Methotrexate negatively acts on inflammatory responses triggered in Drosophila larva with hyperactive JAK/STAT pathway.

Drosophila is a valuable paradigm for studying tumorigenesis and cancer. Mutations causing hematopoietic aberrations and melanotic-blood-tumors found in Drosophila mutants are vastly studied. Clear understanding about the blood cells, signaling pathways and the tissues affected during hematopoietic tumor formation provide an opportunity to delineate the effects of cancer therapeutics. Using this simple hematopoietic archetype, we elucidated the effects of the anti-cancer drug, Methotrexate (MTX) on immune responses in two scenarios i.e. against wasp infection and in hematopoietic mutant, hopTum-l. Through this in vivo study we show that MTX impedes the immune responses against wasp infection including the encapsulation response. We further observed that MTX reduces the tumor penetrance in gain-of-function mutants of JAK/STAT pathway, hopTum-l. MTX is anti-inflammatory as it hinders not only the immune responses of acute inflammation as observed after wasp infestation, but also chronic inflammatory responses associated with constitutively activated JAK/STAT pathway mutant (hopTum-l) carrying blood tumors.

Integrins Modulate Extracellular Matrix Organization to Control Cell Signaling during Hematopoiesis.

During hematopoiesis, progenitor cells receive and interpret a diverse array of regulatory signals from their environment. These signals control the maintenance of the progenitors and regulate the production of mature blood cells. Integrins are well known in vertebrates for their roles in hematopoiesis, particularly in assisting in the migration to, as well as the physical attachment of, progenitors to the niche. However, whether and how integrins are also involved in the signaling mechanisms that control hematopoiesis remains to be resolved. Here, we show that integrins play a key role during fly hematopoiesis in regulating cell signals that control the behavior of hematopoietic progenitors. Integrins can regulate hematopoiesis directly, via focal adhesion kinase (FAK) signaling, and indirectly, by directing extracellular matrix (ECM) assembly and/or maintenance. ECM organization and density controls blood progenitor behavior by modulating multiple signaling pathways, including bone morphogenetic protein (BMP) and Hedgehog (Hh). Furthermore, we show that integrins and the ECM are reduced following infection, which may assist in activating the immune response. Our results provide mechanistic insight into how integrins can shape the signaling environment around hematopoietic progenitors.

The prophenoloxidase system in Drosophila participates in the anti-nematode immune response.

Drosophila melanogaster relies on an evolutionarily conserved innate immune system to protect itself from potentially deadly pathogens. One of the earliest pathways activated after injury or infection is the melanization pathway, which is responsible for synthesizing and depositing melanin at the site of injury, or onto invading microbes. Three genes, PPO1-3, encoding prophenoloxidase (PPO), an inactive precursor of phenoloxidase (PO), are responsible for the production of melanin after their activation via immune challenge. One pathogen capable of infecting D. melanogaster are entomopathogenic nematodes. Steinernema carpocapsae nematodes exist in a mutualistic relationship with Xenorhabdus nematophila bacteria and are an important biological control agent for controlling insect pests. The nematode-bacteria complex (symbiotic nematodes) can be separated, creating "axenic" nematodes, devoid of their associated bacteria, which are still capable of infecting and killing D. melanogaster. In order to investigate how the D. melanogaster melanization pathway contributes to the anti-nematode immune response, symbiotic and axenic S. carpocapsae were used to study D. melanogaster survival, PPO gene expression, and activation of PPO to PO. Our research suggests that the expression of all three D. melanogaster PPO genes contributes to survival, however only PPO1 or PPO3 appear to be up-regulated during axenic or symbiotic nematode infection. Additionally, we present data suggesting that a complex regulatory system exists between PPOs, potentially allowing for the compensation of PPOs by one another. Further, we found that axenic nematode infection leads to higher levels of PO, suggesting that X. nematophila suppresses this activation. We also report for the first time the differentiation of lamellocytes, a specialized type of hemocytes in D. melanogaster, in response to symbiotic S. carpocapsae nematode infection. Our results suggest an important role played by the melanization pathway in response to nematode infection, and demonstrate how this response can be manipulated by S. carpocapsae nematodes and their mutualistic X. nematophila bacteria.

Intestinal NF-κB and STAT signalling is important for uptake and clearance in a Drosophila-Herpetomonas interaction model.

Dipteran insects transmit serious diseases to humans, often in the form of trypanosomatid parasites. To accelerate research in more difficult contexts of dipteran-parasite relationships, we studied the interaction of the model dipteran Drosophila melanogaster and its natural trypanosomatid Herpetomonas muscarum. Parasite infection reduced fecundity but not lifespan in NF-κB/Relish-deficient flies. Gene expression analysis implicated the two NF-κB pathways Toll and Imd as well as STAT signalling. Tissue specific knock-down of key components of these pathways in enterocytes (ECs) and intestinal stem cells (ISCs) influenced initial numbers, infection dynamics and time of clearance. Herpetomonas triggered STAT activation and proliferation of ISCs. Loss of Relish suppressed ISCs, resulting in increased parasite numbers and delayed clearance. Conversely, overexpression of Relish increased ISCs and reduced uptake. Finally, loss of Toll signalling decreased EC numbers and enabled parasite persistence. This network of signalling may represent a general mechanism with which dipteran respond to trypanosomatids.

Drosophila species learn dialects through communal living.

Many species are able to share information about their environment by communicating through auditory, visual, and olfactory cues. In Drosophila melanogaster, exposure to parasitoid wasps leads to a decline in egg laying, and exposed females communicate this threat to naïve flies, which also depress egg laying. We find that species across the genus Drosophila respond to wasps by egg laying reduction, activate cleaved caspase in oocytes, and communicate the presence of wasps to naïve individuals. Communication within a species and between closely related species is efficient, while more distantly related species exhibit partial communication. Remarkably, partial communication between some species is enhanced after a cohabitation period that requires exchange of visual and olfactory signals. This interspecies "dialect learning" requires neuronal cAMP signaling in the mushroom body, suggesting neuronal plasticity facilitates dialect learning and memory. These observations establish Drosophila as genetic models for interspecies social communication and evolution of dialects.

Mechanical stress to Drosophila larvae stimulates a cellular immune response through the JAK/STAT signaling pathway.

Acute inflammation can cause serious tissue damage and disease in physiologically-challenged organisms. The precise mechanisms leading to these detrimental effects remain to be determined. In this study, we utilize a reproducible means to induce cellular immune activity in Drosophila larvae in response to mechanical stress. That is, forceps squeeze-administered stress induces lamellocytes, a defensive hemocyte type that normally appears in response to wasp infestation of larvae. The posterior signaling center (PSC) is a cellular microenvironment in the larval hematopoietic lymph gland that is vital for lamellocyte induction upon parasitoid attack. However, we found the PSC was not required for mechanical stress-induced lamellocyte production. In addition, we observed that mechanical injury caused a systemic expression of Unpaired3. This cytokine is both necessary and sufficient to activate the cellular immune response to the imposed stress. These findings provide new insights into the communication between injured tissues and immune system induction, using stress-challenged Drosophila larvae as a tractable model system.

Adipokinetic hormone and adenosine interfere with nematobacterial infection and locomotion in Drosophila melanogaster.

This study examined how adipokinetic hormone (AKH) and adenosine affect defense responses in Drosophila melanogaster larvae infected with entomopathogenic nematodes (EPN, Steinernema carpocapsae and Heterorhabditis bacteriophora). Three loss-of-function mutant larvae were tested: Akh1, AdoR1 (adenosine receptor), and Akh1 AdoR1. Mortality decreased in all mutants post-EPN infection compared with the control (w1118). Additionally, co-application of external AKH with EPN significantly increased mortality beyond rates observed in EPN-only treatment, while also elevating carbon dioxide production, a measure of metabolism. Furthermore trehalose levels increased in both w1118 and Akh1 larvae post-EPN infection, but the latter group exhibited a lower increase and total trehalose levels. Interestingly, baseline trehalose was relatively high in untreated AdoR1 and Akh1 AdoR1 mutants, with levels remaining unaffected by infection. Infection also elevated haemolymph lipid content overall, but the different mutations did not substantially influence this change. In contrast, haemolymph protein content dropped after EPN infection in all tested groups, but this decline was more intense among Akh1. In uninfected larvae mutations decreased antioxidative capacity in Akh1 and increased in AdoR1, however, its post-infection increases were similar in all mutants, suggesting that antioxidant response in Drosophila involves mechanisms also beyond AKH and adenosine. Furthermore, AKH application in w1118 larvae significantly increased movement distance and percentage of larval activity, but reduced velocity. Mutations of Akh and AdoR did not strongly affect locomotion.

Drosophila muscles regulate the immune response against wasp infection via carbohydrate metabolism.

We recently found that JAK/STAT signaling in skeletal muscles is important for the immune response of Drosophila larvae against wasp infection, but it was not clear how muscles could affect the immune response. Here we show that insulin signaling is required in muscles, but not in fat body or hemocytes, during larval development for an efficient encapsulation response and for the formation of lamellocytes. This effect requires TOR signaling. We show that muscle tissue affects the immune response by acting as a master regulator of carbohydrate metabolism in the infected animal, via JAK/STAT and insulin signaling in the muscles, and that there is indirect positive feedback between JAK/STAT and insulin signaling in the muscles. Specifically, stimulation of JAK/STAT signaling in the muscles can rescue the deficient immune response when insulin signaling is suppressed. Our results shed new light on the interaction between metabolism, immunity, and tissue communication.

Screening and Analysis of Janelia FlyLight Project Enhancer-Gal4 Strains Identifies Multiple Gene Enhancers Active During Hematopoiesis in Normal and Wasp-Challenged Drosophila Larvae.

A GFP expression screen has been conducted on >1000 Janelia FlyLight Project enhancer-Gal4 lines to identify transcriptional enhancers active in the larval hematopoietic system. A total of 190 enhancers associated with 87 distinct genes showed activity in cells of the third instar larval lymph gland and hemolymph. That is, gene enhancers were active in cells of the lymph gland posterior signaling center (PSC), medullary zone (MZ), and/or cortical zone (CZ), while certain of the transcriptional control regions were active in circulating hemocytes. Phenotypic analyses were undertaken on 81 of these hematopoietic-expressed genes, with nine genes characterized in detail as to gain- and loss-of-function phenotypes in larval hematopoietic tissues and blood cells. These studies demonstrated the functional requirement of the cut gene for proper PSC niche formation, the hairy, Btk29A, and E2F1 genes for blood cell progenitor production in the MZ domain, and the longitudinals lacking, dFOXO, kayak, cap-n-collar, and delilah genes for lamellocyte induction and/or differentiation in response to parasitic wasp challenge and infestation of larvae. Together, these findings contribute substantial information to our knowledge of genes expressed during the larval stage of Drosophila hematopoiesis and newly identify multiple genes required for this developmental process.

Transdifferentiation and Proliferation in Two Distinct Hemocyte Lineages in Drosophila melanogaster Larvae after Wasp Infection.

Cellular immune responses require the generation and recruitment of diverse blood cell types that recognize and kill pathogens. In Drosophila melanogaster larvae, immune-inducible lamellocytes participate in recognizing and killing parasitoid wasp eggs. However, the sequence of events required for lamellocyte generation remains controversial. To study the cellular immune system, we developed a flow cytometry approach using in vivo reporters for lamellocytes as well as for plasmatocytes, the main hemocyte type in healthy larvae. We found that two different blood cell lineages, the plasmatocyte and lamellocyte lineages, contribute to the generation of lamellocytes in a demand-adapted hematopoietic process. Plasmatocytes transdifferentiate into lamellocyte-like cells in situ directly on the wasp egg. In parallel, a novel population of infection-induced cells, which we named lamelloblasts, appears in the circulation. Lamelloblasts proliferate vigorously and develop into the major class of circulating lamellocytes. Our data indicate that lamellocyte differentiation upon wasp parasitism is a plastic and dynamic process. Flow cytometry with in vivo hemocyte reporters can be used to study this phenomenon in detail.

Drosophila innate immunity: regional and functional specialization of prophenoloxidases.

BACKGROUND: The diversification of immune systems during evolution involves the expansion of particular gene families in given phyla. A better understanding of the metazoan immune system requires an analysis of the logic underlying such immune gene amplification. This analysis is now within reach due to the ease with which we can generate multiple mutations in an organism. In this paper, we analyze the contribution of the three Drosophila prophenoloxidases (PPOs) to host defense by generating single, double and triple mutants. PPOs are enzymes that catalyze the production of melanin at the site of infection and around parasites. They are the rate-limiting enzymes that contribute to the melanization reaction, a major immune mechanism of arthropods. The number of PPO-encoding genes is variable among insects, ranging from one in the bee to ten in the mosquito. RESULTS: By analyzing mutations alone and in combination, we ascribe a specific function to each of the three PPOs of Drosophila. Our study confirms that two PPOs produced by crystal cells, PPO1 and PPO2, contribute to the bulk of melanization in the hemolymph, upon septic or clean injury. In contrast, PPO3, a PPO restricted to the D. melanogaster group, is expressed in lamellocytes and contributes to melanization during the encapsulation process. Interestingly, another overlapping set of PPOs, PPO2 and PPO3, achieve melanization of the capsule upon parasitoid wasp infection. CONCLUSIONS: The use of single or combined mutations allowed us to show that each PPO mutant has a specific phenotype, and that knocking out two of three genes is required to abolish fully a particular function. Thus, Drosophila PPOs have partially overlapping functions to optimize melanization in at least two conditions: following injury or during encapsulation. Since PPO3 is restricted to the D. melanogaster group, this suggests that production of PPO by lamellocytes emerged as a recent defense mechanism against parasitoid wasps. We conclude that differences in spatial localization, immediate or late availability, and mode of activation underlie the functional diversification of the three Drosophila PPOs, with each of them having non-redundant but overlapping functions.

Apoptosis in Hemocytes Induces a Shift in Effector Mechanisms in the Drosophila Immune System and Leads to a Pro-Inflammatory State.

Apart from their role in cellular immunity via phagocytosis and encapsulation, Drosophila hemocytes release soluble factors such as antimicrobial peptides, and cytokines to induce humoral responses. In addition, they participate in coagulation and wounding, and in development. To assess their role during infection with entomopathogenic nematodes, we depleted plasmatocytes and crystal cells, the two classes of hemocytes present in naïve larvae by expressing proapoptotic proteins in order to produce hemocyte-free (Hml-apo, originally called Hemoless) larvae. Surprisingly, we found that Hml-apo larvae are still resistant to nematode infections. When further elucidating the immune status of Hml-apo larvae, we observe a shift in immune effector pathways including massive lamellocyte differentiation and induction of Toll- as well as repression of imd signaling. This leads to a pro-inflammatory state, characterized by the appearance of melanotic nodules in the hemolymph and to strong developmental defects including pupal lethality and leg defects in escapers. Further analysis suggests that most of the phenotypes we observe in Hml-apo larvae are alleviated by administration of antibiotics and by changing the food source indicating that they are mediated through the microbiota. Biochemical evidence identifies nitric oxide as a key phylogenetically conserved regulator in this process. Finally we show that the nitric oxide donor L-arginine similarly modifies the response against an early stage of tumor development in fly larvae.

An unexpected link between notch signaling and ROS in restricting the differentiation of hematopoietic progenitors in Drosophila.

A fundamental question in hematopoietic development is how multipotent progenitors achieve precise identities, while the progenitors themselves maintain quiescence. In Drosophila melanogaster larvae, multipotent hematopoietic progenitors support the production of three lineages, exhibit quiescence in response to cues from a niche, and from their differentiated progeny. Infection by parasitic wasps alters the course of hematopoiesis. Here we address the role of Notch (N) signaling in lamellocyte differentiation in response to wasp infection. We show that Notch activity is moderately high and ubiquitous in all cells of the lymph gland lobes, with crystal cells exhibiting the highest levels. Wasp infection reduces Notch activity, which results in fewer crystal cells and more lamellocytes. Robust lamellocyte differentiation is induced even in N mutants. Using RNA interference knockdown of N, Serrate, and neuralized (neur), and twin clone analysis of a N null allele, we show that all three genes inhibit lamellocyte differentiation. However, unlike its cell-autonomous function in crystal cell development, Notch's inhibitory influence on lamellocyte differentiation is not cell autonomous. High levels of reactive oxygen species in the lymph gland lobes, but not in the niche, accompany N(RNAi)-induced lamellocyte differentiation and lobe dispersal. Our results define a novel dual role for Notch signaling in maintaining competence for basal hematopoiesis: while crystal cell development is encouraged, lamellocytic fate remains repressed. Repression of Notch signaling in fly hematopoiesis is important for host defense against natural parasitic wasp infections. These findings can serve as a model to understand how reactive oxygen species and Notch signals are integrated and interpreted in vivo.

Expression of Drosophila adenosine deaminase in immune cells during inflammatory response.

Extra-cellular adenosine is an important regulator of inflammatory responses. It is generated from released ATP by a cascade of ectoenzymes and degraded by adenosine deaminase (ADA). There are two types of enzymes with ADA activity: ADA1 and ADGF/ADA2. ADA2 activity originates from macrophages and dendritic cells and is associated with inflammatory responses in humans and rats. Drosophila possesses a family of six ADGF proteins with ADGF-A being the main regulator of extra-cellular adenosine during larval stages. Herein we present the generation of a GFP reporter for ADGF-A expression by a precise replacement of the ADGF-A coding sequence with GFP using homologous recombination. We show that the reporter is specifically expressed in aggregating hemocytes (Drosophila immune cells) forming melanotic capsules; a characteristic of inflammatory response. Our vital reporter thus confirms ADA expression in sites of inflammation in vivo and demonstrates that the requirement for ADA activity during inflammatory response is evolutionary conserved from insects to vertebrates. Our results also suggest that ADA activity is achieved specifically within sites of inflammation by an uncharacterized post-transcriptional regulation based mechanism. Utilizing various mutants that induce melanotic capsule formation and also a real immune challenge provided by parasitic wasps, we show that the acute expression of the ADGF-A protein is not driven by one specific signaling cascade but is rather associated with the behavior of immune cells during the general inflammatory response. Connecting the exclusive expression of ADGF-A within sites of inflammation, as presented here, with the release of energy stores when the ADGF-A activity is absent, suggests that extra-cellular adenosine may function as a signal for energy allocation during immune response and that ADGF-A/ADA2 expression in such sites of inflammation may regulate this role.

Lineage tracing of lamellocytes demonstrates Drosophila macrophage plasticity.

Leukocyte-like cells called hemocytes have key functions in Drosophila innate immunity. Three hemocyte types occur: plasmatocytes, crystal cells, and lamellocytes. In the absence of qimmune challenge, plasmatocytes are the predominant hemocyte type detected, while crystal cells and lamellocytes are rare. However, upon infestation by parasitic wasps, or in melanotic mutant strains, large numbers of lamellocytes differentiate and encapsulate material recognized as "non-self". Current models speculate that lamellocytes, plasmatocytes and crystal cells are distinct lineages that arise from a common prohemocyte progenitor. We show here that over-expression of the CoREST-interacting transcription factor Chn in plasmatocytes induces lamellocyte differentiation, both in circulation and in lymph glands. Lamellocyte increases are accompanied by the extinction of plasmatocyte markers suggesting that plasmatocytes are transformed into lamellocytes. Consistent with this, timed induction of Chn over-expression induces rapid lamellocyte differentiation within 18 hours. We detect double-positive intermediates between plasmatocytes and lamellocytes, and show that isolated plasmatocytes can be triggered to differentiate into lamellocytes in vitro, either in response to Chn over-expression, or following activation of the JAK/STAT pathway. Finally, we have marked plasmatocytes and show by lineage tracing that these differentiate into lamellocytes in response to the Drosophila parasite model Leptopilina boulardi. Taken together, our data suggest that lamellocytes arise from plasmatocytes and that plasmatocytes may be inherently plastic, possessing the ability to differentiate further into lamellocytes upon appropriate challenge.

Ontogeny of the Drosophila larval hematopoietic organ, hemocyte homeostasis and the dedicated cellular immune response to parasitism.

Over the years, the fruit fly Drosophila melanogaster has become a major invertebrate model to study developmental and evolutionary aspects of both humoral and cellular aspects of innate immunity. Drosophila hematopoiesis which supplies three types of circulating hemocytes, occurs in two spatially and temporally distinct phases during development. The first embryonic phase is described in detail in accompanying reviews in this Int. J. Dev. Biol. Special Issue. The second phase takes place at the end of larval development in a specialised hematopoietic organ, termed the lymph gland. We review here recent studies on the ontogeny of the lymph gland, focusing on the formation and role of the Posterior Signalling Center which acts as a niche for hematopoietic progenitors. We then report recent progress in understanding the dedicated cellular immune response of Drosophila larvae against parasitization by Hymenopterae, a common threat for many Dipterae. This response involves the differentiation of lamellocytes, a cryptic cell fate, revealing the high degree of plasticity of Drosophila hematopoiesis. We end up by integrating studies in Drosophila within a more general picture of insect hematopoiesis and hemocyte homeostasis.

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.

Control of blood cell homeostasis in Drosophila larvae by the posterior signalling centre.

Drosophila haemocytes (blood cells) originate from a specialized haematopoietic organ-the lymph gland. Larval haematopoietic progenitors (prohaemocytes) give rise to three types of circulating haemocytes: plasmatocytes, crystal cells and lamellocytes. Lamellocytes, which are devoted to encapsulation of large foreign bodies, only differentiate in response to specific immune threats, such as parasitization by wasps. Here we show that a small cluster of signalling cells, termed the PSC (posterior signalling centre), controls the balance between multipotent prohaemocytes and differentiating haemocytes, and is necessary for the massive differentiation of lamellocytes that follows parasitization. Communication between the PSC and haematopoietic progenitors strictly depends on the PSC-restricted expression of Collier, the Drosophila orthologue of mammalian early B-cell factor. PSC cells act, in a non-cell-autonomous manner, to maintain JAK/STAT signalling activity in prohaemocytes, preventing their premature differentiation. Serrate-mediated Notch signalling from the PSC is required to maintain normal levels of col transcription. The key role of the PSC in controlling blood cell homeostasis is reminiscent of interactions between haematopoietic progenitors and their micro-environment in vertebrates, thus further highlighting the interest of Drosophila as a model system for studying the evolution of haematopoiesis and cellular innate immunity.

Rac1 signalling in the Drosophila larval cellular immune response.

The Drosophila larval cellular immune response involves cells (hemocytes) that can be recruited from a hematopoietic organ located behind the brain, as well as a sessile population of cells found just underneath the larval cuticle arranged in a segmental pattern. By using two Rac1 GTPase effector-loop mutants together with epistasis studies, we show that Rac1 requires the Drosophila melanogaster Jun N-terminal kinase Basket (Bsk), as well as stable actin formation to recruit the sessile hemocyte population. We show that actin stabilization is necessary for Rac1-induced hemocyte activation by lowering cofilin (encoded by the twinstar gene tsr) expression in blood cells. Removing Bsk by RNAi suppressed Rac1-induced release of sessile hemocytes. RNAi against Bsk also suppressed Rac1 induction of lamellocytes, a specialized population of hemocytes necessary for the encapsulation of invading pathogens. Furthermore, Rac1 and Bsk are involved in regulating the formation of actin- and focal adhesion kinase (FAK)-rich placodes in hemocytes. Lastly, Rac1 and Bsk are both required for the proper encapsulation of eggs from the parasitoid wasp Leptipolina boulardi. From these data we conclude that Rac1 induces Bsk activity and stable actin formation for cellular immune activation, leading to sessile hemocyte release and an increase in the number of circulating hemocytes.

Plasmodium berghei ookinetes bind to Anopheles gambiae and Drosophila melanogaster annexins.

Using a proteomic approach we identified polypeptides from Anopheles gambiae and Drosophila melanogaster protein extracts that selectively bind purified Plasmodium berghei ookinetes in vitro; these were two and three distinct polypeptides, respectively, with an apparent molecular weight of about 36 kDa. Combining two-dimensional electrophoresis and MALDI-TOF (matrix-associated laser desorption ionization time of flight) mass spectrometry we determined that the polypeptides correspond to isomorphs of the annexin B11 protein of the fruit fly. When protein extracts derived from A. gambiae and D. melanogaster tissue culture cells were further fractionated, the binding activity matching the annexin protein could be localized in the fraction derived from cell membranes in both diptera. Antibody staining showed that annexin also binds to ookinetes during the invasion of the mosquito midgut. Finally, inclusion of antiannexin antisera in a mosquito blood meal impaired parasite development, suggesting a facilitating role for annexins in the infection of the mosquito by Plasmodium.