The raspberry Gene Is Involved in the Regulation of the Cellular Immune Response in Drosophila melanogaster

Abstract

Drosophila is an extremely useful model organism for understanding how innate immune mechanisms defend against microbes and parasitoids. Large foreign objects trigger a potent cellular immune response in Drosophila larva. In the case of endoparasitoid wasp eggs, this response includes hemocyte proliferation, lamellocyte differentiation and eventual encapsulation of the egg. The encapsulation reaction involves the attachment and spreading of hemocytes around the egg, which requires cytoskeletal rearrangements, changes in adhesion properties and cell shape, as well as melanization of the capsule. Guanine nucleotide metabolism has an essential role in the regulation of pathways necessary for this encapsulation response. Here, we show that the Drosophila inosine 5'-monophosphate dehydrogenase (IMPDH), encoded by raspberry (ras), is centrally important for a proper cellular immune response against eggs from the parasitoid wasp Leptopilina boulardi. Notably, hemocyte attachment to the egg and subsequent melanization of the capsule are deficient in hypomorphic ras mutant larvae, which results in a compromised cellular immune response and increased survival of the parasitoid.

Fig 1. The eclosion of D. melanogaster versus L. boulardi G486 in ras² mutant and after RNAi silencing.

(A) The eclosion of flies (blue) and wasps (brown) from the ras² mutant or from the Oregon-R after parasitization and from the (B) non-infested control. (C, D) Number of hatching flies from the L. boulardi G486 infested progenies (msnF9mo-Gal4>P{TRIP.JF01446} and msnF9mo-Gal4>P{TRIP.HMC03250} attP2}) and parental lines (P{TRIP.JF01446}/+, P{TRIP>HMC03250/+ and msnF9mo/Gal4/+). The numbers in parenthesis indicate the number of the examined D. melanogaster pupae. The error bars indicate the standard error of the mean. *p<0.05, **p<0.01.

Fig 2. The phenotype and proportion of living wasp larvae, partially or completely encapsulated and melanized wasp eggs in ras² mutant or after RNAi silencing.

(A) The phenotypic categories of L. boulardi G486 larvae and capsules in D. melanogaster larvae after parasitization. Proportion of living wasp larvae (blue) and partially (red) or completely melanized (green) capsules in the ras² mutant or in the Oregon-R control (B, C) and from the parental lines (P{TRIP.JF01446}/+, P{TRIP>HMC03250/+ and msnF9mo/Gal4/+) and progenies (P{TRIP.JF01446}>msnF(mo/Gal4 and P{TRIP>HMC03250>msnF9mo/Gal4 72h after the L. boulardi G486 infestation. The numbers in parenthesis indicate the number of the examined Drosophila larvae. The error bars indicate the standard error of the mean.*p<0.05, **p<0.01, ***p<0.001.

Fig 3. Attachment of hemocytes to the wasp egg.

(A) Lamellocyte attachment to the wasp egg in the Oregon-R and in the (B) ras² larvae. (C) Proportion of partially or completely encapsulated and melanized wasp eggs 72h after immune induction in the ras² mutant or (D, E) after RNAi silencing. The numbers in parenthesis indicate the number of the examined Drosophila larvae. The error bars indicate the standard error of the mean **p<0.01, ***p<0.001. The scale bars indicate 20 μm.

Fig 4. The number and morphology of circulating hemocytes in non-infested and immune induced Oregon-R and ras² larvae.

(A) Total hemocyte number of non-infested and L. boulardi G486 infested (72h) larvae originated from Oregon-R or ras² mutant. n.s. means non-significant difference. (B) Hemocytes of non-infested or (C) L. boulardi G486 infested larvae (72h) were stained with plasmatocyte specific anti-NimC1 or with lamellocyte specific anti-Atilla antibody [38]. The scale bar indicates 20 μm.

Fig 5. Formation of pseudopod-like cytoplasmic extensions in hemocytes.

(A) The proportion of the filopodia-like extensions in hemocytes. (B) The morphology of circulating hemocytes in immune induced larvae 24 h after infestation. Arrowheads indicate the cytoplasmic extensions in hemocytes. The statistical analysis was performed counting at least 100 hemocytes derived from six larvae from each genotype. The experiment was repeated at least three times. The arrows show phalloidin-binding cytoplasmic extensions. The error bars indicate the standard error of the mean ***p<0.001. The scale bar indicates 20 μm.