Background

Parasitoid wasps attack the Drosophila genus with an infection rate of 90% in the natural population (Fleury et al., 2004). Depending on the species, wasps lay their eggs in the developing stages of fruit fly larvae or pupae. If the parasitized host fails to encapsulate the newly injected parasitoid egg, these developing eggs hatch into larval wasps that devour the fly progeny innards before emerging from a Drosophila pupal case. After mating, the adult female parasitoid searches for a new host and thus begins the parasitoid life cycle (Carton and Nappi, 1997).

Drosophila have evolved various defensive behavioral and cellular strategies to reduce the risk of parasitoid infection. For instance, while fly larvae display a rolling response (Hwang et al., 2007; Robertson et al., 2013) and parasitoid avoidance behavior to escape the wasp attack (Ebrahim et al., 2015), Drosophila adults show accelerated mating behavior (Ebrahim et al., 2021), avoidance response (Ebrahim et al., 2015), oviposition depression (Lynch et al., 2016; Sadanandappa et al., 2021), and ethanol-seeking behavior (Kacsoh et al., 2013; Kacsoh et al., 2015) in the presence of parasitoids. These host defensive strategies lead to parasitoid’s counter-offensive tactics, resulting in more substantial selection pressure on the host. In the wild, both the host and the parasitoid seem to be locked in a perpetual arms race to best one another's newly evolved adaptations (Wertheim, 2022). Beyond the ecological and evolutionary perspective, studying host–parasitoid biology in the laboratory is significant in economic value. Since parasitoids are used in the biocontrol of fruit flies and other insects, understanding the strategies employed by parasitoid wasps helps to reduce agricultural loss by pests.

Compared to Drosophila , parasitoid wasps culturing in the laboratory can be labor intensive and demands careful planning and gentle handling of the wasps (Small et al., 2012). Here, we provide the step-by-step procedure for culturing parasitoid wasp species—Leptopilina and Trichopria—on Drosophila larvae and pupae, respectively, in laboratory conditions that already exist in a typical fruit fly research or teaching laboratory. Once established, the maintenance and scaling up to larger productions of the parasitoid cultures do not present significant challenges; optimized culture conditions are generally transferable to many parasitoid species. We also summarize the egg-laying assay used by Sadanandappa et al. (2021) to study the parasitoid-induced reproductive modifications in female fruit flies and discuss the adaptability of this behavioral prototype to examine the influence of environmental and genetic factors in germline development.

Materials and Reagents

1. Narrow Drosophila vials (Genesee Scientific, catalog number: 32-116SB)

2. Flugs^® narrow plastic vials (Genesee Scientific, catalog number: 49-102)

3. Square bottom Drosophila bottle (Genesee Scientific, catalog number: 32-130)

4. Flugs^® plastic fly bottles (Genesee Scientific, catalog number: 49-100)

5. Round synthetic paintbrush (Lowell hillcrest, size 4/0 or 3/0)

6. Kimtech^TM science kimwipes (Kimberly-Clark Professional, catalog number: 34155, 4.4” × 8.4”)

7. Cheesecloth (Genesee Scientific, catalog number: 53-100)

8. Host cultures: larvae and pupae of Drosophila melanogaster (strain Canton S, Bloomington Stock Center)

9. Larval parasitoid wasps: Leptopilina boulardi (strain Lb17) or L. heterotoma (strain Lh14) (obtained from Todd Schlenke’s laboratory at the University of Arizona)

10. Pupal parasitoid wasp: Trichopria drosophilae (strain Trical) (from Todd Schlenke’s laboratory)

11. Raw and unfiltered honey (Nature Nate’s Honey Co.)

12. Industrial grade carbon dioxide (CO₂) (Airgas, an Air Liquide company)

13. Fly morgue (glass beaker containing Wescodyne^® Plus diluted in water and plastic funnel)

14. Ethanol, 70%, laboratory grade

15. Drosophila medium (cornmeal molasses yeast medium) (see Recipes)

    1. Gelidium agar (MoorAgar, catalog number: 41076)

    2. Yeast (Phileo by Lesaffre, catalog number: 73050 SafPro Relax+YF)

    3. Corn (MP Biomedicals, catalog number: 0290141180)

    4. Molasses (Reinhart Foodservice, catalog number: DW816)

    5. Propionic acid (Fisher Scientific, catalog number: A258-500)

    6. Methyl 4-hydrobenzoate (NIPAGIN) (Sigma-Aldrich, catalog number: H5501)

Equipment

1. Stereomicroscope (Zeiss, model: Stemi 2000)

2. Fiber optic light source (Fisher Scientific, model: Laxco^TM PIFOS150IB)

3. Incubator with controlled temperature (25 °C), humidity (60%), and 12:12 h light/dark cycles (Percival Scientific, model: DR41VL)

4. Fly room with controlled temperature (25 °C), humidity (60%), and 12:12 h light/dark cycles

5. Fly pad, CO₂ anesthetizing apparatus (Genesee Scientific, catalog number: 59-119)

Software

1. Prism 9 (GraphPad Software, LLC, San Diego, CA, https://www.graphpad.com)

2. OriginPro (OriginLab Corporation, Northampton, MA, https://www.originlab.com)

3. BioRender (BioRender, https://biorender.com)

Procedure