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The authors would like to thank Kanata Tachibana, Shion Kudo, Toshiya Makino, Shunta Yorimoto, Shuji Shigenobu, Akiko Kawamura, Ari Fujinoki, and Masako Iida for technical assistance. We also thank all other members in our laboratory.

A schematic overview of the synchronized rearing method for D. melanogaster and A. japonica&#160;is presented in Figure 1.
1. Rearing the host fruit fly Drosophila melanogaster&#160;in the laboratory
<ol>
	<li>Maintain all experimental animals at 25 &#176;C&#160;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&#160;(Figure 2A).</li>
	<li>Transfer adult flies to a new food vial containing standard cornmeal-yeast-agar medium (0.55% agar,&#160;Table 1). Label the vial with the date of transfer and the strain name &#34;OR&#34;.</li>
	<li>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 (&#966;30&#215;100 mm). Higher egg densities may reduce parasitism success and increase variation in host and wasp development.</li>
	<li>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.</li>
</ol>
2. Rearing the endoparasitoid wasp Asobara japonica in the laboratory
<ol>
	<li>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.</li>
	<li>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 &#34;TK&#34; or &#34;IR&#34;. Maintain the wasps in the same conditions as Step 1.1.</li>
	<li>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&#160;10 or&#160;more days after Step 2.2.</li>
	<li>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 &#176;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&#160;studies11.</li>
</ol>
3. Single-Oviposition Infection Assay
<ol>
	<li>Synchronize the developmental stage of fly larvae used for the infection experiment.
	<ol>
		<li>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.</li>
		<li>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 &#176;C.</li>
	</ol>
	</li>
	<li>At the appropriate time point, prepare a 4&#160;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.</li>
	<li>Collect staged larvae and transfer them to a dish containing deionized water (resistivity = 15 M&#937;&#183;cm, 25 &#176;C). Rinse the larvae in the dish with gentle agitation to remove residual food from their bodies.</li>
	<li>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.</li>
	<li>Anesthetize adult wasps with CO2 gas at a flow rate of 5 L&#183;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.</li>
	<li>Observe the oviposition behavior of these wasps under a stereomicroscope (zoom setting: 10&#215; to 15&#215;). 
	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.</li>
	<li>After infection, open the lid and transfer the infected fly larva to the&#160;new small vial containing fly food paste prepared in Step 3.2. To standardize infection timing, perform the infection experiment within 15 min.</li>
	<li>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 &#730;C.</li>
</ol>
4. Generation of RNA interference (RNAi) wasps by dsRNA injection
<ol>
	<li>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 &#176;C until use. Detailed protocols are as previously described9.</li>
	<li>Prepare a 5.5&#160;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.</li>
	<li>Place a lint-free paper wipe moistened with water on the lid of a plastic dish.</li>
	<li>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.</li>
	<li>Under the stereomicroscope (zoom setting: 20&#215;), 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.</li>
	<li>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.</li>
	<li>Following the manufacturer&#8217;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.&#160;Place the agar plate in which the wasp heads are oriented toward the tip of the injection needle. The injection volume is 50 nL&#183;wasp-1 and the injection speed is 50 nL&#183;sec-1 on the injector.</li>
	<li>Prepare glass capillary needles using a needle puller. The shape of glass capillary is adjustable with heater temperature.</li>
	<li>Place the glass capillary needle on the needle grinder at an angle of 30-35&#176;. Grind the needle until the tip length reaches 0.04 mm (four divisions on the eyepiece reticle, Figure 3B).</li>
	<li>Just before injection, thaw dsRNA. The concentration of dsRNA is 0.25-1 &#181;g&#183;&#181;L-1. For visualization, the dsRNA solution is colored with a blue dye (Erioglaucine disodium salt, 2% weight&#183;volume-1). Using a microloader pipette tip, fill the glass capillary with dsRNA solution with blue dye. 4 &#181;L 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.</li>
	<li>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.</li>
	<li>Attach the glass capillary needle containing dsRNA solution and mineral oil to the needle holder and then mount it onto the injection device.</li>
	<li>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&#176; angle relative to the wasp bodies (Figure 3A).</li>
	<li>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&#160;(Figure 3C).</li>
	<li>After the injection, place the agar plates in a humid&#160;container (70-80% relative humidity) and maintain them in a 25 &#176;C&#160;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.</li>
</ol>

<ol>
	<li>Hasik AZ, et al. Parasitism as a driver of host diversification. Nat Rev Biodivers. 2025;1(6):401-410.</li>
	<li>Pennacchio F, Strand MR. Evolution of developmental strategies in parasitic Hymenoptera. Annu Rev Entomol. 2006;51:233-258.</li>
	<li>Quicke DL. Parasitic Wasps. 1997:221-255.</li>
	<li>Moreau S, Asgari S. Venom proteins from parasitoid wasps and their biological functions. Toxins. 2015;7(7):2385-2412.</li>
	<li>Huang J, Chen XX, Zhan S. Molecular mechanisms underlying parasitoid-derived host manipulation strategies. Annu Rev Entomol. 2026;71:51-68.</li>
	<li>Belokobylskij SA. Key to the Insects of Russian Far East. Tribe Alysiini. In: Ler PA, ed. 1998;4(3):163-298.</li>
	<li>Ideo S, Watada M, Mitsui H, Kimura MT. Host range of Asobara japonica (Hymenoptera: Braconidae), a larval parasitoid of drosophilid flies. Entomol Sci. 2008;11(1):1-6.</li>
	<li>Mitsui H, Van Achterberg K, Nordlander G, Kimura MT. Geographical distributions and host associations of larval parasitoids of frugivorous Drosophilidae in Japan. J Nat Hist. 2010;41(25-28):1731-1738.</li>
	<li>Kamiyama T, et al. Whole-genome sequencing analysis and protocol for RNA interference of the endoparasitoid wasp Asobara japonica. DNA Res. 2022;29(4):1-14.</li>
	<li>Kamiyama T, et al. Parasitoid wasp venoms degrade Drosophila imaginal discs for successful parasitism. Sci Adv. 2025;11(5).</li>
	<li>Shrestha B, Aryal B, Lee Y. The taste of vitamin C in Drosophila. EMBO Rep. 2023;24(6).</li>
	<li>Murata Y, Ideo S, Watada M, Mitsui H, Kimura MT. Genetic and physiological variation among sexual and parthenogenetic populations of Asobara japonica (Hymenoptera: Braconidae), a larval parasitoid of drosophilid flies. Eur J Entomol. 2009;106(2):171-178.</li>
	<li>Werren JH, Baldo L, Clark ME. Wolbachia: master manipulators of invertebrate biology. Nat Rev Microbiol. 2008;6(10):741-751.</li>
	<li>Furihata S, Hirata M, Matsumoto H, Hayakawa Y.&#160;Bacteria endosymbiont,&#160;Wolbachia, promotes parasitism of parasitoid wasp Asobara japonica. PLoS One. 2015;10(10).</li>
	<li>Yamashita S, Takahashi KH. Artificial transfer of a thelytoky-inducing Wolbachia endosymbiont between strains of the endoparasitoid wasp Asobara japonica (Hymenoptera: Braconidae). Appl Entomol Zool. 2018;53(3):403-409.</li>
	<li>Wang X, Biondi A, Daane KM. Functional responses of three candidate Asian larval parasitoids evaluated for classical biological control of Drosophila suzukii (Diptera: Drosophilidae). J Econ Entomol. 2020;113(1):73-80.</li>
	<li>Niwa R, Niwa YS. Molecular mechanisms of ecdysteroid biosynthesis. Compr Mol Insect Sci. 2026:106-137.</li>
	<li>Zhang J, et al. A set of novel venom proteins enables parasitoid wasps to exploit older hosts and coexist with competitors. Adv Sci. 2026;13(4).</li>
	<li>Schott S, et al. A fluorescent toolkit for spatiotemporal tracking of apoptotic cells in living Drosophila tissues. Development. 2017;144(20):3840-3846.</li>
	<li>Kacsoh BZ, Bozler J, Ramaswami M, Bosco G. Social communication of predator-induced changes in Drosophila behavior and germ line physiology. eLife. 2015;4.</li>
	<li>Sadanandappa MK, Sathyanarayana SH, Kondo S, Bosco G. Neuropeptide F signaling regulates parasitoid-specific germline development and egg-laying in Drosophila. PLoS Genet. 2021;17(3).</li>
	<li>Amano S, Akada T, Takahashi KH. In vivo and in vitro developmental profiling of Asobara japonica, a larval endoparasitoid of drosophilid flies. Entomol Exp Appl. 2019;167(5).12790.</li>
	<li>Shirai Y, Piulachs MD, Belles X, Daimon T. DIPA-CRISPR is a simple and accessible method for insect gene editing. Cell Rep Methods. 2022;2(5):100215.</li>
	<li>Zhang X, Singh A, Soriano Martinez K, Ferree PM. Direct parental (DIPA) CRISPR in the jewel wasp, Nasonia vitripennis. G3 (Bethesda). 2024;14(7).</li>
</ol>

We have no conflict of interest to be disclosed.

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....

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 &#8220;arms races.&#8221; In particular, endoparasitoid wasps deploy a diverse array of factors, including venoms, symbiotic viruses, and teratocytes4,5. 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&#160;(Hymenoptera: Braconidae), a species originally identified in Japan6,7,8. 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,&#160;which&#160;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.

<table><tbody><tr><td>50 mL tube</td><td>Greiner Bio-One</td><td>210261</td><td>Infection assay&amp;nbsp;</td></tr><tr><td>1 mL syringe</td><td>TERUMO</td><td>SS-01T</td><td>dsRNA injection</td></tr><tr><td>Agar</td><td>Daisin, Ltd.</td><td>P-700</td><td>Rearing</td></tr><tr><td>Asnol Petri Dish &amp;phi;40&amp;times;13.5mm</td><td>As One Corporation</td><td>1-8549-01</td><td>Infection assay&amp;nbsp;</td></tr><tr><td>Asnol Petri Dish &amp;phi;55&amp;times;17mm</td><td>As One Corporation</td><td>1-8549-02</td><td>dsRNA injection</td></tr><tr><td>Blue dye (Erioglaucine disodium salt)</td><td>Sigma-Aldrich Co. Llc.</td><td>861146-25G</td><td>dsRNA injection</td></tr><tr><td>Butyl p-Hydroxybenzoate</td><td>Nacalai Tesque, Inc.</td><td>06327-15</td><td>Rearing</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; pressure regulator</td><td>YAMATOSANGYO</td><td>YR-507F-2</td><td>Infection assay</td></tr><tr><td>Confocal laser scanning microscope</td><td>Zeiss</td><td>LSM700</td><td>Image analysis</td></tr><tr><td>Corn meal</td><td>Sunny Maize Co., Ltd.</td><td>No.4M</td><td>Rearing</td></tr><tr><td>DAPI</td><td>Thermo Fisher Scientific Inc.</td><td>PI62247</td><td>Nuclei staining, 1:10000</td></tr><tr><td>Deionized water</td><td>Millipore Inc.</td><td>ZLXEV030WW</td><td>Infection assay</td></tr><tr><td>Dual-Stage Glass Micropipette Puller</td><td>Narishige</td><td>PC-10</td><td>dsRNA injection</td></tr><tr><td>Fiji</td><td>NA</td><td>&lt;a href=&quot;https://fiji.sc/&quot;&gt;https://fiji.sc&lt;/a&gt;</td><td>Image analysis</td></tr><tr><td>Filter paper</td><td>Advantec</td><td>ITEM 526</td><td>Rearing</td></tr><tr><td>Fly food vial</td><td>Chiyoda Science Co., Ltd.</td><td>KFB-3M</td><td>Rearing</td></tr><tr><td>Fly food vial plug</td><td>Chiyoda Science Co., Ltd.</td><td>AS-275</td><td>Rearing</td></tr><tr><td>Forceps</td><td>Dumont Biologie</td><td>11252-20</td><td>Dissection</td></tr><tr><td>Freezer, -20 &amp;deg;C</td><td>Nihon Freezer</td><td>GS-3120HC</td><td>Sample storage</td></tr><tr><td>Freezer, -80 &amp;deg;C</td><td>Nihon Freezer</td><td>CLN-52UD2</td><td>Sample storage</td></tr><tr><td>Glass slides</td><td>Matsunami Glass Ind., Ltd</td><td>S7213</td><td>Infection assay&amp;nbsp;</td></tr><tr><td>Glucose</td><td>Showa Sangyo Co., Ltd.&amp;nbsp;</td><td>Not availavle</td><td>Rearing</td></tr><tr><td>KOD Plus Neo</td><td>Toyobo</td><td>KOD-401</td><td>dsRNA synthesis</td></tr><tr><td>Incubator</td><td>Panasonic</td><td>MIR-254-PJ</td><td>Rearing</td></tr><tr><td>Ligation high Ver.2</td><td>Toyobo</td><td>LGK-201</td><td>dsRNA synthesis</td></tr><tr><td>Micropipette Grinder</td><td>Narishige</td><td>EG-401</td><td>dsRNA injection</td></tr><tr><td>Mineral oil</td><td>Nacalai Tesque, Inc.</td><td>23306-84</td><td>dsRNA injection</td></tr><tr><td>Nanoject III</td><td>Drummond Scientific Company</td><td>3-000-207</td><td>dsRNA injection</td></tr><tr><td>Nanoject Glass Capillaries</td><td>Drummond Scientific Company</td><td>3-000-203-G/X</td><td>dsRNA injection</td></tr><tr><td>pBluescript KS (+) plasmid</td><td>Not availavle</td><td>Not availavle</td><td>dsRNA synthesis</td></tr><tr><td>pBluescript SK (-) plasmid</td><td>Not availavle</td><td>Not availavle</td><td>dsRNA synthesis</td></tr><tr><td>PrimeScript reverse Transcriptase</td><td>Takara</td><td>2680A</td><td>dsRNA synthesis</td></tr><tr><td>Propionic acid</td><td>Nacalai Tesque, Inc.</td><td>29018-55</td><td>Rearing</td></tr><tr><td>PROWIPE</td><td>Daio Paper Corporation</td><td>2-2624-02</td><td>Infection assay, dsRNA injection.</td></tr><tr><td>ReverTra Ace qPCR RT Master Mix with gDNA Remover</td><td>Toyobo</td><td>FSQ-301</td><td>qRT-PCR</td></tr><tr><td>RNAiso Plus reagent</td><td>Takara</td><td>9108</td><td>qRT-PCR</td></tr><tr><td>Small vial (Test tube 12 mL)</td><td>Sarstedt</td><td>REF 58.487</td><td>Infection assay&amp;nbsp;</td></tr><tr><td>Small vial plug</td><td>Chiyoda Science Co., ltd.</td><td>QD-S4</td><td>Infection assay&amp;nbsp;</td></tr><tr><td>Spatula</td><td>As One Corporation</td><td>6-522-02</td><td>Rearing</td></tr><tr><td>Square petri dish</td><td>Eiken Chemical Co., Ltd.</td><td>64-2192-55, AW2000</td><td>dsRNA injection</td></tr><tr><td>Stereomicroscope</td><td>Leica Microsystems</td><td>lvesta3 (C-Mount)</td><td>Infection assay&amp;nbsp;</td></tr><tr><td>Stereomicroscope</td><td>Nikon Solutions Co., Ltd.</td><td>SMZ1000</td><td>dsRNA injection</td></tr><tr><td>T7 RiboMAX Express RNAi System</td><td>Promega</td><td>P1700</td><td>dsRNA synthesis</td></tr><tr><td>Thermal Cycler Dice Real Time System</td><td>Takara</td><td>TP815</td><td>qRT-PCR</td></tr><tr><td>Thermal Cycler GeneAtlas</td><td>Astel</td><td>G02</td><td>dsRNA synthesis, qRT-PCR</td></tr><tr><td>THUNDERBIRD SYBR qPCR Mix</td><td>Toyobo</td><td>QPS-201</td><td>qRT-PCR</td></tr><tr><td>TissueLyser II</td><td>Qiagen</td><td>Not availavle</td><td>qRT-PCR</td></tr><tr><td>Vitamin C, L-Ascorbic Acid</td><td>Nacalai Tesque, Inc.</td><td>03420-65</td><td>Rearing</td></tr><tr><td>Welch&#039;s grape 100 juice</td><td>Asahi Soft drinks Co., Ltd.</td><td>32390</td><td>Infection assay&amp;nbsp;</td></tr><tr><td>Yeast</td><td>Asahi Group Foods, Ltd.</td><td>HB-P02</td><td>Rearing</td></tr></tbody></table>

protocols for efficient rearing and functional analysis of the endoparasitoid wasp asobara japonica and its host drosophila melanogaster

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

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&#160;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.

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 islands8,12. In sexual strains, males emerge earlier than females by a few days so that the timing of adult transfer is distinct between t...

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.

Parasitism is a biological interaction in which one organism exploits the body or resources of another (the host), resulting in substantial damage or ...

protocols-for-efficient-rearing-functional-analysis-endoparasitoid

Research

JoVE Journal

Developmental Biology

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An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

The feeding circuit in Drosophila melanogaster larvae serves a simple yet powerful model that allows changes in feeding rate to be correlated with alterations in the stomatogastric neural circuitry. This circuit is composed of central serotonergic neurons that send projections to the mouth hooks as well as the foregut.

Functional Analysis of the Larval Feeding Circuit in Drosophila

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

Drosophila melanogaster adult flies have been extensively utilized as model organisms to investigate the molecular mechanisms underlying host antimicrobial innate immune responses and microbial infection strategies. To promote the D. melanogaster larva stage as an additional or alternative model system, a larval injection technique is described.

Drosophila melanogaster Larva Injection Protocol

The present protocol describes the rearing method of tortricid pest insects in the laboratories. The procedures to distinguish insects' sex and extract nucleic acids for high throughput sequencing are established using two tortricid pests.

Mass-Rearing and Molecular Studies in Tortricidae Pest Insects

Ganaspis brasiliensis-a larval parasitoid of Drosophila suzukii (a global invasive fruit crop pest)-has been approved or is considered for introduction into Europe and the United States for biological control of this pest. This article provides protocols for both small-scale and large-scale rearing of this parasitoid.

Methods for Rearing the Parasitoid Ganaspis brasiliensis, a Promising Biological Control Agent for the Invasive Drosophila suzukii

Nasonia wasp embryos were dissected from Lucillia sericata pupae after parasitization for 12-24 h and washed with alcohol and 10% sodium hypochlorite solution to obtain germ-free embryos. After rearing the germ-free embryos and supplying them with Nasonia rearing medium to grow and develop in vitro, germ-free Nasonia adults were obtained.

Optimizing the Rearing Procedure of Germ-Free Wasps

This paper outlines the laboratory maintenance (including mating and feeding) of the lower dipteran fly Bradysia (Sciara) coprophila.

Laboratory Maintenance of the Lower Dipteran Fly Bradysia (Sciara) coprophila: A New/Old Emerging Model Organism

University of Tsukuba.  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.

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

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