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The interactions between Drosophila melanogaster and several species of parasitoid wasps are an interesting system for studying immune response and immunosuppressive activities in nature. Parasitoid wasps succeed against insect immune systems through the suppression of melanotic capsule formation via the use of virus-like particles and other venom proteins, as well as through passive immune evasion techniques. Although D. melanogaster is a genetic model system, the genetics of D. melanogaster parasitoid wasps are largely uncharacterized. In order to understand the molecular basis of this particular host-pathogen interaction, it is important that genetic tools are developed in the most common species of D. melanogaster parasitoid wasps. To address this issue, a chromosome squash technique was developed, allowing for the visualization of metaphase chromosomes in five species of parasitoid wasps. Visualizing metaphase chromosomes allowed for the construction of karyograms, which provide basic genetic information about each species and insight into karyotype evolution across species.
Up to 80% of D. melanogaster larvae in natural populations are parasitized by wasps.
Parasitoid wasps are extremely successful at suppressing insect immune systems through passive immune evasion techniques and the use of virus-like particles and venom proteins.
Once wasp eggs hatch inside the fly, the wasp larvae begin to feed on fly tissues and eventually eclose from the fly pupal case.
No genetic studies have been performed on D. melanogaster parasitoid wasps.
Develop a protocol for visualizing metaphase chromosomes of parasitoid wasps.
Successfully karyotype five species of D. melanogaster parasitoid wasps; Leptopilina boulardi, Leptopilina heterotoma, Ganaspis xanathopoda, Asobara tabida, and L. victoria Philippines.
D. melanogaster parasitization
Second and third instar D. melanogaster larvae, strain Oregon R, were placed in petri dishes containing food and yeast. Four experienced female wasps were placed in the petri dish overnight, ensuring that all larvae were attacked. The next morning, the wasps were removed.
Dissecting Out Wasp Larvae
D. melanogaster pupal cases were dissected six days post wasp attack. The pupal casing was carefully removed with forceps, exposing D. melanogaster tissue. These tissues were carefully picked apart, revealing a 2nd or 3rd instar wasp larvae.
Chromosome Squashes
Wasp larvae were placed in 0.5% sodium citrate with 4 μg colchicine per ml for five minutes. Wasp larvae were then transferred to fixative solution, composed of 3:1 ethanol to acetic acid, for five minutes. A slide was cleaned with ethanol and warmed on a 40oC heat plate. Several drops of 60% acetic acid were placed on the warm slide, and five wasp larvae were transferred to the acetic acid. Wasp larvae were dissected into small pieces to ensure that a monolayer of cells formed on the slide. After the acetic acid evaporated, 4% giemsa was used to stain the slide for twenty minutes. A cover slip was firmly placed on the slide after 20 minutes, and chromosomes were visualized with a light microscope at 1000x magnification.
L. boulardi, L. heterotoma, G. xanathopoda, and L. victoria Philippines each have 10 chromosomes.
A. tabida has 17 chromosomes.
Future studies will examine the presence of “pathogenicity islands” in the wasps using fluorescent in situ hybridization.
We would like to thank members of the Schlenke lab for all of their help and support. This research is supported by the Howard Hughes Medical Institute under grant No. 52005873.
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