In Drosophila Hemolymph, Serine Proteases Are the Major Gelatinases and Caseinases.

After separation on gel zymography, Drosophila melanogaster hemolymph displays gelatinase and caseinase bands of varying sizes, ranging from over 140 to 25 kDa. Qualitative and quantitative variations in these bands were observed during larval development and between different D. melanogaster strains and Drosophila species. The activities of these Drosophila hemolymph gelatinase and caseinase were strongly inhibited by serine protease inhibitors, but not by EDTA. Mass spectrometry identified over 60 serine proteases (SPs) in gel bands corresponding to the major D. melanogaster gelatinases and caseinases, but no matrix metalloproteinases (MMPs) were found. The most abundant proteases were tequila and members of the Jonah and trypsin families. However, the gelatinase bands did not show any change in the tequila null mutant. Additionally, no clear changes could be observed in D. melanogaster gel bands 24 h after injection of bacterial lipopolysaccharides (LPS) or after oviposition by Leptopilina boulardi endoparasitoid wasps. It can be concluded that the primary gelatinases and caseinases in Drosophila larval hemolymph are serine proteases (SPs) rather than matrix metalloproteinases (MMPs). Furthermore, the gelatinase pattern remains relatively stable even after short-term exposure to pathogenic challenges.

Amount of venom that Leptopilina species inject into Drosophila melanogaster larvae in relation to parasitic success.

The Drosophila endoparasitoid wasps Leptopilina boulardi and L. heterotoma (Hymenoptera: Cynipidae) are pro-ovigenic species, i.e., females contain their lifetime number of mature eggs at emergence. They are therefore able to immediately parasitize many hosts when present. In response to parasitoid oviposition, the larval host D. melanogaster can mount an immune response, encapsulation, that can destroy the parasitoid eggs. This response is counteracted by the venom the wasp injects during oviposition. Here, we estimated the amount of venom injected into a D. melanogaster host larva using immunodetection of venom proteins and we attempted to correlate this amount with the number of eggs a female can lay on successive days. The venom reservoir of L. boulardi contains enough venom for at least 100 ovipositions while that of L. heterotoma contains venom for about 16 ovipositions. While a female L. boulardi may have enough venom for three days of parasitism when 20 or 40 larval hosts were presented each day, L. heterotoma certainly needs to synthesize new venom to parasitize the number of hosts offered. Interestingly, parasitism stopped (L. boulardi), egg protection (L. heterotoma) and egg hatching decreased (both species) after three days of parasitism. Thus, although venom does not appear to be a limiting factor for parasitism, our data suggest that it may have less effectiveness on the egg protection and on egg/host development after high repetitive egg laying.

Proteomics of purified lamellocytes from Drosophila melanogaster HopTum-l identifies new membrane proteins and networks involved in their functions.

In healthy Drosophila melanogaster larvae, plasmatocytes and crystal cells account for 95% and 5% of the hemocytes, respectively. A third type of hemocytes, lamellocytes, are rare, but their number increases after oviposition by parasitoid wasps. The lamellocytes form successive layers around the parasitoid egg, leading to its encapsulation and melanization, and finally the death of this intruder. However, the total number of lamellocytes per larva remains quite low even after parasitoid infestation, making direct biochemical studies difficult. Here, we used the HopTum-l mutant strain that constitutively produces large numbers of lamellocytes to set up a purification method and analyzed their major proteins by 2D gel electrophoresis and their plasma membrane surface proteins by 1D SDS-PAGE after affinity purification. Mass spectrometry identified 430 proteins from 2D spots and 344 affinity-purified proteins from 1D bands, for a total of 639 unique proteins. Known lamellocyte markers such as PPO3 and the myospheroid integrin were among the components identified with specific chaperone proteins. Affinity purification detected other integrins, as well as a wide range of integrin-associated proteins involved in the formation and function of cell-cell junctions. Overall, the newly identified proteins indicate that these cells are highly adapted to the encapsulation process (recognition, motility, adhesion, signaling), but may also have several other physiological functions (such as secretion and internalization of vesicles) under different signaling pathways. These results provide the basis for further in vivo and in vitro studies of lamellocytes, including the development of new markers to identify coexisting populations and their respective origins and functions in Drosophila immunity.

Hosting certain facultative symbionts modulates the phenoloxidase activity and immune response of the pea aphid Acyrthosiphon pisum.

The pea aphid Acyrthosiphon pisum hosts different facultative symbionts (FS) which provide it with various benefits, such as tolerance to heat or protection against natural enemies (e.g., fungi, parasitoid wasps). Here, we investigated whether and how the presence of certain FS could affect phenoloxidase (PO) activity, a key component of insect innate immunity, under normal and stressed conditions. For this, we used clones of A. pisum of different genetic backgrounds (LL01, YR2 and T3-8V1) lacking FS or harboring one or two (Regiella insecticola, Hamiltonella defensa, Serratia symbiotica + Rickettsiella viridis). Gene expression and proteomics analyses of the aphid hemolymph indicated that the two A. pisum POs, PPO1 and PPO2, are expressed and translated into proteins. The level of PPO genes expression as well as the amount of PPO proteins and phenoloxidase activity in the hemolymph depended on both the aphid genotype and FS species. In particular, H. defensa and R. insecticola, but not S. symbiotica + R. viridis, caused a sharp decrease in PO activity by interfering with both transcription and translation. The microinjection of different types of stressors (yeast, Escherichia coli, latex beads) in the YR2 lines hosting different symbionts affected the survival rate of aphids and, in most cases, also decreased the expression of PPO genes after 24 h. The amount and activity of PPO proteins varied according to the type of FS and stressor, without clear corresponding changes in gene expression. These data demonstrate that the presence of certain FS influences an important component of pea aphid immunity.

Venom Atypical Extracellular Vesicles as Interspecies Vehicles of Virulence Factors Involved in Host Specificity: The Case of a Drosophila Parasitoid Wasp.

Endoparasitoid wasps, which lay eggs inside the bodies of other insects, use various strategies to protect their offspring from the host immune response. The hymenopteran species of the genus Leptopilina, parasites of Drosophila, rely on the injection of a venom which contains proteins and peculiar vesicles (hereafter venosomes). We show here that the injection of purified L. boulardi venosomes is sufficient to impair the function of the Drosophila melanogaster lamellocytes, a hemocyte type specialized in the defense against wasp eggs, and thus the parasitic success of the wasp. These venosomes seem to have a unique extracellular biogenesis in the wasp venom apparatus where they acquire specific secreted proteins/virulence factors and act as a transport system to deliver these compounds into host lamellocytes. The level of venosomes entry into lamellocytes of different Drosophila species was correlated with the rate of parasitism success of the wasp, suggesting that this venosome-cell interaction may represent a new evolutionary level of host-parasitoid specificity.

Biochemical characterization and comparison of aspartylglucosaminidases secreted in venom of the parasitoid wasps Asobara tabida and Leptopilina heterotoma.

Aspartylglucosaminidase (AGA) is a low-abundance intracellular enzyme that plays a key role in the last stage of glycoproteins degradation, and whose deficiency leads to human aspartylglucosaminuria, a lysosomal storage disease. Surprisingly, high amounts of AGA-like proteins are secreted in the venom of two phylogenetically distant hymenopteran parasitoid wasp species, Asobara tabida (Braconidae) and Leptopilina heterotoma (Cynipidae). These venom AGAs have a similar domain organization as mammalian AGAs. They share with them key residues for autocatalysis and activity, and the mature α- and ß-subunits also form an (αß)2 structure in solution. Interestingly, only one of these AGAs subunits (α for AtAGA and ß for LhAGA) is glycosylated instead of the two subunits for lysosomal human AGA (hAGA), and these glycosylations are partially resistant to PGNase F treatment. The two venom AGAs are secreted as fully activated enzymes, they have a similar aspartylglucosaminidase activity and are both also efficient asparaginases. Once AGAs are injected into the larvae of the Drosophila melanogaster host, the asparaginase activity may play a role in modulating their physiology. Altogether, our data provide new elements for a better understanding of the secretion and the role of venom AGAs as virulence factors in the parasitoid wasps' success.

Development of RNAi in a Drosophila endoparasitoid wasp and demonstration of its efficiency in impairing venom protein production.

Endoparasitoid wasps are essential regulators of insect pests in ecosystems as well as important biological control auxiliaries. Traits important for parasitism success, such as the injection of venom proteins at oviposition, have thus been mainly studied. However, identification of the key genes involved among the large number of genes identified was still prevented by the lack of functional approaches. Here, we report the development of RNA interference (RNAi) in Leptopilina boulardi, a figitid endoparasitoid that performs its entire development inside the Drosophila host. Having set up conditions for in vitro development of parasitoid late larval stages or pupae, we first targeted the cinnabar gene by microinjecting double-stranded RNA (dsRNA), leading to its silencing and production of red-eyed individuals. We then demonstrated that expression of the gene encoding LbGAP, a virulence factor found in a high amount in L. boulardi venom, could be specifically and almost completely silenced. Finally, a time-course analysis revealed that LbGAP silencing lasted during the entire lifetime of L. boulardi. This is the first report of the efficient silencing of venom protein-encoding genes in parasitoid wasps. Overall, RNAi opens the way for a large-scale functional analysis of parasitoid venom factors as well as other traits involved in parasitism success and more largely in the biology of these ecologically important organisms.