Drosophila parasitoids go to space: Unexpected effects of spaceflight on hosts and their parasitoids.

While fruit flies (Drosophila melanogaster) and humans exhibit immune system dysfunction in space, studies examining their immune systems' interactions with natural parasites in space are lacking. Drosophila parasitoid wasps modify blood cell function to suppress host immunity. In this study, naive and parasitized ground and space flies from a tumor-free control and a blood tumor-bearing mutant strain were examined. Inflammation-related genes were activated in space in both fly strains. Whereas control flies did not develop tumors, tumor burden increased in the space-returned tumor-bearing mutants. Surprisingly, control flies were more sensitive to spaceflight than mutant flies; many of their essential genes were downregulated. Parasitoids appeared more resilient than fly hosts, and spaceflight did not significantly impact wasp survival or the expression of their virulence genes. Previously undocumented mutant wasps with novel wing color and wing shape were isolated post-flight and will be invaluable for host-parasite studies on Earth.

In Silico Analysis of a Drosophila Parasitoid Venom Peptide Reveals Prevalence of the Cation-Polar-Cation Clip Motif in Knottin Proteins.

As generalist parasitoid wasps, Leptopilina heterotoma are highly successful on many species of fruit flies of the genus Drosophila. The parasitoids produce specialized multi-strategy extracellular vesicle (EV)-like structures in their venom. Proteomic analysis identified several immunity-associated proteins, including the knottin peptide, LhKNOT, containing the structurally conserved inhibitor cysteine knot (ICK) fold, which is present in proteins from diverse taxa. Our structural and docking analysis of LhKNOT's 36-residue core knottin fold revealed that in addition to the knottin motif itself, it also possesses a Cation-Polar-Cation (CPC) clip. The CPC clip motif is thought to facilitate antimicrobial activity in heparin-binding proteins. Surprisingly, a majority of ICKs tested also possess the CPC clip motif, including 75 bona fide plant and arthropod knottin proteins that share high sequence and/or structural similarity with LhKNOT. Like LhKNOT and these other 75 knottin proteins, even the Drosophila Drosomycin antifungal peptide, a canonical target gene of the fly's Toll-NF-kappa B immune pathway, contains this CPC clip motif. Together, our results suggest a possible defensive function for the parasitoid LhKNOT. The prevalence of the CPC clip motif, intrinsic to the cysteine knot within the knottin proteins examined here, suggests that the resultant 3D topology is important for their biochemical functions. The CPC clip is likely a highly conserved structural motif found in many diverse proteins with reported heparin binding capacity, including amyloid proteins. Knottins are targets for therapeutic drug development, and insights into their structure-function relationships will advance novel drug design.

DROP: Molecular voucher database for identification of Drosophila parasitoids.

Molecular identification is increasingly used to speed up biodiversity surveys and laboratory experiments. However, many groups of organisms cannot be reliably identified using standard databases such as GenBank or BOLD due to lack of sequenced voucher specimens identified by experts. Sometimes a large number of sequences are available, but with too many errors to allow identification. Here, we address this problem for parasitoids of Drosophila by introducing a curated open-access molecular reference database, DROP (Drosophila parasitoids). Identifying Drosophila parasitoids is challenging and poses a major impediment to realize the full potential of this model system in studies ranging from molecular mechanisms to food webs, and in biological control of Drosophila suzukii. In DROP, genetic data are linked to voucher specimens and, where possible, the voucher specimens are identified by taxonomists and vetted through direct comparison with primary type material. To initiate DROP, we curated 154 laboratory strains, 856 vouchers, 554 DNA sequences, 16 genomes, 14 transcriptomes, and six proteomes drawn from a total of 183 operational taxonomic units (OTUs): 114 described Drosophila parasitoid species and 69 provisional species. We found species richness of Drosophila parasitoids to be heavily underestimated and provide an updated taxonomic catalogue for the community. DROP offers accurate molecular identification and improves cross-referencing between individual studies that we hope will catalyse research on this diverse and fascinating model system. Our effort should also serve as an example for researchers facing similar molecular identification problems in other groups of organisms.

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.

Reactive oxygen species signaling in primordial germ cell development in Drosophila embryos.

REDOX mechanisms that induce biosynthesis of the reactive oxygen species (ROS) have attracted considerable attention due to both the deleterious and beneficial responses elicited by the reactive radical. In several organisms including Drosophila melanogaster, modulation of ROS activity is thought to be crucial for the maintenance of cell fates in developmental contexts. Interestingly, REDOX mechanisms have been shown to be involved in maintaining progenitor fate of stem cells as well as their proliferation and differentiation. Here, we have explored the possible functions of ROS during proper specification and developmental progression of embryonic primordial germ cells (PGCs). Indicating its potential involvement in these processes, ROS can be detected in the embryonic PGCs and the surrounding somatic cells from very early stages of embryogenesis. Using both "loss" and "gain" of function mutations in two different components of the REDOX pathway, we show that ROS levels are likely to be critical in maintaining germ cell behavior, including their directed migration. Altering the activity of a putative regulator of ROS also adversely influences the ability of PGCs to adhere to one another in cellular blastoderm embryos, suggesting potential involvement of this pathway in orchestrating different phases of germ cell migration.

Immune Suppressive Extracellular Vesicle Proteins of Leptopilina heterotoma Are Encoded in the Wasp Genome.

Leptopilina heterotoma are obligate parasitoid wasps that develop in the body of their Drosophila hosts. During oviposition, female wasps introduce venom into the larval hosts' body cavity. The venom contains discrete, 300 nm-wide, mixed-strategy extracellular vesicles (MSEVs), until recently referred to as virus-like particles. While the crucial immune suppressive functions of L. heterotoma MSEVs have remained undisputed, their biotic nature and origin still remain controversial. In recent proteomics analyses of L. heterotoma MSEVs, we identified 161 proteins in three classes: conserved eukaryotic proteins, infection and immunity related proteins, and proteins without clear annotation. Here we report 246 additional proteins from the L. heterotoma MSEV proteome. An enrichment analysis of the entire proteome supports vesicular nature of these structures. Sequences for more than 90% of these proteins are present in the whole-body transcriptome. Sequencing and de novo assembly of the 460 Mb-sized L. heterotoma genome revealed 90% of MSEV proteins have coding regions within the genomic scaffolds. Altogether, these results explain the stable association of MSEVs with their wasps, and like other wasp structures, their vertical inheritance. While our results do not rule out a viral origin of MSEVs, they suggest that a similar strategy for co-opting cellular machinery for immune suppression may be shared by other wasps to gain advantage over their hosts. These results are relevant to our understanding of the evolution of figitid and related wasp species.

Discovery of aspirin-triggered eicosanoid-like mediators in a Drosophila metainflammation blood tumor model.

Epidemiologic studies have linked the use of aspirin to a decline in chronic inflammation that underlies many human diseases, including some cancers. Aspirin reduces the levels of cyclooxygenase-mediated pro-inflammatory prostaglandins, promotes the production of pro-resolution molecules, and triggers the production of anti-inflammatory electrophilic mono-oxygenated (EFOX) lipid mediators. We investigated the effects of aspirin in fruit fly models of chronic inflammation. Ectopic Toll/NF-κB and JAK/STAT signaling in mutant D. melanogaster results in overproliferation of hematopoietic blood progenitors resulting in the formation of granuloma-like tumors. Ectopic JAK-STAT signaling also leads to metabolic inflammation. We report that aspirin-treated mutant flies experience reduction in metabolic inflammation, mitosis, ectopic immune signaling, and macrophage infiltration. Moreover, these flies synthesize 13-HODE, and aspirin triggers 13-oxoODE (13-EFOX-L2) production. Providing the precursor of 13-HODE, linoleic acid, or performing targeted knockdown of the transcription factor STAT in inflammatory blood cells, boosts 13-EFOX-L2 levels while decreasing metabolic inflammation. Thus, hematopoietic cells regulate metabolic inflammation in flies, and their effects can be reversed by pharmaceutical or dietary intervention, suggesting deep phylogenetic conservation in the ability of animals to resolve inflammation and repair tissue damage. These findings can help identify novel treatment targets in humans.

A combined computational strategy of sequence and structural analysis predicts the existence of a functional eicosanoid pathway in Drosophila melanogaster.

This study reports on a putative eicosanoid biosynthesis pathway in Drosophila melanogaster and challenges the currently held view that mechanistic routes to synthesize eicosanoid or eicosanoid-like biolipids do not exist in insects, since to date, putative fly homologs of most mammalian enzymes have not been identified. Here we use systematic and comprehensive bioinformatics approaches to identify most of the mammalian eicosanoid synthesis enzymes. Sensitive sequence analysis techniques identified candidate Drosophila enzymes that share low global sequence identities with their human counterparts. Twenty Drosophila candidates were selected based upon (a) sequence identity with human enzymes of the cyclooxygenase and lipoxygenase branches, (b) similar domain architecture and structural conservation of the catalytic domain, and (c) presence of potentially equivalent functional residues. Evaluation of full-length structural models for these 20 top-scoring Drosophila candidates revealed a surprising degree of conservation in their overall folds and potential analogs for functional residues in all 20 enzymes. Although we were unable to identify any suitable candidate for lipoxygenase enzymes, we report structural homology models of three fly cyclooxygenases. Our findings predict that the D. melanogaster genome likely codes for one or more pathways for eicosanoid or eicosanoid-like biolipid synthesis. Our study suggests that classical and/or novel eicosanoids mediators must regulate biological functions in insects-predictions that can be tested with the power of Drosophila genetics. Such experimental analysis of eicosanoid biology in a simple model organism will have high relevance to human development and health.

Novel Organelles with Elements of Bacterial and Eukaryotic Secretion Systems Weaponize Parasites of Drosophila.

The evolutionary success of parasitoid wasps, a highly diverse group of insects widely used in biocontrol, depends on a variety of life history strategies in conflict with those of their hosts [1]. Drosophila melanogaster is a natural host of parasitic wasps of the genus Leptopilina. Attack by L. boulardi (Lb), a specialist wasp to flies of the melanogaster group, activates NF-κB-mediated humoral and cellular immunity. Inflammatory blood cells mobilize and encapsulate Lb eggs and embryos [2-5]. L. heterotoma (Lh), a generalist wasp, kills larval blood cells and actively suppresses immune responses. Spiked virus-like particles (VLPs) in wasp venom have clearly been linked to wasps' successful parasitism of Drosophila [6], but the composition of VLPs and their biotic nature have remained mysterious. Our proteomics studies reveal that VLPs lack viral coat proteins but possess a pharmacopoeia of (1) the eukaryotic vesicular transport system, (2) immunity, and (3) previously unknown proteins. These novel proteins distinguish Lh from Lb VLPs; notably, some proteins specific to Lh VLPs possess sequence similarities with bacterial secretion system proteins. Structure-informed analyses of an abundant Lh VLP surface and spike-tip protein, p40, reveal similarities to the needle-tip invasin proteins SipD and IpaD of Gram-negative bacterial type-3 secretion systems that breach immune barriers and deliver virulence factors into mammalian cells. Our studies suggest that Lh VLPs represent a new class of extracellular organelles and share pathways for protein delivery with both eukaryotic microvesicles and bacterial surface secretion systems. Given their mixed prokaryotic and eukaryotic properties, we propose the term mixed-strategy extracellular vesicle (MSEV) to replace VLP.

A comparative cytogenetic study of Drosophila parasitoids (Hymenoptera, Figitidae) using DNA-binding fluorochromes and FISH with 45S rDNA probe.

Karyotypes of Leptopilina boulardi (Barbotin, Carton et Keiner-Pillault, 1979) (n = 9), L. heterotoma (Thomson, 1862) (n = 10), L. victoriae Nordlander, 1980 (n = 10) and Ganaspis xanthopoda (Ashmead, 1896) (n = 9) (Hymenoptera, Figitidae) were studied using DNA-binding ligands with different base specificity [propidium iodide (PI), chromomycin A3 (CMA3) and 4',6-diamidino-2-phenylindole (DAPI)], and fluorescence in situ hybridization (FISH) with a 45S rDNA probe. Fluorochrome staining was similar between the different fluorochromes, except for a single CMA3- and PI-positive and DAPI-negative band per haploid karyotype of each species. FISH with 45S rDNA probe detected a single rDNA site in place of the bright CMA3-positive band, thus identifying the nucleolus organizing region (NOR). Chromosomal locations of NORs were similar for both L. heterotoma and L. victoriae, but strongly differed in L. boulardi as well as in G. xanthopoda. Phylogenetic aspects of NOR localization in all studied species are briefly discussed.

An extensive allelic series of Drosophila kae1 mutants reveals diverse and tissue-specific requirements for t6A biogenesis.

N(6)-threonylcarbamoyl-adenosine (t6A) is one of the few RNA modifications that is universally present in life. This modification occurs at high frequency at position 37 of most tRNAs that decode ANN codons, and stabilizes cognate anticodon-codon interactions. Nearly all genetic studies of the t6A pathway have focused on single-celled organisms. In this study, we report the isolation of an extensive allelic series in the Drosophila ortholog of the core t6A biosynthesis factor Kae1. kae1 hemizygous larvae exhibit decreases in t6A that correlate with allele strength; however, we still detect substantial t6A-modified tRNAs even during the extended larval phase of null alleles. Nevertheless, complementation of Drosophila Kae1 and other t6A factors in corresponding yeast null mutants demonstrates that these metazoan genes execute t6A synthesis. Turning to the biological consequences of t6A loss, we characterize prominent kae1 melanotic masses and show that they are associated with lymph gland overgrowth and ectopic generation of lamellocytes. On the other hand, kae1 mutants exhibit other phenotypes that reflect insufficient tissue growth. Interestingly, whole-tissue and clonal analyses show that strongly mitotic tissues such as imaginal discs are exquisitely sensitive to loss of kae1, whereas nonproliferating tissues are less affected. Indeed, despite overt requirements of t6A for growth of many tissues, certain strong kae1 alleles achieve and sustain enlarged body size during their extended larval phase. Our studies highlight tissue-specific requirements of the t6A pathway in a metazoan context and provide insights into the diverse biological roles of this fundamental RNA modification during animal development and disease.

A central support system can facilitate implementation and sustainability of a Classroom-based Undergraduate Research Experience (CURE) in Genomics.

In their 2012 report, the President's Council of Advisors on Science and Technology advocated "replacing standard science laboratory courses with discovery-based research courses"-a challenging proposition that presents practical and pedagogical difficulties. In this paper, we describe our collective experiences working with the Genomics Education Partnership, a nationwide faculty consortium that aims to provide undergraduates with a research experience in genomics through a scheduled course (a classroom-based undergraduate research experience, or CURE). We examine the common barriers encountered in implementing a CURE, program elements of most value to faculty, ways in which a shared core support system can help, and the incentives for and rewards of establishing a CURE on our diverse campuses. While some of the barriers and rewards are specific to a research project utilizing a genomics approach, other lessons learned should be broadly applicable. We find that a central system that supports a shared investigation can mitigate some shortfalls in campus infrastructure (such as time for new curriculum development, availability of IT services) and provides collegial support for change. Our findings should be useful for designing similar supportive programs to facilitate change in the way we teach science for undergraduates.

A course-based research experience: how benefits change with increased investment in instructional time.

There is widespread agreement that science, technology, engineering, and mathematics programs should provide undergraduates with research experience. Practical issues and limited resources, however, make this a challenge. We have developed a bioinformatics project that provides a course-based research experience for students at a diverse group of schools and offers the opportunity to tailor this experience to local curriculum and institution-specific student needs. We assessed both attitude and knowledge gains, looking for insights into how students respond given this wide range of curricular and institutional variables. While different approaches all appear to result in learning gains, we find that a significant investment of course time is required to enable students to show gains commensurate to a summer research experience. An alumni survey revealed that time spent on a research project is also a significant factor in the value former students assign to the experience one or more years later. We conclude: 1) implementation of a bioinformatics project within the biology curriculum provides a mechanism for successfully engaging large numbers of students in undergraduate research; 2) benefits to students are achievable at a wide variety of academic institutions; and 3) successful implementation of course-based research experiences requires significant investment of instructional time for students to gain full benefit.

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.

Harnessing the natural Drosophila-parasitoid model for integrating insect immunity with functional venomics.

Drosophila species lack most hallmarks of adaptive immunity yet are highly successful against an array of natural microbial pathogens and metazoan enemies. When attacked by figitid parasitoid wasps, fruit flies deploy robust, multi-faceted innate immune responses and overcome many attackers. In turn, parasitoids have evolved immunosuppressive strategies to match, and more frequently to overcome, their hosts. We present methods to examine the evolutionary dynamics underlying anti-parasitoid host defense by teasing apart the specialized immune-modulating venoms of figitid parasitoids and, in turn, possibly delineating the roles of individual venom molecules. This combination of genetic, phylogenomic, and "functional venomics" methods in the Drosophila-parasitoid model should allow entomologists and immunologists to tackle important outstanding questions with implications across disciplines and to pioneer translational applications in agriculture and medicine.

Polydnaviral ankyrin proteins aid parasitic wasp survival by coordinate and selective inhibition of hematopoietic and immune NF-kappa B signaling in insect hosts.

Polydnaviruses are mutualists of their parasitoid wasps and express genes in immune cells of their Lepidopteran hosts. Polydnaviral genomes carry multiple copies of viral ankyrins or vankyrins. Vankyrin proteins are homologous to IκB proteins, but lack sequences for regulated degradation. We tested if Ichnoviral Vankyrins differentially impede Toll-NF-κB-dependent hematopoietic and immune signaling in a heterologous in vivo Drosophila, system. We first show that hematopoiesis and the cellular encapsulation response against parasitoid wasps are tightly-linked via NF-κB signaling. The niche, which neighbors the larval hematopoietic progenitors, responds to parasite infection. Drosophila NF-κB proteins are expressed in the niche, and non cell-autonomously influence fate choice in basal and parasite-activated hematopoiesis. These effects are blocked by the Vankyrin I²-vank-3, but not by P-vank-1, as is the expression of a NF-κB target transgene. I²-vank-3 and P-vank-1 differentially obstruct cellular and humoral inflammation. Additionally, their maternal expression weakens ventral embryonic patterning. We propose that selective perturbation of NF-κB-IκB interactions in natural hosts of parasitic wasps negatively impacts the outcome of hematopoietic and immune signaling and this immune deficit contributes to parasite survival and species success in nature.

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.

Sumoylation is tumor-suppressive and confers proliferative quiescence to hematopoietic progenitors in Drosophila melanogaster larvae.

How cell-intrinsic regulation of the cell cycle and the extrinsic influence of the niche converge to provide proliferative quiescence, safeguard tissue integrity, and provide avenues to stop stem cells from giving rise to tumors is a major challenge in gene therapy and tissue engineering. We explore this question in sumoylation-deficient mutants of Drosophila. In wild type third instar larval lymph glands, a group of hematopoietic stem/progenitor cells acquires quiescence; a multicellular niche supports their undifferentiated state. However, how proliferative quiescence is instilled in this population is not understood. We show that Ubc9 protein is nuclear in this population. Loss of the SUMO-activating E1 enzyme, Aos1/Uba2, the conjugating E2 enzyme, Ubc9, or the E3 SUMO ligase, PIAS, results in a failure of progenitors to quiesce; progenitors become hyperplastic, misdifferentiate, and develop into microtumors that eventually detach from the dorsal vessel. Significantly, dysplasia and lethality of Ubc9 mutants are rescued when Ubc9(wt) is provided specifically in the progenitor populations, but not when it is provided in the niche or in the differentiated cortex. While normal progenitors express high levels of the Drosophila cyclin-dependent kinase inhibitor p21 homolog, Dacapo, the corresponding overgrown mutant population exhibits a marked reduction in Dacapo. Forced expression of either Dacapo or human p21 in progenitors shrinks this population. The selective expression of either protein in mutant progenitor cells, but not in other hematopoietic populations, limits overgrowth, blocks tumorogenesis, and restores organ integrity. We discuss an essential and complex role for sumoylation in preserving the hematopoietic progenitor states for stress response and in the context of normal development of the fly.

Association of a new Wolbachia strain with, and its effects on, Leptopilina victoriae, a virulent wasp parasitic to Drosophila spp.

Wolbachia bacteria are ubiquitous intracellular bacteria of arthropods. Often considered reproductive parasites, they can benefit certain host species. We describe a new Wolbachia strain from Leptopilina victoriae, a Drosophila wasp. The strain is closely related to Wolbachia from Culex sp. Located to the posterior poles of oocytes, it manipulates its host's reproduction by inducing a male development type of cytoplasmic incompatibility. We also report its diverse effects on the wasp's life history traits.

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 hosts. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited. Strains of L. boulardi also produce virus-like particles although they differ significantly in their ability to succeed on D. melanogaster. Some of these L. boulardi strains are difficult to grow on D. melanogaster 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. 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). 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). 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).