Protocols for Efficient Rearing and Functional Analysis of the Endoparasitoid Wasp Asobara japonica and its Host Drosophila melanogaster

Summary

The goal of this protocol is to facilitate functional analysis of parasitoid-host interactions in Asobara japonica and Drosophila melanogaster through optimized rearing methods, a single-oviposition infection assay, and RNAi-mediated gene knockdown.

Abstract

Parasitism is a biological interaction in which one organism exploits the body or resources of another (the host), resulting in substantial damage or death to the host. Among parasitic animals, parasitoid wasps are one of the most species-rich lineages, accounting for nearly 20% of all insect species. In particular, endoparasitoid wasps oviposit directly into host bodies and deploy a diverse array of venom factors to manipulate host development, immunity, and physiology. In response to wasp attack, hosts attempt to eliminate parasitoid eggs through innate immune mechanisms. However, the molecular and cellular mechanisms by which individual venom components modulate host biology and promote successful parasitism remain poorly understood. Here, we describe a standardized laboratory protocol for rearing the endoparasitoid wasp Asobara japonica and its host Drosophila melanogaster. Both parthenogenetic and sexual strains of A. japonica are available, and the parthenogenetic strain exhibits a high parasitism success rate, allowing stable maintenance of laboratory stocks for genome analysis and parasitism assays. A single-oviposition infection assay and a double-stranded RNA-based gene knockdown method were optimized for functional analysis of venom genes. Together, these protocols provide a practical experimental framework for dissecting the molecular mechanisms underlying parasitoid-host interactions and will facilitate future research in developmental biology, immunology, and physiology.

Introduction

Parasitism is a lifestyle in which one organism exploits the nutritional resources of another and is widespread across taxa. It is estimated that up to half of all known organisms exhibit parasitism in their life cycle¹. Among parasites, parasitoid wasps are a particularly diverse group, accounting for approximately 20% of all insects². They exploit a wide range of arthropod hosts, such as insects, spiders, and mites, and exhibit remarkable diversity in their life histories and parasitic strategies³. These features make parasitoid wasps one of the most evolutionarily and ecologically successful insect groups.

Interactions between parasitoids and their hosts have been extensively studied as model systems of evolutionary “arms races.” In particular, endoparasitoid wasps deploy a diverse array of factors, including venoms, symbiotic viruses, and teratocytes^4,5. These factors manipulate host physiology and development, thereby ensuring successful development within the host. Despite these advances, the molecular mechanisms underlying parasitism remain poorly understood across most parasitoid species. One major limitation is their small body size, which has hindered the biochemical identification of venom components. In addition, it is technically challenging to maintain parasitoids and their hosts with synchronized developmental stages under laboratory conditions.

Recent advances in next-generation sequencing and other omics technologies now enable genome-wide analyses even in very small parasitoid species. These approaches have opened new avenues for investigating previously inaccessible molecular mechanisms, allowing parasitoid-host interactions to be studied at unprecedented resolution. In this context, our laboratory focuses on Asobara japonica Belokobylskij (Hymenoptera: Braconidae), a species originally identified in Japan^6,7,8. A. japonica includes a thelytokous parthenogenetic strain, in which females produce offspring without mating. This feature allows us to collect substantial amounts of genetically uniform genomic DNA for whole-genome sequencing⁹. In addition, this species parasitizes a broad range of Drosophila species, including the model organism D. melanogaster, which offers major experimental advantages due to its well-established rearing conditions and precisely defined developmental stages, enabling reproducible infection assays and accurate control of parasitoid development¹⁰.

During parasitism of A. japonica, a female wasp injects venom together with a single egg into a fly larva, inside which a wasp larva grows alongside its host. Notably, A. japonica exhibits a high parasitism success rate on the host D. melanogaster, making this parasitoid-host pair a powerful experimental model. Here, we describe protocols for stable rearing, a single-oviposition infection assay, and a double-stranded RNA (dsRNA)-based gene knockdown method in A. japonica. These protocols provide a reliable framework for dissecting molecular mechanisms underlying parasitoid-host interactions.

Protocol

A schematic overview of the synchronized rearing method for D. melanogaster and A. japonica is presented in Figure 1.

1. Rearing the host fruit fly Drosophila melanogaster in the laboratory

1. Maintain all experimental animals at 25 °C and 50-60% relative humidity under a 12:12 h light/dark cycle. Use the wild-type strain Oregon-R (OR) as a host for A. japonica (Figure 2A).
2. Transfer adult flies to a new food vial containing standard cornmeal-yeast-agar medium (0.55% agar, Table 1). Label the vial with the date of transfer and the strain name "OR".
3. Clear these adult flies one day after the transfer to prevent an excessive number of eggs in the food. Make sure that offspring larvae are feeding on the surface of the food.
   NOTE: As a guideline, maintain approximately 200-250 eggs per food vial (φ30×100 mm). Higher egg densities may reduce parasitism success and increase variation in host and wasp development.
4. Four days after Step 1.2, transfer a spoonful of fly larvae (4 days after egg laying, dAEL) to a new food vial. Keep these larvae unexposed to parasitoids for the next generation. This procedure also helps to control the host larval density in a vial.

2. Rearing the endoparasitoid wasp Asobara japonica in the laboratory

1. Use a parthenogenetic strain Tokyo (TK) and a sexual strain Iriomote-jima (IR) (Figure 2B). The former consists of females, whereas the latter consists of females and males. Males eclose two-four days earlier than females. Therefore, confirm the presence of females by their ovipositors before the transfer.
2. Transfer 10-20 adult wasps directly from the stock vial into the vial containing fly larvae at 4 dAEL. Label the vial with the date of transfer and the strain name "TK" or "IR". Maintain the wasps in the same conditions as Step 1.1.
3. Allow adult wasps to oviposit in fly larvae. Clear these wasps one to two days later to prevent superparasitism, which can lead to host death. Remove eclosed adult flies that escaped from parasitism 10 or more days after Step 2.2.
4. Two weeks after Step 2.2, adult wasps begin to eclose from the host fly pupal case. Transfer these newly-eclosed adult wasps to a new food vial with filter paper containing 25% glucose and 50 mM Vitamin C solution. The pieces of filter paper should be sterilized at 130 °C for 2 h. Providing the solution on filter paper promotes oviposition behaviors and prolongs the lifespan of adult wasps. Our approach to using vitamin C to enhance oviposition was inspired by observations in Drosophila studies¹¹.

3. Single-Oviposition Infection Assay

1. Synchronize the developmental stage of fly larvae used for the infection experiment.
   1. Place adult flies in a 50-mL tube containing a grape-juice agar plate (3% agar) spread with fresh yeast paste and allow them to lay eggs for 24 h.
   2. Collect newly-hatched first-instar larvae on the grape-juice agar plate and transfer them to a small vial (12 mL) containing 2-3 g fly food paste (fly food mixed with an appropriate amount of water). 20-30 larvae are placed in the vial. The fly larvae are allowed to grow at 25 °C.
2. At the appropriate time point, prepare a 4 cm Petri dish containing a thin, evenly spread layer of fly food paste covering an area approximately 1.5-2.0 cm in diameter (hereafter referred to as infection assay arena). In addition to the infection assay arena, prepare a new small vial (12 mL) containing an appropriate amount of fly food paste. This vial is used for rearing the host larvae after the infection assay.
3. Collect staged larvae and transfer them to a dish containing deionized water (resistivity = 15 MΩ·cm, 25 °C). Rinse the larvae in the dish with gentle agitation to remove residual food from their bodies.
4. After rinsing, gently remove excess moisture from the larvae using lint-free paper wipes, as excess water interferes with wasp oviposition behavior on the infection assay arena. Then transfer the larvae to the arena. This step increases larval activity during the infection experiment.
5. Anesthetize adult wasps with CO₂ gas at a flow rate of 5 L·min^-1 and a pressure of 0.2 MPa. Then, gently place approximately three female wasps into the infection assay arena. Close the lid of the dish and allow them to recover on fly food paste.
6. Observe the oviposition behavior of these wasps under a stereomicroscope (zoom setting: 10× to 15×).
   NOTE: When a wasp touches a moving fly larval body, it quickly inserts its ovipositor into the fly body and injects venom components. Although the fly larva writhes and thrashes its body, it immediately becomes paralyzed. Subsequently, the wasp vibrates its abdomen and deposits an egg into the host larval body, after which the ovipositor is withdrawn. This oviposition behavior is referred to as infection.
7. After infection, open the lid and transfer the infected fly larva to the new small vial containing fly food paste prepared in Step 3.2. To standardize infection timing, perform the infection experiment within 15 min.
8. Label the small vial with experimental information, such as the date, sample name, infection time, and number of individuals. These vials are plugged and then placed in a humid container and kept at 25 ˚C.

4. Generation of RNA interference (RNAi) wasps by dsRNA injection

1. Synthesize dsRNAs of 400 bp or longer for dsRNA-mediated gene knockdown (RNA interference, RNAi) using an in vitro transcription-based dsRNA synthesis system. The dsRNA samples should be kept at -20 to -80 °C until use. Detailed protocols are as previously described⁹.
2. Prepare a 5.5 cm petri dish containing 2% agar for placing wasp bodies. Fill two-thirds of each dish with agar to generate a thick layer of agar plate for injection.
3. Place a lint-free paper wipe moistened with water on the lid of a plastic dish.
4. Collect host fly pupae at 7 days post-infection (dpi) from the vial and place them on the lint-free paper wipes prepared in Step 4.3. Adding a drop of water to the vial wall helps to detach host fly pupal cases from the vial.
5. Under the stereomicroscope (zoom setting: 20×), remove the host fly pupal case and expose a wasp body with forceps. At 7 dpi, most wasps are in the pupal stage, in which the head, thorax, and abdomen parts are distinguishable. A wasp body is immobile.
6. Place the retrieved wasp bodies on the agar plate prepared in Step 4.2, aligning their body axes in the same orientation. Arrange 20-30 wasp bodies with their dorsal side up in one to two lines. Remove excess moisture on the agar plate using a lint-free paper wipe to prevent wasps from slipping.
7. Following the manufacturer’s instructions, set up the injection apparatus and stereomicroscope (Figure 3A). Use a microinjection system equipped with a glass capillary needle that allows precise control of injection volume and injection speed. Place the agar plate in which the wasp heads are oriented toward the tip of the injection needle. The injection volume is 50 nL·wasp^-1 and the injection speed is 50 nL·sec^-1 on the injector.
8. Prepare glass capillary needles using a needle puller. The shape of glass capillary is adjustable with heater temperature.
9. Place the glass capillary needle on the needle grinder at an angle of 30-35°. Grind the needle until the tip length reaches 0.04 mm (four divisions on the eyepiece reticle, Figure 3B).
10. Just before injection, thaw dsRNA. The concentration of dsRNA is 0.25-1 µg·µL^-1. For visualization, the dsRNA solution is colored with a blue dye (Erioglaucine disodium salt, 2% weight·volume^-1). Using a microloader pipette tip, fill the glass capillary with dsRNA solution with blue dye. 4 µL is sufficient for injecting 30 wasp bodies. Ensure that no air bubbles are present inside the solution.
    NOTE: After loading the dsRNA solution, gently tap the side of the needle with a fingernail to remove any trapped air bubbles. Residual air bubbles can obstruct the flow of the solution and interfere with successful injection.
11. Using a 1 mL syringe and a needle, fill the unoccupied portion of the glass capillary with mineral oil, ensuring that no air bubbles remain inside the capillary.
12. Attach the glass capillary needle containing dsRNA solution and mineral oil to the needle holder and then mount it onto the injection device.
13. Adjust the position of the agar plate and the glass capillary needle on the injector, so that the glass needle is positioned at a 30-45° angle relative to the wasp bodies (Figure 3A).
14. Insert the glass capillary needle into the dorsal side of the wasp body, with the slightly lateral side to the dorsal midline. Microinject the dsRNA solution inside the body. The injected dsRNA solution is visible with blue dye (Figure 3C).
15. After the injection, place the agar plates in a humid container (70-80% relative humidity) and maintain them in a 25 °C incubator for a week, until adult wasps eclose. Injured wasp bodies should be removed from the agar plate to prevent bacterial contamination.
    PAUSE POINT: In Step 4.6, the wasp pupae can be kept on the agar plate for several hours before injection. Needles can be prepared in advance (Step 4.8 and 4.9); however, they are prone to clogging over time. Freshly prepared needles generally provide more reliable injection performance.

Results

The asexual and sexual strains of Asobara japonica
A. japonica is widely distributed throughout Japan. Thelytokous parthenogenetic strains are predominant in the main islands, while arrhenotokous sexual strains are found in the subtropical islands, including Amami-Oshima and Iriomote-jima islands^8,12. In sexual strains, males emerge earlier than females by a few days so that the timing of adult transfer is distinct between t...

Discussion

In this study, we established a versatile experimental platform for investigating parasitoid-host interactions at the molecular level. By optimizing rearing conditions, a single-oviposition infection assay, and the RNAi protocol, we provide a robust system for functional studies of parasitoid-derived factors. This platform will facilitate future studies aimed at uncovering the mechanisms by which parasitoid wasps manipulate host development and may also contribute to the development of biological pest control strategies....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
50 mL tube	Greiner Bio-One	210261	Infection assay
1 mL syringe	TERUMO	SS-01T	dsRNA injection
Agar	Daisin, Ltd.	P-700	Rearing
Asnol Petri Dish φ40×13.5mm	As One Corporation	1-8549-01	Infection assay
Asnol Petri Dish φ55×17mm	As One Corporation	1-8549-02	dsRNA injection
Blue dye (Erioglaucine disodium salt)	Sigma-Aldrich Co. Llc.	861146-25G	dsRNA injection
Butyl p-Hydroxybenzoate	Nacalai Tesque, Inc.	06327-15	Rearing
CO2 pressure regulator	YAMATOSANGYO	YR-507F-2	Infection assay
Confocal laser scanning microscope	Zeiss	LSM700	Image analysis
Corn meal	Sunny Maize Co., Ltd.	No.4M	Rearing
DAPI	Thermo Fisher Scientific Inc.	PI62247	Nuclei staining, 1:10000
Deionized water	Millipore Inc.	ZLXEV030WW	Infection assay
Dual-Stage Glass Micropipette Puller	Narishige	PC-10	dsRNA injection
Fiji	NA	https://fiji.sc	Image analysis
Filter paper	Advantec	ITEM 526	Rearing
Fly food vial	Chiyoda Science Co., Ltd.	KFB-3M	Rearing
Fly food vial plug	Chiyoda Science Co., Ltd.	AS-275	Rearing
Forceps	Dumont Biologie	11252-20	Dissection
Freezer, -20 °C	Nihon Freezer	GS-3120HC	Sample storage
Freezer, -80 °C	Nihon Freezer	CLN-52UD2	Sample storage
Glass slides	Matsunami Glass Ind., Ltd	S7213	Infection assay
Glucose	Showa Sangyo Co., Ltd.	Not availavle	Rearing
KOD Plus Neo	Toyobo	KOD-401	dsRNA synthesis
Incubator	Panasonic	MIR-254-PJ	Rearing
Ligation high Ver.2	Toyobo	LGK-201	dsRNA synthesis
Micropipette Grinder	Narishige	EG-401	dsRNA injection
Mineral oil	Nacalai Tesque, Inc.	23306-84	dsRNA injection
Nanoject III	Drummond Scientific Company	3-000-207	dsRNA injection
Nanoject Glass Capillaries	Drummond Scientific Company	3-000-203-G/X	dsRNA injection
pBluescript KS (+) plasmid	Not availavle	Not availavle	dsRNA synthesis
pBluescript SK (-) plasmid	Not availavle	Not availavle	dsRNA synthesis
PrimeScript reverse Transcriptase	Takara	2680A	dsRNA synthesis
Propionic acid	Nacalai Tesque, Inc.	29018-55	Rearing
PROWIPE	Daio Paper Corporation	2-2624-02	Infection assay, dsRNA injection.
ReverTra Ace qPCR RT Master Mix with gDNA Remover	Toyobo	FSQ-301	qRT-PCR
RNAiso Plus reagent	Takara	9108	qRT-PCR
Small vial (Test tube 12 mL)	Sarstedt	REF 58.487	Infection assay
Small vial plug	Chiyoda Science Co., ltd.	QD-S4	Infection assay
Spatula	As One Corporation	6-522-02	Rearing
Square petri dish	Eiken Chemical Co., Ltd.	64-2192-55, AW2000	dsRNA injection
Stereomicroscope	Leica Microsystems	lvesta3 (C-Mount)	Infection assay
Stereomicroscope	Nikon Solutions Co., Ltd.	SMZ1000	dsRNA injection
T7 RiboMAX Express RNAi System	Promega	P1700	dsRNA synthesis
Thermal Cycler Dice Real Time System	Takara	TP815	qRT-PCR
Thermal Cycler GeneAtlas	Astel	G02	dsRNA synthesis, qRT-PCR
THUNDERBIRD SYBR qPCR Mix	Toyobo	QPS-201	qRT-PCR
TissueLyser II	Qiagen	Not availavle	qRT-PCR
Vitamin C, L-Ascorbic Acid	Nacalai Tesque, Inc.	03420-65	Rearing
Welch's grape 100 juice	Asahi Soft drinks Co., Ltd.	32390	Infection assay
Yeast	Asahi Group Foods, Ltd.	HB-P02	Rearing