin acts directly and destructively on lamellocytes.     

Levels of encapsulation and melanization in two larval instars of Ostrinia furnacalis Guenée (Lep., Pyralidae) during simulation of parasitization by Macrocentrus cingulum Brischke (Hym., Braconidae)

Abstract:  The hymenopteran Macrocentrus cingulum is a polyembryonic endoparasitoid that uses larvae of the lepidopteran Ostrinia furnacalis as one of its host insects. Previous studies indicated that although this parasitoid does not transmit polydnaviruses when it lays its eggs, a layer of fibrous tissue on the surface of the eggs helps them to avoid being encapsulated by the immune system of the host insect. However, as eggs of M. cingulum that are laid into late instar larvae of O . furnacalis often do not survive, there is a tendency for the adults to lay their eggs in earlier instar larvae. We studied the amounts of encapsulation and melanization around beads of DEAE-Sephadex A-25 injected into the haemoceol of fourth and fifth larval instars of O. furnacalis . The beads were injected to simulate the presence of eggs of the parasitoid M. cingulum . We found that the levels of encapsulation and melanization around the beads increased with the age of the O. furnacalis larvae. Likewise, the total counts of the haemocytes circulating within the haemolymph increased with the age of the O. furnacalis larvae and were correlated negatively with the percentage of larvae parasitized by M. cingulum . It appears that young O. furnacalis possess a weak cellular defence, and as a result are more susceptible to being parasitized. Hence, the correlation between the levels of encapsulation and the age of the host insect appears simply to reflect passive evasion.     

敲低piwi基因对黑腹果蝇血细胞增殖及分化的影响

【目的】探究敲低piwi基因对黑腹果蝇Drosophila melanogaster血细胞增殖及分化的影响。【方法】利用黑腹果蝇e33C-Gal4和Hml-Gal4-UAS-2×EGFP品系分别与野生型w¹¹¹⁸和UAS-piwi RNAi品系杂交,实现在黑腹果蝇游离血细胞或淋巴腺中降低piwi基因的表达;采用免疫荧光染色方法检测Piwi蛋白在血细胞中的定位及其对黑腹果蝇血细胞增殖与分化的影响。【结果】Piwi蛋白在黑腹果蝇游离血细胞及整个淋巴腺中都表达,且主要定位在细胞质;敲低piwi基因导致游离血细胞数量明显增加,处于有丝分裂M期的细胞数量增加,但未影响游离血细胞中浆细胞及薄层细胞的分化;敲低piwi基因对淋巴腺血细胞增殖无影响,但导致浆细胞过度分化及薄层细胞的产生。【结论】piwi基因在果蝇游离血细胞中的缺失可引起血细胞过度增殖,而在淋巴腺中敲低可引起血细胞的异常分化。