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.

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.

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).

A database for the analysis of immunity genes in Drosophila: PADMA database.

While microarray experiments generate voluminous data, discerning trends that support an existing or alternative paradigm is challenging. To synergize hypothesis building and testing, we designed the Pathogen Associated Drosophila MicroArray (PADMA) database for easy retrieval and comparison of microarray results from immunity-related experiments (www.padmadatabase.org). PADMA also allows biologists to upload their microarray-results and compare it with datasets housed within PADMA. We tested PADMA using a preliminary dataset from Ganaspis xanthopoda-infected fly larvae, and uncovered unexpected trends in gene expression, reshaping our hypothesis. Thus, the PADMA database will be a useful resource to fly researchers to evaluate, revise, and refine hypotheses.

Genomic and karyotypic variation in Drosophila parasitoids (Hymenoptera, Cynipoidea, Figitidae).

Drosophila melanogaster Meigen, 1830 has served as a model insect for over a century. Sequencing of the 11 additional Drosophila Fallen, 1823 species marks substantial progress in comparative genomics of this genus. By comparison, practically nothing is known about the genome size or genome sequences of parasitic wasps of Drosophila. Here, we present the first comparative analysis of genome size and karyotype structures of Drosophila parasitoids of the Leptopilina Förster, 1869 and Ganaspis Förster, 1869 species. The gametic genome size of Ganaspis xanthopoda (Ashmead, 1896) is larger than those of the three Leptopilina species studied. The genome sizes of all parasitic wasps studied here are also larger than those known for all Drosophila species. Surprisingly, genome sizes of these Drosophila parasitoids exceed the average value known for all previously studied Hymenoptera. The haploid chromosome number of both Leptopilina heterotoma (Thomson, 1862) and Leptopilina victoriae Nordlander, 1980 is ten. A chromosomal fusion appears to have produced a distinct karyotype for Leptopilina boulardi (Barbotin, Carton et Keiner-Pillault, 1979)(n = 9), whose genome size is smaller than that of wasps of the Leptopilina heterotoma clade. Like Leptopilina boulardi, the haploid chromosome number for Ganaspis xanthopoda is also nine. Our studies reveal a positive, but non linear, correlation between the genome size and total chromosome length in Drosophila parasitoids. These Drosophila parasitoids differ widely in their host range, and utilize different infection strategies to overcome host defense. Their comparative genomics, in relation to their exceptionally well-characterized hosts, will prove to be valuable for understanding the molecular basis of the host-parasite arms race and how such mechanisms shape the genetic structures of insectcommunities.

Virulence factors and strategies of Leptopilina spp.: selective responses in Drosophila hosts.

To ensure survival, parasitic wasps of Drosophila have evolved strategies to optimize host development to their advantage. They also produce virulence factors that allow them to overcome or evade host defense. Wasp infection provokes cellular and humoral defense reactions, resulting in alteration in gene expression of the host. The activation of these reactions is controlled by conserved mechanisms shared by other invertebrate and vertebrate animals. Application of genomics and bioinformatics approaches is beginning to reveal comparative host gene expression changes after infection by different parasitic wasps. We analyze this comparison in the context of host physiology and immune cells, as well as the biology of the venom factors that wasps introduce into their hosts during oviposition. We compare virulence strategies of Leptopilina boulardi and L. heterotoma, in relation to genome-wide changes in gene expression in the fly hosts after infection. This analysis highlights fundamental differences in the changes that the host undergoes in its immune and general physiology in response to the two parasitic wasps. Such a comparative approach has the potential of revealing mechanisms governing the evolution of pathogenicity and how it impacts host range.

Quantitative analysis of phenol oxidase activity in insect hemolymph.

We describe a simple, inexpensive, and robust protocol for the quantification of phenol oxidase activity in insect hemolymph. Discrete volumes of hemolymph from Drosophila melanogaster larvae are applied to pieces of filter paper soaked in an L-3, 4-dihydroxyphenylalanine (L-DOPA) solution. Phenol oxidase present in the samples catalyzes melanin synthesis from the L-DOPA precursor, resulting in the appearance of a roughly circular melanized spot on the filter paper. The filter paper is then scanned and analyzed with image-processing software. Each pixel in an image is assigned a grayscale value. The mean of the grayscale values for a circular region of pixels at the center of the image of each spot is used to compute a melanization index (MI) value, the computation is based on a comparison to an external standard (India ink). Numerical MI values for control and experimental larvae can then be pooled and subjected to statistical analysis. This protocol was used to evaluate phenol oxidase activity in larvae of different backgrounds: wild-type, lozenge, hopscotch(Tumorous-lethal) (which induces the formation of large melanotic tumors), and body-color mutations ebony and yellow. Our results demonstrate that this assay is sensitive enough for use in genetic screens with D. melanogaster and could conceivably be used for evaluation of MI from hemolymph of other insects.