Haemocyte changes in resistant and susceptible strains of D. melanogaster caused by virulent and avirulent strains of the parasitic wasp Leptopilina boulardi

Two strains of Drosophila melanogaster (resistant and susceptible) were parasitized by a virulent or avirulent strain of the parasitoid wasp Leptopilina boulardi. The success of encapsulation depends on both the genetic status of the host strain and the genetic status of the parasitoid strain: the immune cellular reaction (capsule) is observed only with the resistant strain-avirulent strain combination. The total numbers of host haemocytes increased in all 4 combinations, suggesting that an immune reaction was triggered in all hosts. Resistant host larvae infected with the virulent or avirulent strains of parasitoid wasp had slightly more haemocytes per mm(3) than did susceptible host larvae at the beginning of the reaction (less than 15 h post-parasitization). This difference disappeared later. Only the virulent parasitoid strain caused the production of a high percentage of altered lamellocytes (from a discoid shape to a bipolar shape), half the total number of lamellocytes are altered. This suggests that the alteration of lamellocyte shape alone is not sufficient to explain the lack of capsule formation seen in resistant hosts parasitized by the virulent strain. Lastly, there were very few altered lamellocytes in resistant or susceptible hosts parasitized by the avirulent parasitoid strain, two combinations in which no capsule was formed. As is now established for Drosophila-parasitoid interactions, virus-like particles contained in the long gland of the female wasp affect the morphology of the lamellocytes. The results presented here are further proof of the action (direct or indirect) of virus like particles of the virulent strain on lamellocytes.     

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.     

Active suppression of D. melanogaster immune response by long gland products of the parasitic wasp Leptopilina boulardi

To develop inside their insect hosts, endoparasitoid wasps must either evade or overcome the host's immune system. Several ichneumonid and braconid wasps inject polydnaviruses that display well-studied immune suppressive effects. However, little is known about the strategies of immunoevasion used by other parasitoid families, such as figitid wasps. The present study provides experimental evidence, based on superparasitism and injection experiments, that the figitid species Leptopilina boulardi uses an active mechanism to suppress the Drosophila melanogaster host immune response, i.e. the encapsulation of the parasitoid eggs. The immune suppressive factors are localised in the long gland and reservoir of the female genital tractus, where virus-like particles (VLPs) have been observed. Parasitism experiments using a host tumorous strain indicate that these factors do not destroy host lamellocytes but that they impair the melanisation pathway. Interestingly, they are not susceptible to heating and are not depleted with prolonged oviposition experience, in contrast to observations reported for L. heterotoma, another figitid species. The mechanisms that prevent encapsulation of eggs from L. boulardi and L. heterotoma differ in several respects, suggesting that different physiological strategies of immunosuppression might be used by specialised and generalist parasitoids.     

An inherited virus influences the coexistence of parasitoid species through behaviour manipulation

The potential role of pathogens or parasites in maintaining species coexistence is well documented. However, the impact of vertically transmitted symbionts, that can markedly modify their host's biology, is largely unknown. Some females of the Drosophila parasitoid Leptopilina boulardi are infected with an inherited virus (LbFV). The virus forces females to lay supernumerary eggs in already parasitised hosts, thus allowing its horizontal transmission. Using two independent experimental procedures, we found that LbFV impacts inter-specific competition between L. boulardi and the related L. heterotoma. While L. boulardi rapidly outcompetes L. heterotoma in the absence of the virus, L. heterotoma was able to maintain or even to eliminate L. boulardi in the presence of LbFV. By forcing females to superparasitise, LbFV induced egg wastage in L. boulardi thus explaining its impact on the competition outcome. We conclude that this symbiont whose transmission is L. boulardi-density-dependant may affect the coexistence of Leptopilina species.     

Genetic analysis of contributions of dorsal group and JAK-Stat92E pathway genes to larval hemocyte concentration and the egg encapsulation response in Drosophila

Drosophila larvae defend themselves against parasitoid wasps by completely surrounding the egg with layers of specialized hemocytes called lamellocytes. Similar capsules of lamellocytes, called melanotic capsules, are also formed around "self" tissues in larvae carrying gain-of-function mutations in Toll and hopscotch. Constitutive differentiation of lamellocytes in larvae carrying these mutations is accompanied by high concentrations of plasmatocytes, the major hemocyte class in uninfected control larvae. The relative contributions of hemocyte concentration vs. lamellocyte differentiation to wasp egg encapsulation are not known. To address this question, we used Leptopilina boulardi to infect more than a dozen strains of host larvae harboring a wide range of hemocyte densities. We report a significant correlation between hemocyte concentration and encapsulation capacity among wild-type larvae and larvae heterozygous for mutations in the Hopscotch-Stat92E and Toll-Dorsal pathways. Larvae carrying loss-of-function mutations in Hopscotch, Stat92E, or dorsal group genes exhibit significant reduction in encapsulation capacity. Larvae carrying loss-of-function mutations in dorsal group genes (including Toll and tube) have reduced hemocyte concentrations, whereas larvae deficient in Hopscotch-Stat92E signaling do not. Surprisingly, unlike hopscotch mutants, Toll and tube mutants are not compromised in their ability to generate lamellocytes. Our results suggest that circulating hemocyte concentration and lamellocyte differentiation constitute two distinct physiological requirements of wasp egg encapsulation and Toll and Hopscotch proteins serve distinct roles in this process.     

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.     

A RhoGAP protein as a main immune suppressive factor in the Leptopilina boulardi (Hymenoptera, Figitidae)-Drosophila melanogaster interaction

To protect its eggs, the endoparasitoid wasp Leptopilina boulardi injects immune suppressive factors into Drosophila melanogaster host larvae. These factors are localized in the female long gland and reservoir. We analyzed the protein content of these tissues and found that it strongly differed between virulent and avirulent parasitoid strains. Four protein bands present in virulent long glands were eluted and their immune suppressive effect was assessed in vivo, allowing demonstrating a major effect of one of these proteins. The corresponding cDNA encodes a predicted 30 kDa subunit containing a Ras homologous GTPase Activating Protein (RhoGAP) domain, suggesting a possible involvement in the regulation of actin cytoskeleton changes. Using Western-blot experiments, we showed that this protein is abundant in virulent female long glands but is undetectable in virulent females deprived of long glands or in long glands from avirulent wasps. Its potential role in modifying the morphology and the adhesive properties of the host lamellocytes, involved in Drosophila cellular immune responses, is discussed.     

Haemocyte changes in D. Melanogaster in response to long gland components of the parasitoid wasp Leptopilina boulardi: a Rho-GAP protein as an important factor

The hymenopteran wasp Leptopilina boulardi (Figitidae) is a larval solitary parasitoid of Drosophila larvae of the melanogaster sub-group. The factors used by parasitoid females to prevent encapsulation of their eggs by the host are localized in the female long gland and reservoir. We report here the physiological effects of these factors on host haemocytes using in vivo injection experiments. The total number of haemocytes, the number of plasmatocytes and the number of crystal cells were not modified by injection of long gland extracts. In contrast, long gland extracts either from virulent or avirulent strains had a significant effect on the lamellocyte number. Compared to the Ringer control, the avirulent long gland products induced an increase of the lamellocyte number while virulent extracts induced a drastic decrease together with an alteration of the morphology of these cells. Interestingly, changes in the lamellocyte morphology were also observed following injection of the P4 protein, a major component of L. boulardi female long glands that displays a strong immune suppressive effect on Drosophila larvae. The implication of the P4 protein in suppressing the host cellular immunity is discussed in correlation with its predicted molecular function as a Rho-GAP protein.     

Convergent use of RhoGAP toxins by eukaryotic parasites and bacterial pathogens

Inactivation of host Rho GTPases is a widespread strategy employed by bacterial pathogens to manipulate mammalian cellular functions and avoid immune defenses. Some bacterial toxins mimic eukaryotic Rho GTPase-activating proteins (GAPs) to inactivate mammalian GTPases, probably as a result of evolutionary convergence. An intriguing question remains whether eukaryotic pathogens or parasites may use endogenous GAPs as immune-suppressive toxins to target the same key genes as bacterial pathogens. Interestingly, a RhoGAP domain-containing protein, LbGAP, was recently characterized from the parasitoid wasp Leptopilina boulardi, and shown to protect parasitoid eggs from the immune response of Drosophila host larvae. We demonstrate here that LbGAP has structural characteristics of eukaryotic RhoGAPs but that it acts similarly to bacterial RhoGAP toxins in mammals. First, we show by immunocytochemistry that LbGAP enters Drosophila immune cells, plasmatocytes and lamellocytes, and that morphological changes in lamellocytes are correlated with the quantity of LbGAP they contain. Demonstration that LbGAP displays a GAP activity and specifically interacts with the active, GTP-bound form of the two Drosophila Rho GTPases Rac1 and Rac2, both required for successful encapsulation of Leptopilina eggs, was then achieved using biochemical tests, yeast two-hybrid analysis, and GST pull-down assays. In addition, we show that the overall structure of LbGAP is similar to that of eukaryotic RhoGAP domains, and we identify distinct residues involved in its interaction with Rac GTPases. Altogether, these results show that eukaryotic parasites can use endogenous RhoGAPs as virulence factors and that despite their differences in sequence and structure, eukaryotic and bacterial RhoGAP toxins are similarly used to target the same immune pathways in insects and mammals.     

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.     

Cellular immune competence of a Drosophila mutant with neoplastic hematopoietic organs

In the Tum^l mutant of Drosophila melanogaster, the larval hematopoietic organs undergo neoplastic changes and release into circulation large numbers of blood cells. The lamellocytes, and to a lesser extent the plasmatocytes from which they are derived, are the cells that encapsulate various endogenous tissues and form melanotic tumors. The mutation is temperature sensitive, with maximum gene expression manifested at 29°C. The ability of Tum^l larvae to encapsulate eggs of the wasp parasite Leptopilina heterotoma is dependent not only on temperature, with host larvae much more immune reactive at 29°C than at lower temperatures (15° or 21°C), but also on the interval of time following infection when temperature shift experiments are performed. When the shift of parasitized larvae from 21° to 29°C is delayed by 18 hr the hosts are not as immune reactive as those shifted immediately after infection. Since Tum^l larvae are potentially highly immune reactive at the time of infection (with sufficient numbers of lamellocytes in circulation to encapsulate parasites), the low degree of immune competence in hosts shifted to 29°C after 18 hr or maintained at lower temperatures suggests that the increased capacity of blood cells to react against foreign surfaces is dependent on the cells acquiring new or altered recognition and adherence properties at 29°C. The 18-hr delay may provide the parasite with an opportunity to interfere with the acquisition of these specific cellular alterations. Differential hemocyte counts from parasitized larvae show abnormally low lamellocyte counts in susceptible hosts, indicating that successfully developing parasites interfere with the differentiation of hemocytes.     

Parasite-induced enhancement of hemolymph tyrosinase activity in a selected immune reactive strain of Drosophila melanogaster

Larval hemolymph tyrosinase activity in Drosophila melanogaster was detected with high performance liquid chromatography with electrochemical detection. The enzyme hydroxylated L-tyrosine, and oxidized the diphenol substrates L-dopa and dopamine. In larvae of a selected immune-reactive strain the rates of tyrosine hydroxylation, dopa oxidation, and dopamine oxidation were markedly increased during the early stages of melanotic encapsulation of the eggs of the parasitic wasp Leptopilina boulardi. Tyrosinase activity was not modified in parasitized larvae of a selected susceptible strain of D. melanogaster, in which hosts the parasitoids developed unmolested. During the same period of parasitization, the amount of free tyrosine in immune reactive larvae was approximately three times higher than in susceptible hosts. These data indicate that the tyrosinase system of the immune reactive strain is activated during parasitization, and this results in the synthesis of some precursors which ultimately produce a melanotic and sclerotic capsule around the eggs of the parasite. Based on known genetic information of the enzyme system in Drosophila, it appears that at least two genes may be involved in the activation process, one associated with the proenzyme for monophenol oxidase activity, and the second with the proenzyme for diphenol oxidase activity.     

Hemocyte responses to implanted tissues inDrosophila melanogaster larvae

Summary At 26° C temperature-sensitivetu(1) Sz ^ts larvae ofDrosophila melanogaster develop melanotic tumors consisting of aberrant caudal adipose tissue encapsulated by precociously differentiated hemocytes (lamellocytes). Whentu-Sz ^ts larvae are grown at 18° C, lamellocytes are present but the caudal fat body surfaces remain normal and melanotic tumors do not develop (Rizki and Rizki, preceding paper). In this paper we demonstrate that the lamellocytes intu-Sz ^ts larvae at 18° C encapsulate implants of mechanically-damaged fat bodies and adipose cells devoid of basement membrane, while leaving host fat bodies or implanted fat bodies with intact basement membrane unencapsulated. Therefore, low temperature blocks melanotic tumor formation by normalizing the surfaces of the prospective tumor-forming sites intu-Sz ^ts.The discriminatory ability oftu-Sz ^ts lamellocytes was examined by challenging them with undamaged heterospecific tissues. Tissues from sibling species ofD. melanogaster were not encapsulated whereas tissues fromDrosophila species outside theD. melanogaster species subgroup were. Ultrastructural examination of encapsulated heterospecific tissues showed intact basement membrane, so we propose that distinction between self and not self by lamellocytes depends upon the molecular architecture of the basement membrane. In similar series of experiments usingD. virilis donor tissues inOre-R wild type larval hosts, fat bodies remained unencapsulated and imaginal disks metamorphosed. These studies suggest that continued presence of lamellocytes in the larval host is a prerequisite for encapsulation.     

Biogenesis, structure, and immune-suppressive effects of virus-like particles of a Drosophila parasitoid, Leptopilina victoriae

Drosophila melanogaster larvae are attacked by virulent strains of parasitoid wasps. Females of Leptopilina heterotoma produce virus-like particles (VLPs) that efficiently destroy lamellocytes, a major larval immune effector cell type. We report here that L. victoriae, a closely related wasp species, also produces VLPs that trigger immune suppression responses in fly hosts. We compare the ability of immune suppression of the two parasitoids using a mutant host strain hopscotch^{Tumorous-lethal} (hop^Tum-l). hop^Tum-l larvae have two defects of hematopoietic origin: overproliferation of hemocytes and constitutive encapsulation of self-tissue by lamellocytes. The encapsulation phenotype is suppressed weakly by L. victoriae and strongly by L. heterotoma. In vitro studies on hop^Tum-l lamellocytes show that VLP-containing fluid from either wasp species induces lamellocyte lysis, but with different kinetics.Previously undocumented precursors of L. victoriae VLPs are synthesized in the long gland and are first visible within canals connecting secretory cells to the long gland lumen. VLP assembly occurs in the lumen. VLPs show multiple electron-dense projections surrounding a central core. Maturing particles appear segmented, singly or in arrays, embedded in the reservoir matrix. In sections, mature particles are pentagonal or hexagonal; the polygon vertices extending into spikes. Our results suggest that L. victoriae is likely to promote immune suppression by an active mechanism that is mediated by VLPs, similar to that used by L. heterotoma.     

Cellular immune response of brown soft scale Coccus hesperidum L. (Hemiptera: Coccidae) to eggs of Metaphycus luteolus Timberlake (Hymenoptera: Encyrtidae)

The parasitic wasp, Metaphycus luteolus Timberlake, is an endoparasitoid of various soft scale insects including brown soft scale, Coccus hesperidum L. Development of this parasitoid in scale hosts is hindered by encapsulation. In the present study, using transmission and scanning electron microscopy, we show that hemocytes are responsible for encapsulation, which is mediated by the direct deposition of cells and melanin on the surface of M. luteolus eggs. By 12 h post-oviposition, scale hemocytes, presumably granulocytes, aggregate, spread and directly lyse on the surface of parasitoid eggs. This process continues for at least 1 day and results in the gradual formation of a capsule. Two to three days post-oviposition, a melanized capsule is well formed and signs of chemical deposition are evident by examination of the outer surface of the capsule. These results demonstrate that soft scale insects are fully capable of melanotic encapsulation of foreign material mediated by hemocytes.     

Influence of the virus LbFV and of Wolbachia in a host-parasitoid interaction

Symbionts are widespread and might have a substantial effect on the outcome of interactions between species, such as in host-parasitoid systems. Here, we studied the effects of symbionts on the outcome of host-parasitoid interactions in a four-partner system, consisting of the parasitoid wasp Leptopilina boulardi, its two hosts Drosophila melanogaster and D. simulans, the wasp virus LbFV, and the endosymbiotic bacterium Wolbachia. The virus is known to manipulate the superparasitism behavior of the parasitoid whereas some Wolbachia strains can reproductively manipulate and/or confer pathogen protection to Drosophila hosts. We used two nuclear backgrounds for both Drosophila species, infected with or cured of their respective Wolbachia strains, and offered them to L. boulardi of one nuclear background, either infected or uninfected by the virus. The main defence mechanism against parasitoids, i.e. encapsulation, and other important traits of the interaction were measured. The results showed that virus-infected parasitoids are less frequently encapsulated than uninfected ones. Further experiments showed that this viral effect involved both a direct protective effect against encapsulation and an indirect effect of superparasitism. Additionally, the Wolbachia strain wAu affected the encapsulation ability of its Drosophila host but the direction of this effect was strongly dependent on the presence/absence of LbFV. Our results confirmed the importance of heritable symbionts in the outcome of antagonistic interactions.