A parasitoid wasp of Drosophila employs preemptive and reactive strategies to deplete its host’s blood cells

The wasps Leptopilina heterotoma parasitize and ingest their Drosophila hosts. They produce extracellular vesicles (EVs) in the venom that are packed with proteins, some of which perform immune suppressive functions. EV interactions with blood cells of host larvae are linked to hematopoietic depletion, immune suppression, and parasite success. But how EVs disperse within the host, enter and kill hematopoietic cells is not well understood. Using an antibody marker for L. heterotoma EVs, we show that these parasite-derived structures are readily distributed within the hosts’ hemolymphatic system. EVs converge around the tightly clustered cells of the posterior signaling center (PSC) of the larval lymph gland, a small hematopoietic organ in Drosophila. The PSC serves as a source of developmental signals in naïve animals. In wasp-infected animals, the PSC directs the differentiation of lymph gland progenitors into lamellocytes. These lamellocytes are needed to encapsulate the wasp egg and block parasite development. We found that L. heterotoma infection disassembles the PSC and PSC cells disperse into the disintegrating lymph gland lobes. Genetically manipulated PSC-less lymph glands remain non-responsive and largely intact in the face of L. heterotoma infection. We also show that the larval lymph gland progenitors use the endocytic machinery to internalize EVs. Once inside, L. heterotoma EVs damage the Rab7- and LAMP-positive late endocytic and phagolysosomal compartments. Rab5 maintains hematopoietic and immune quiescence as Rab5 knockdown results in hematopoietic over-proliferation and ectopic lamellocyte differentiation. Thus, both aspects of anti-parasite immunity, i.e., (a) phagocytosis of the wasp’s immune-suppressive EVs, and (b) progenitor differentiation for wasp egg encapsulation reside in the lymph gland. These results help explain why the lymph gland is specifically and precisely targeted for destruction. The parasite’s simultaneous and multipronged approach to block cellular immunity not only eliminates blood cells, but also tactically blocks the genetic programming needed for supplementary hematopoietic differentiation necessary for host success. In addition to its known functions in hematopoiesis, our results highlight a previously unrecognized phagocytic role of the lymph gland in cellular immunity. EV-mediated virulence strategies described for L. heterotoma are likely to be shared by other parasitoid wasps; their understanding can improve the design and development of novel therapeutics and biopesticides as well as help protect biodiversity.     

An introduction to parasitic wasps of Drosophila and the antiparasite immune response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts'' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts¹. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response². Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited¹. Strains of L. boulardi also produce virus-like particles³ although they differ significantly in their ability to succeed on D. melanogaster¹. Some of these L. boulardi strains are difficult to grow on D. melanogaster¹ as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms^1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.Wasp infection activates immune signaling (Fig. 4)⁴. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)^5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).We present representative data with Toll signal transduction pathway components Dorsal and Spätzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).     

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.     

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.     

Partial venom gland transcriptome of a Drosophila parasitoid wasp, Leptopilina heterotoma, reveals novel and shared bioactive profiles with stinging Hymenoptera

Analysis of natural host-parasite relationships reveals the evolutionary forces that shape the delicate and unique specificity characteristic of such interactions. The accessory long gland-reservoir complex of the wasp Leptopilina heterotoma (Figitidae) produces venom with virus-like particles. Upon delivery, venom components delay host larval development and completely block host immune responses. The host range of this Drosophila endoparasitoid notably includes the highly-studied model organism, Drosophila melanogaster. Categorization of 827 unigenes, using similarity as an indicator of putative homology, reveals that approximately 25% are novel or classified as hypothetical proteins. Most of the remaining unigenes are related to processes involved in signaling, cell cycle, and cell physiology including detoxification, protein biogenesis, and hormone production. Analysis of L. heterotoma's predicted venom gland proteins demonstrates conservation among endo- and ectoparasitoids within the Apocrita (e.g., this wasp and the jewel wasp Nasonia vitripennis) and stinging aculeates (e.g., the honey bee and ants). Enzyme and KEGG pathway profiling predicts that kinases, esterases, and hydrolases may contribute to venom activity in this unique wasp. To our knowledge, this investigation is among the first functional genomic studies for a natural parasitic wasp of Drosophila. Our findings will help explain how L. heterotoma shuts down its hosts' immunity and shed light on the molecular basis of a natural arms race between these insects.     

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.     

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.     

Mgat1-dependent N-glycosylation of Membrane Components Primes Drosophila melanogaster Blood Cells for the Cellular Encapsulation Response

In nature, larvae of the fruitfly Drosophila melanogaster are commonly infected by parasitoid wasps, and so have evolved a robust immune response to counter wasp infection. In this response, fly immune cells form a multilayered capsule surrounding the wasp egg, leading to death of the parasite. Many of the molecular mechanisms underlying this encapsulation response are conserved with human immune responses. Our findings suggest that protein N-glycosylation, a common protein post-translational modification of human immune proteins, may be one such conserved mechanism. We found that membrane proteins on Drosophila immune cells are N-glycosylated in a temporally specific manner following wasp infection. Furthermore we have identified mutations in eight genes encoding enzymes of the N-glycosylation pathway that decrease fly resistance to wasp infection. More specifically, loss of protein N-glycosylation in immune cells following wasp infection led to the formation of defective capsules, which disintegrated over time and were thereby unsuccessful at preventing wasp development. Interestingly, we also found that one species of Drosophila parasitoid wasp, Leptopilina victoriae, targets protein N-glycosylation as part of its virulence mechanism, and that overexpression of an N-glycosylation enzyme could confer resistance against this wasp species to otherwise susceptible flies. Taken together, these findings demonstrate that protein N-glycosylation is a key player in Drosophila cellular encapsulation and suggest that this response may provide a novel model to study conserved roles of protein glycosylation in immunity.     

Presence of a novel small RNA-containing virus in a laboratory culture of the endoparasitic wasp Venturia canescens (Hymenoptera: Ichneumonidae)

Parasitoid wasps use a variety of mechanisms to alter their host's physiology to the benefit of the developing endoparasite inside the host larva. Association of certain wasps with viruses and virus-like particles (VLPs) that contribute to their success in parasitism is one of the fascinating evolutionary adaptations conferring active or passive protection for the endoparasite from the host immune system. Venturia canescens has been shown to produce VLPs that provide protection for the developing parasitoid egg inside the host, Ephestia kuehniella. Here, we report on the presence of a novel small RNA-containing virus from V. canescens, designated as VcSRV, occurring in the ovaries of the wasp. The virus particles are found together with VcVLPs in the lumen of the calyx region of the ovaries and are injected together with the egg and VcVLPs into E. kuehniella larvae where they enter hemocytes. Alignment of the RNA-dependent RNA polymerase gene of VcSRV indicates that the virus most likely belongs to the recently described genus Iflavirus.     

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.     

Immunosuppression induced by entomopathogens is rescued by addition of apolipophorin III in the diamondback moth, Plutella xylostella

Apolipophorin III (ApoLpIII) has been known to play critical roles in lipid transport and immune activation in insects. This study reports a partial ApoLpIII gene cloned from the diamondback moth, Plutella xylostella. It showed that the gene was expressed in all developmental stages of P. xylostella. In larval stage, it was expressed in all tested tissues of hemocyte, fat body, gut, and epidermis. In response to bacterial challenge, the larvae showed an enhanced level of ApoLpIII expression by a quantitative real-time RT-PCR. RNA interference of ApoLpIII by its specific double stranded RNA (dsRNA) caused significant knockdown of its expression level and resulted in significant suppression in hemocyte nodule formation in response to bacterial challenge. However, larvae treated with the dsRNA exhibited a significant recovery in the cellular immune response by addition of a recombinant ApoLpIII. Parasitization by an endoparasitoid wasp, Cotesia plutellae, suppressed expression of ApoLpIII and resulted in a significant suppression in the hemocyte nodule formation. The addition of the recombinant ApoLpIII to the parasitized larvae significantly restored the hemocyte activity. Infection of an entomopathogenic bacterium, Xenorhabdus nematophila, caused potent pathogenicity of P. xylostella. However, the addition of the recombinant ApoLpIII to the infected larvae significantly prevented the lethal pathogenicity. This study suggests that ApoLpIII limits pathogenicity induced by parasitization or bacterial infection in P. xylostella.     

The Rho-family GTPase Rac1 regulates integrin localization in Drosophila immunosurveillance cells

Background

When the parasitoid wasp Leptopilina boulardi lays an egg in a Drosophila larva, phagocytic cells called plasmatocytes and specialized cells known as lamellocytes encapsulate the egg. The Drosophila β-integrin Myospheroid (Mys) is necessary for lamellocytes to adhere to the cellular capsule surrounding L. boulardi eggs. Integrins are heterodimeric adhesion receptors consisting of α and β subunits, and similar to other plasma membrane receptors undergo ligand-dependent endocytosis. In mammalian cells it is known that integrin binding to the extracellular matrix induces the activation of Rac GTPases, and we have previously shown that Rac1 and Rac2 are necessary for a proper encapsulation response in Drosophila larvae. We wanted to test the possibility that Myospheroid and Rac GTPases interact during the Drosophila anti-parasitoid immune response.

Results

In the current study we demonstrate that Rac1 is required for the proper localization of Myospheroid to the cell periphery of haemocytes after parasitization. Interestingly, the mislocalization of Myospheroid in Rac1 mutants is rescued by hyperthermia, involving the heat shock protein Hsp83. From these results we conclude that Rac1 and Hsp83 are required for the proper localization of Mys after parasitization.

Significance

We show for the first time that the small GTPase Rac1 is required for Mysopheroid localization. Interestingly, the necessity of Rac1 in Mys localization was negated by hyperthermia. This presents a problem, in Drosophila we quite often raise larvae at 29°C when using the GAL4/UAS misexpression system. If hyperthermia rescues receptor endosomal recycling defects, raising larvae in hyperthermic conditions may mask potentially interesting phenotypes.     

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.     

A directed miniscreen for genes involved in the Drosophila anti-parasitoid immune response

Drosophila larvae react against eggs from the endoparasitoid wasp Leptopilina boulardi by surrounding them in a multilayered cellular capsule. Once a wasp egg is recognized as foreign, circulating macrophage-like cells, known as plasmatocytes, adhere to the invader. After spreading around the wasp egg, plasmatocytes form cellular junctions between the cells, effectively separating the egg from the hemocoel. Next, a second sub-type of circulating immunosurveillance cell (hemocyte), known as lamellocytes, adhere to either the wasp egg or more likely the plasmatocytes surrounding the egg. From these events, it is obvious that adhesion and cell shape change are an essential part of Drosophila's cellular immune response against parasitoid wasp eggs. To date, very few genes have been described as being necessary for a proper anti-parasitization response in Drosophila. With this in mind, we performed a directed genetic miniscreen to discover new genes required for this response. Many of the genes with an encapsulation defect have mammalian homologues involved in cellular adhesion, wound healing, and thrombosis, including extracellular matrix proteins, cellular adhesion molecules, and small GTPases.     

Edin Expression in the Fat Body Is Required in the Defense Against Parasitic Wasps in Drosophila melanogaster

The cellular immune response against parasitoid wasps in Drosophila involves the activation, mobilization, proliferation and differentiation of different blood cell types. Here, we have assessed the role of Edin (elevated during infection) in the immune response against the parasitoid wasp Leptopilina boulardi in Drosophila melanogaster larvae. The expression of edin was induced within hours after a wasp infection in larval fat bodies. Using tissue-specific RNAi, we show that Edin is an important determinant of the encapsulation response. Although edin expression in the fat body was required for the larvae to mount a normal encapsulation response, it was dispensable in hemocytes. Edin expression in the fat body was not required for lamellocyte differentiation, but it was needed for the increase in plasmatocyte numbers and for the release of sessile hemocytes into the hemolymph. We conclude that edin expression in the fat body affects the outcome of a wasp infection by regulating the increase of plasmatocyte numbers and the mobilization of sessile hemocytes in Drosophila larvae.     

Deadly venom of Asobara japonica parasitoid needs ovarian antidote to regulate host physiology

Asobara japonica (Braconidae) is an endophagous parasitoid developing in Drosophila larvae. The present study shows that A. japonica was never encapsulated in Drosophila melanogaster, and that it caused an overall inhibition of the host encapsulation reaction since injected foreign bodies were never encapsulated in parasitized hosts. Both the number of circulating hemocytes and the phenoloxidase activity decreased in parasitized larvae, and the hematopoietic organ appeared highly disrupted. We also found that A. japonica venom secretions had atypical effects on hosts compared to other braconid wasps. A. japonica venom secretions induced permanent paralysis followed by death of D. melanogaster larvae, whether injected by the female wasp during an interrupted oviposition, or manually injected into unparasitized larvae. More remarkably, these effects could be reversed by injection of ovarian extracts from female wasps. This is the first report that the venom of an endophagous braconid parasitoid can have a deadly effect on hosts, and moreover, that ovarian extracts can act as an antidote to reverse the effects of the wasp's venom. These results also demonstrate that A. japonica secretions from both venom gland and ovary are required to regulate synergistically the host physiology for the success of the parasitoid.