This research was funded by the Projects of the National Natural Science Foundation of China (32172476, 32102275), the Agricultural Science and Technology Innovation Program (CAAS-ZDRW202203, CAAS-ZDRW202108), and Central Funds Guiding the Local Science and Technology Development (XZ202301YD0042C).

NOTE: See the Table of Materials for details related to all materials, instruments, software, and reagents used in this protocol.
1. Collection and maintenance of insect culture
<ol>
	<li>Adhere a group of ~5,000 eggs of Corcyra cephalonica (Stainton) onto a 9 cm by 16 cm card using a 1:5 (v/v) solution of gum arabic powder and water25,26. 
	NOTE: Avoid attaching too many host eggs to the card as overcrowding makes it impractical for subsequent transfer and dissection procedures.</li>
	<li>To prevent the hatching of host eggs, subject the host egg cards to 30 min of ultraviolet (UV) irradiation (Figure 1-i). Subsequently, cut the paper with inactivated eggs into egg cards measuring 1 cm thick and 8 cm in diameter, each containing approximately 300-500 eggs (Figure 1-ii)25,26,27.</li>
	<li>Introduce a cohort of 80-120 T. denrolimi wasps into a glass tube measuring 2 cm in diameter and 8 cm in length. Seal the tube with cotton. 
	NOTE: Maintain the wasps at a temperature of 25 &#177; 1 &#176;C, with a light/dark cycle of 16 h of light and 8 h of darkness and maintain a relative humidity of approximately 75%.</li>
	<li>Present a host egg card to the wasps for parasitization (Figure 1-ii). Permit the wasps to deposit their eggs into the host eggs for 6 h, after which promptly remove the wasps. 
	&#8203;NOTE: Avoid prolonging the parasitization period excessively as this can lead to overcrowding of T. denrolimi offspring within a host egg, resulting in increased offspring wasp mortality28,29.</li>
</ol>
2. Synthesis of dsRNA
<ol>
	<li>Design primer sets containing a T7 promoter for the synthesis of double-stranded RNA (dsRNA). Choose a 300-400 bp segment from the targeted gene (Ferhch) for dsRNA synthesis.</li>
	<li>Acquire commercially synthesized primer sets for the production of dsRNA for the targeted gene and dsGFP for use as a negative control.</li>
	<li>Extract RNA content from 100 T. denrolimi wasps using the referenced kit following the manufacturer&#39;s protocol9. Check the quality of RNA content; proceed if the value of OD260/OD280 ranges from 1.8 to 2.09.</li>
	<li>Purify the RNA content with 2 &#181;L of 5x buffer (1 U/50 &#181;L), 1 &#181;L of DNAse (1 U/50 &#181;L), and 6.5 &#181;L of RNA (~100 ng/&#181;L), 0.5 &#181;L of H2O at 42 &#176;C for 2 min.</li>
	<li>Perform reverse transcriptase polymerase chain reaction (RT-PCR) to generate a cDNA template with 10 &#181;L of purified RNA (~100 ng/&#181;L), 4 &#181;L of 5x buffer (1 U/50 &#181;L), 1 &#181;L of Enzyme Mix I (1 U/50 &#181;L), and 4 &#181;L of H2O. Perform RT-PCR using the following settings: 37 &#176;C for 15 min, 85 &#176;C for 5 s. Store the cDNA product at -4 &#176;C until use.</li>
	<li>Perform polymerase chain reaction (PCR) to amplify the dsRNA sequences in accordance with the referenced master mix (10 &#181;L of Taq II [1 U/50 &#181;L], 0.8 &#181;L of forward primer [0.4 &#181;M], 0.8 &#181;L of reverse primer [0.4 &#181;M], 0.8 &#181;L of DNA template [40 ng/&#181;L], and 7.6 &#181;L of H2O). Perform PCR using the following settings: 94 &#176;C for 2 min; 35 cycles with 94 &#176;C for 30 s, 60 &#176;C for 30 s, 72 &#176;C for 30; 72 &#176;C for 2 min.</li>
	<li>Assess the quality of PCR products by electrophoresis on a 2.0% agarose gel5,9 and confirm the target sequence through Sanger sequencing.</li>
	<li>Purify the PCR product using the Gel Extraction Kit as per the manufacturer&#39;s guidelines9,23.</li>
	<li>Utilize an RNAi System to synthesize dsRNA for the target gene with 10 &#181;L of 2x buffer (0.02 U/&#181;L), 8 &#181;L of DNA template (75 ng/&#181;L), and 2 &#181;L of enzyme mix (1 U/50 &#181;L) with the following settings: 37 &#176;C for 30 min, 70 &#176;C for 10 min, and 25 &#176;C for 20 min. Elute the synthesized dsRNA with 30 &#181;L of Nuclease-Free Water.</li>
	<li>Assess the quality of the dsRNA product by visualizing it on a 1.0% agarose gel5,9. Dilute the dsRNA to 7,000 ng/&#181;L. Check the quality of the dsRNA product; proceed if the value of OD260/OD280 ranges from 1.8 to 2.09. Store the dsRNA product at -20 &#176;C until use.</li>
</ol>
3. Transplantation of T. denrolimi pupae
<ol>
	<li>Cultivate the parasitized host eggs for approximately 8 days at 25 &#177; 1&#176;C, maintaining a 16 h light/8 h dark cycle and a relative humidity of ~75%. The parasitized host eggs will blacken after 5 days30 (Figure 1-iv,v).</li>
	<li>Transfer the host egg card to a dissecting microscope. Employ a pair of tungsten needles (Figure 1-vi) to meticulously remove the chorion from the host eggs (Figure 1-vii) and retrieve the pupa from within (Figure 1-viii)5,17,18. 
	NOTE: The tip of the dissecting needle should be less than 0.05 mm.</li>
	<li>Prepare a clean plate for the substrate. Pour out 10-15 mL of a 15 g/L agar solution, allowing it to naturally cool (Figure 2-i). 
	NOTE: Mix the agar solution with 0.5 g of streptomycin to inhibit the growth of contaminants in vitro.</li>
	<li>Employ a disinfected graver to etch several grooves measuring 0.2-0.3 mm in depth and 0.4-0.8 mm in width (Figure 2-ii).</li>
	<li>Use a small brush (Figure 2-iii) to transplant a pupa from the dissected host egg into one of the grooves on the agar substrate (Figure 2-iv).</li>
	<li>Repeat steps 3.4 and 3.5, transplanting 100-300 pupae individually into the grooves one by one (Figure 2-v).</li>
</ol>
4. Micro-injecting dsRNA into T. denrolimi pupa
<ol>
	<li>Use a glass needle puller to pull a glass capillary (Figure 3-i,ii). Set the Heat to 280, the Velocity to 170, the Delay to 250, and the Pull to 30 on a needle puller (Figure 3-iii). Prepare multiple capillary glass needles for microinjection (Figure 3-iv,v).</li>
	<li>Bevel the tip of the glass needle using an abrasive plate (Figure 3-vi). 
	NOTE: Ensure that the injection needle&#39;s tip remains sharp; otherwise, it may result in pupal mortality or impede reagent flow through the needle (Figure 3-vii).</li>
	<li>Load approximately 2 &#181;L of the dsRNA injection solution with 7,000 ng/&#181;L into the prepared injection needle using a micro-injection pump (Figure 3-vii).</li>
	<li>Transfer the agar substrate containing hundreds of pupae onto the platform of a compound microscope (Figure 3-viii).</li>
	<li>Carefully insert the injection needle into the abdomen of a T. denrolimi pupa at a ~30&#176; angle to the abdomen under the lens (Figure 3-ix).</li>
	<li>Gradually inject ~5 nL of the injection solution (step 4.3) into the T. denrolimi pupa as gently as possible. 
	NOTE: The T. denrolimi pupa may slightly swell as the dsRNA solution is injected. Injecting an excessive amount of solution into the pupa or using needles with overly large openings may result in premature pupal death. Change the needles when they become clogged after injecting several to tens of pupae.</li>
	<li>Repeat steps 4.5 and 4.6, injecting dsRNA into 600 pupae one by one. 
	&#8203;NOTE: For reliable RT-qPCR analysis, RNA content should be isolated from at least 100 pupae. Inject at least 600 pupae for three replicates in the dsRNA treatment and three replicates in the dsGFP negative control9.</li>
</ol>
5. Incubation of T. denrolimi pupae
<ol>
	<li>Transfer the substrate containing injected pupae to an incubator at 25 &#177; 1 &#176;C with a 16 h/8 h light/dark cycle and ~75% RH.</li>
	<li>Incubate approximately 700 T. denrolimi pupae per treatment for either 24 h or 48 h. Extract RNA content from a group of 100 pupae per replicate9. 
	&#8203;NOTE: Remove the shrunk pupae (dead pupae) from the samples; otherwise, it may result in errors in the detection of gene expression.</li>
	<li>Incubate ~50 T. denrolimi pupae per treatment until the wasps emerge (Figure 3-x). Record the emergence rate and deformity rate.</li>
</ol>
6. Detection of targeted gene expression
<ol>
	<li>Design the primer set for the target gene and reference genes for RT-qPCR using a design tool. 
	NOTE: Ensure that the sequences of the RT-qPCR primers do not overlap with the targeted region of dsRNA.</li>
	<li>Employ the gene forkhead box O (FoxO) as the reference gene in the RT-qPCR analysis; the primers of FoxO have been reported previously9.</li>
	<li>Obtain commercially synthesized primer sets. Perform RT-qPCR with 10 &#181;L of Taq II (1 U/50 &#181;L), 0.8 &#181;L of forward primer (0.4 &#181;M), 0.8 &#181;L of reverse primer (0.4 &#181;M), 0.8 &#181;L of template (10 ng/&#181;L), and 7.6 &#181;L of H2O. Perform RT-qPCR using the following settings: 95 &#176;C for 30 s; 35 cycles with 95 &#176;C for 5 s, 57 &#176;C for 30 s, 72 &#176;C for 30; 95 &#176;C for 10 s; 60 &#176;C for 5 s, and 95 &#176;C for 5 s.</li>
	<li>Calculate the expression value of the targeted gene by using the 2-&#916;&#916;Ct method9,25.</li>
	<li>Employ the Kruskal-Wallis test to analyze the expression of the target gene under the influence of different treatments (dsFerhch, dsGFP, non-injection). 
	NOTE: Avoid using parametric tests (e.g., t-test) for post-hoc comparisons as the data&#39;s heteroscedasticity and non-normality may lead to erroneous conclusions25.</li>
</ol>

Advancing RNAi Techniques in Trichogramma dendrolimi for Gene Function Analysis

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The authors declare that they have no conflicts of interest.

Trichogramma wasps are recognized as effective biological control agents, specifically targeting a range of lepidopteran pests in agriculture and forestry1. These diminutive wasps undergo their immature stages within the host egg, a characteristic that presents challenges in conducting RNAi experiments5,18. This study offers a comprehensive visual guide for conducting RNAi experiments in T. denrolimi. Given the shared biological traits among Trichogramma species, the procedure can be readily adapted for RNAi studies or micro-injections in other Trichogramma species and endoparasitic species with some adjustments.
The procedure introduces several innovative approaches, facilitating efficient micro-injections of dsRNA content, streamlining the RT-qPCR process for T. denrolimi, and optimizing the transplantation and in vitro incubation of T. denrolimi pupae. According to this study, the emergence rate of transplanted pupae reached 90% without any injection. When the transplanted pupae were subjected to dsGFP injection, the emergence rate of these pupae was 78% (Table 1). Successful RNAi experiments in Trichogramma hinge significantly on precise micro-injections and the careful incubation of pupae in vitro. The susceptibility of isolated Trichogramma pupae to damage during injection may result from oversized micro-injection needles, hasty injection techniques, or microbial contamination on the agar substrate. The detailed visual method presented here also offers insights into the microinjection technique in T. denrolimi, which is not only essential for other genetic editing tools (e.g., CRISPR-Cas9 system) but also for conducting various physiological experiments (e.g., artificial transinfection of symbionts and the delivery of inhibitors or activators of physiological responses)1,14,15,16.
For RT-qPCR analysis, approximately 1,500 pupae were injected in this study. Given the small size of T. denrolimi, a minimum of 100 pupae per replicate is necessary to ensure sufficient RNA quality and quantity for RT-qPCR analysis9. Consequently, injecting dsRNA into hundreds of pupae is imperative. Additionally, improvements in the RNA isolation procedure (e.g., employing a specific RNA extraction kit, refining PCR programs) are required to obtain eligible RNA content from a reduced number of Trichogramma individuals per replicate. An alternative approach involves the delivery of dsRNA through soaking or feeding, which may be more convenient for obtaining a substantial number of individuals subjected to dsRNA. Additionally, it is essential to note that the current RNAi procedure is applicable only at the pupal stage of T. denrolimi. Future research should place particular emphasis on the development of efficient RNAi methods for the embryo and larva stages15,16.
In summary, this study has delineated an efficient RNAi method in T. denrolimi. For decades, gene functions within the entire Trichogramma genus have been rarely explored. The present methodology provides a robust model for investigating gene regulations during developmental and physiological activities in Trichogramma wasps. Future research should emphasize the development of genetic tools, such as genome editing and RNA interference in Trichogramma wasps. The depth exploration of the molecular biology of Trichogramma wasps offers valuable insights for improving the mass-rearing efficiency and field performance of these biological control agents for the control of pests8,9.

Trichogramma spp. are a group of egg parasitoids that have been extensively utilized as highly efficient biological control agents against a wide spectrum of lepidopteran pests in agricultural and forest ecosystems worldwide1,2,3,4. The application of mass-reared Trichogramma provides an environmentally friendly approach for the sustainable management of pests5,6,7. Understanding the molecular biology of Trichogramma wasps provides valuable insights into enhancing the mass-rearing efficiency and field performance of these biological control agents8,9by investigating the methodology of gene regulation and genome editing10.
Since the discovery of double-stranded RNA (dsRNA)-mediated specific genetic interference in Caenorhabditis elegans in 1998, the RNA-interference (RNAi) method has evolved into a vital genetic toolkit for exploring the regulatory mechanisms of organisms by suppressing the expression of target genes11. RNAi experiments have become a standard methodology widely applied to study gene function in numerous insect species12,13. Nevertheless, the manipulation of RNAi presents a formidable challenge in many parasitoid species, particularly among those belonging to the endoparasitic Chalcidoidea family14,15,16. The RNAi method has been documented in at least 13 parasitoid species14,15,16,17,18,19. Among these, the RNAi approach has been comprehensively conducted in Nasonia wasps and is applicable throughout the developmental stages, including embryos, larvae, pupae, and adults14,15,16. It is noteworthy that Nasonia wasps are ectoparasitoids, with their offspring developing in the interstitial space between the host pupa and the puparium, enabling their cultivation in vitro and making them tolerate certain treatments, such as micro-injection. Unlike Nasonia wasps, Trichogramma individuals undergo their entire embryonic, larval, and pupal development inner the host egg. The layer at embryo and larva stages (which may impede dsRNA permeability), vulnerability to damage, and the difficulty in surviving in vitro present formidable obstacles20,21,22. Additionally, the diminutive size of Trichogramma individuals, approximately ~0.5 mm in adult or pupal length, renders them exceedingly intricate to manipulate20,21,22.
In the present study, we outline a comprehensive procedure for conducting RNA interference (RNAi) experiments in Trichogramma denrolimi Matsumura. This procedure encompasses the following procedures: (1) the design and synthesis of double-stranded RNA (dsRNA), (2) microinjection of T. denrolimi pupae, (3) the transplantation and in vitro incubation of these pupae, and (4) the detection of target gene knockdown through RT-qPCR analysis. The target gene selected for the RNAi experiment is the ferritin heavy chain homology (Ferhch). FerHCH, an iron-binding protein, contains a ferroxidase center endowed with antioxidant capabilities, facilitating the oxidation of Fe2+ to Fe3+. It plays an indispensable role in the growth and development of various organisms by maintaining redox equilibrium and iron homeostasis. Depletion of FerHCH can result in the overaccumulation of iron, leading to irreversible tissue damage, and often culminating in significant phenotypic alterations, including growth defects, deformities, and mortality23,24. This study offers a step-by-step guide for conducting RNAi in T. denrolimi, which will be invaluable for investigating the gene functions within the broader context of Trichogramma wasps.

<table><tbody><tr><td>2x ES Taq MasterMix (Dye)</td><td>Cowin Biotech, China</td><td>CW0690H</td><td>To amplify the dsRNA sequences</td></tr><tr><td>20x PBS Buffer, DEPC treated (7.2-7.6)</td><td>Sangon Biotech, China</td><td>B540627-0500</td><td>To dilute dsRNA</td></tr><tr><td>Agar strip</td><td>Shishi Globe Agar Industries Co.,Ltd, China</td><td>n/a</td><td>To make culture medium</td></tr><tr><td>Ampicillin sodium</td><td>Sangon Biotech, China</td><td>A610028</td><td>To make culture medium</td></tr><tr><td>Bioer Constant temperature metal bath&amp;nbsp;</td><td>BIOER, China</td><td>MB-102</td><td>To synthesis dsRNA</td></tr><tr><td>Borosilicate glass capillary&amp;nbsp;</td><td>WPI, USA</td><td>1B100-4</td><td>To pull capillary glass needle</td></tr><tr><td>Clean bench&amp;nbsp;</td><td>Airtech, China</td><td>SW-CJ-1FD</td><td>To extract RNA</td></tr><tr><td>Double distilled water</td><td>Sangon Biotech, China</td><td>A500197-0500</td><td>To dilute cDNA</td></tr><tr><td>Environmental Testing chamber&amp;nbsp;</td><td>Panasonic, Japan</td><td>MLR-352H-PC</td><td>To culture &lt;em&gt;T. denrolimi&lt;/em&gt;</td></tr><tr><td>Eppendorf Centrifuge&amp;nbsp;</td><td>Eppendrof, Germany</td><td>5418R</td><td>To store RNA content</td></tr><tr><td>Eppendorf FemtoJet 4i</td><td>Eppendrof, Germany</td><td>FemtoJet 4i</td><td>To inject &lt;em&gt;T. denrolimi&lt;/em&gt;</td></tr><tr><td>Eppendorf Refrigerated Centrifuge&amp;nbsp;</td><td>Eppendrof, Germany</td><td>5810R</td><td>Centrifuge</td></tr><tr><td>Ethanol solution (75%, RNase-free)</td><td>Aladdin, China</td><td>M052131-500ml</td><td>To extract RNA</td></tr><tr><td>Gel Extraction Kit</td><td>Omega, USA</td><td>D25000-02</td><td>To extract cDNA</td></tr><tr><td>GUM Arabic</td><td>Solarbio, China</td><td>CG5991-500g</td><td>To make egg card</td></tr><tr><td>Isopropyl alchohol</td><td>Aladdin, China</td><td>80109218</td><td>To extract RNA</td></tr><tr><td>Laser-Based Micropipette Puller&amp;nbsp;</td><td>SUTTER, USA</td><td>P-2000</td><td>To pull capillary glass needle</td></tr><tr><td>Microloader&amp;nbsp;</td><td>Eppendrof, Germany</td><td>20 &amp;micro;L</td><td>To load dsRNA</td></tr><tr><td>Multi-sample tissue grinder&amp;nbsp;</td><td>LICHEN, China</td><td>LC-TG-24</td><td>To grind &lt;em&gt;T. denrolimi&lt;/em&gt;</td></tr><tr><td>Needle Grinder&amp;nbsp;</td><td>SUTTER, USA</td><td>BV-10-E</td><td>To grind capillary glass needle</td></tr><tr><td>Nuclease-Free Water</td><td>Sangon Biotech, China</td><td></td><td>To dilute RNA</td></tr><tr><td>OLYMPUS Microscope</td><td>OLYMPUS, Japan</td><td>XZX16</td><td>To observe T. denrolimi</td></tr><tr><td>PCR machine&amp;nbsp;</td><td>Bio-rad, USA</td><td>S-1000</td><td>For DNA amplification</td></tr><tr><td>PowerPac Basic</td><td>Bio-rad, USA</td><td>PowerPacTM Basic</td><td>To detect the quality of dsRNA&amp;nbsp;</td></tr><tr><td>Primer of &lt;em&gt;dsGFP &lt;/em&gt;(Forward)</td><td></td><td></td><td>[TAATACGACTCACTATAGGG]&lt;br/&gt; ACAAACCAAGGCAAGTAATA</td></tr><tr><td>Primer of &lt;em&gt;dsGFP &lt;/em&gt;(Reverse)</td><td></td><td></td><td>[TAATACGACTCACTATAGGG]&lt;br/&gt; CAGAGGCATCTTCAACG</td></tr><tr><td>Primer of &lt;em&gt;Ferhch &lt;/em&gt;for qPCR (Forward)</td><td></td><td></td><td>TGAAGAGATTCTGCGTTCTGCT</td></tr><tr><td>Primer of &lt;em&gt;Ferhch &lt;/em&gt;for qPCR (Reverse)</td><td></td><td></td><td>CTGTAGGAACATCAGCAGGCTT</td></tr><tr><td>Primer of &lt;em&gt;Ferhch &lt;/em&gt;for RNAi (Reverse)</td><td></td><td></td><td>[TAATACGACTCACTATAGGG]AG&lt;br/&gt; TAGCCATCATCTTTCC</td></tr><tr><td>Primer of &lt;em&gt;Ferhch &lt;/em&gt;for RNAi(Forward)</td><td></td><td></td><td>[TAATACGACTCACTATAGGG]&lt;br/&gt; ACACTGTCAATCGTCCTG</td></tr><tr><td>Primer of &lt;em&gt;FoxO &lt;/em&gt;for qPCR (Forward)</td><td></td><td></td><td>CTACGCCGATCTCATAACGC</td></tr><tr><td>Primer of FoxO for qPCR (Reverse)</td><td></td><td></td><td>TGCTGTCGCCCTTGTCCT</td></tr><tr><td>PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time)</td><td>TaKaRa, Japan</td><td>RR047A</td><td></td></tr><tr><td>Quantitative Real-time PCR&amp;nbsp;</td><td>Bio-rad, USA</td><td>CFX 96 Touch</td><td>To perform reverse transcriptase polymerase chain reaction (RT-PCR)&amp;nbsp;</td></tr><tr><td>Real-time PCR (TaqMan) Primer and Probes Design Tool</td><td></td><td></td><td>https://www.genscript.com/tools/real-time-pcr-taqman-primer-design-tool/</td></tr><tr><td>T7 RiBoMAX Express RNAi System</td><td>Promega, USA</td><td>P1700</td><td>To synthesis dsRNA &amp;nbsp;&lt;em&gt;in vitro&amp;nbsp;&lt;/em&gt;</td></tr><tr><td>TB Green Premix Ex TaqTM &lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/66250/66250eq01.jpg&quot; style=&quot;margin: auto;&quot;/&gt; (Til RnaseH Plus)</td><td>TaKaRa, Japan</td><td>RR820A</td><td>To perform RT-qPCR</td></tr><tr><td>Trichloromethane</td><td>KESHI, China</td><td>GB/T682-2002</td><td>To extract RNA</td></tr><tr><td>TRIzol Reagent</td><td>Ambion, USA</td><td>15596018</td><td>To extract total RNA content from samples</td></tr><tr><td>Ultra-low Temperature Freezer&amp;nbsp;</td><td>Thermo, USA</td><td>Forma 911</td><td></td></tr></tbody></table>

rna interference in the egg parasitoid, trichogramma dendrolimi matsumura

The egg parasitoids, Trichogramma spp, are recognized as efficient biological control agents against various lepidopteran pests in agriculture and forests. The immature stages of Trichogramma offspring develop within the host egg, exhibiting remarkable diminutiveness (approximately 0.5 mm in adult length). RNA-interference (RNAi) methodology has emerged as a crucial tool for elucidating gene functions in numerous organisms. However, manipulating RNAi in certain small parasitoid species, such as Trichogramma, has generally posed significant challenges. In this study, we present an efficient RNAi method in Trichogramma denrolimi. The outlined procedure encompasses the acquisition and isolation of individual T. dendrolimi specimens from host eggs, the design and synthesis of double-stranded RNA (dsRNA), the in vitro transplantation and cultivation of T. dendrolimi pupae, the micro-injection of dsRNA, and the subsequent assessment of target gene knockdown through RT-qPCR analysis. This study furnishes a comprehensive, visually detailed procedure for conducting RNAi experiments in T. dendrolimi, thereby enabling researchers to investigate the gene regulation in this species. Furthermore, this methodology is adaptable for RNAi studies or micro-injections in other Trichogramma species with minor adjustments, rendering it a valuable reference for conducting RNAi experiments in other endoparasitic species.

The emergence rate of T. denrolimi pupae injected with dsFerhch was significantly lower than that of those injected with dsGFP or those without injection (Table 1). Among the emerged wasps, 51.85% of T. denrolimi wasps subjected to dsFerhch developed deformed small wings. The deformed wasps were not observed in the wasps injected with dsGFP or without any injection (Table 1). Moreover, the abdomens of T. denrolimi pupae injected with dsFerhch exhibited melanism which is the phenotype of abnormal iron metabolism23. The melanism was not observed in T. denrolimi pupae injected with dsGFP or those without any injection (Figure 4).
RT-qPCR analysis showed that the expression of Ferhch in T. denrolimi pupae, 24 h after dsFerhch injection (0.6460 ± 0.0056), was significantly downregulated in comparison to those injected with dsGFP (1.4514 ± 0.5402; P = 0.0415). However, it did not differ significantly from the expression in pupae without any injection (1.0000 ± 0.1953; P = 0.8725). Ferhch expression in T. denrolimi pupae, 48 h post dsFerhch injection (0.5170 ± 0.0575), was significantly downregulated compared to pupae injected with dsGFP (0.8515 ± 0.0233; P = 0.0182) and those without any injection (1.0548 ± 0.3006; P = 0.0113) (Figure 5).
<img alt="Insect lifecycle stages, microscopy: egg, larva, pupa, adult. Close-up views, lab setup." class="xfigimg" src="/files/ftp_upload/66250/66250fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Collection and maintenance of Trichogramma denrolimi culture. (i) Expose the host egg cards to ultraviolet (UV) irradiation. (ii) Cut the paper with inactivated eggs into egg cards. (iii) Present a host egg card to the wasps for parasitization. (iv) Cultivate the parasitized host eggs for 8 days. (v) The parasitized host eggs will blacken after 5 days. (vi) The dissecting needle with a 0.04 mm tip. (vii) Remove the chorion from the host eggs. (viii) T. denrolimi pupa. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66250/66250fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Petri dish streaking technique, images i to v; microbiology experiment sequence, bacterial culture." class="xfigimg" src="/files/ftp_upload/66250/66250fig02.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Transplantation and incubation of Trichogramma denrolimi pupae. (i) The agar substrate. (ii) Employ a disinfected graver to etch several grooves on the substrate. (iii) Use a small brush to (iv) transplant the pupae from the dissected host egg into a groove on the substrate. (v) Transplanting 100-300 pupae individually into the grooves one by one. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66250/66250fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microfluidics setup, optical microscopy, cell arrangement; devices include microscope, P-2000 laser." class="xfigimg" src="/files/ftp_upload/66250/66250fig03.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Procedure of micro-injection. (i) Glass capillary. (ii) Glass needle puller. (iii) Settings for needle puller. (iv) Prepare multiple capillary glass needles for microinjection. (v) The tip of the capillary glass needle. (vi) Bevel the tip of the glass needle using an abrasive plate. (vii) Load approximately 2 µL of the dsRNA injection solution. (viii) Compound microscope. (ix) Insert the injection needle into the abdomen and inject the injection solution. (x) Emergence of wasps. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66250/66250fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Insect development stages diagram; wasp pupa and adult under dsGFP, dsFerch, and control." class="xfigimg" src="/files/ftp_upload/66250/66250fig04.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Morphological characteristics of Trichogramma denrolimi pupa and wasp. The pupa and wasp injected with dsFerhch exhibit melanism with blackened body color. The wasp injected with dsFerhch shows a pair of deformed small wings. The melanism and deformed wings are not observed in the pupa and wasp injected with dsGFP or those without any injection. Scale bars = 200 µm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66250/66250fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Gene expression bar chart; Ferhch vs FoxO, dsRNA treatments, 24h, 48h analysis, statistical results." class="xfigimg" src="/files/ftp_upload/66250/66250fig05.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5: Expression of target gene Ferhch relative to FoxO at 24 h and 48 h post dsFerhch or dsGFP injection, or non-injection. Error bars indicate the standard errors. Different lowercase letters indicate a significant difference at P &lt; 0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66250/66250fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Variables</td>
			<td>dsFerhch</td>
			<td>dsGFP</td>
			<td>Non-injection</td>
			<td>χ2</td>
			<td>P</td>
		</tr>
		<tr>
			<td>Emergence rate</td>
			<td>54.00% (27/50) b</td>
			<td>78.00% (39/50) a</td>
			<td>90.00% (45/50) a</td>
			<td>17.46</td>
			<td>&lt;.001</td>
		</tr>
		<tr>
			<td>Deformity rate</td>
			<td>51.85% (14/27) A</td>
			<td>0% (0/39) B</td>
			<td>0% (0/45) B</td>
			<td>49.84</td>
			<td>&lt;.001</td>
		</tr>
	</tbody>
</table>
Table 1: The emergence and deformity of Trichogramma denrolimi injected by dsFerhch, dsGFP, or without injection. Different lowercase letters indicate significant differences in emergence rate at P &lt; 0.05. Different uppercase letters indicate significant differences in deformity rate at P &lt; 0.05. The values of χ2 and P listed in the table are estimated by the χ2 independent test and indicate the total effect of three treatment levels on the emergence rate and deformity rate.

Watch this Scientific Journal Video about RNA Interference in the Egg Parasitoid, Trichogramma dendrolimi Matsumura at JoVE.com

Author Spotlight: Investigating Wolbachia-Induced Thelytokous Parthenogenesis and Genetic Toolkit Development Through RNA Interference

The manipulation of RNA interference (RNAi) presents a formidable challenge in many parasitoid species with diminutive size, such as Trichogramma wasps. This study delineated an efficient RNAi method in Trichogramma denrolimi. The present methodology provides a robust model for investigating gene regulation in Trichogramma wasps.

RNA Interference in the Egg Parasitoid, Trichogramma dendrolimi Matsumura

The egg parasitoids, Trichogramma spp, are recognized as efficient biological control agents against various lepidopteran pests in agriculture and ...

rna-interference-egg-parasitoid-trichogramma-dendrolimi

Nanyang Technological University

Research

JoVE Journal

Biology

1.2K Views.  Shenyang Agricultural University. Trichogramma-wespen zijn een groep eiparasitoïden die worden gebruikt om lepidoptera-plagen in de landbouw te bestrijden. De intracellulaire bacterie Wolbachia induceert delytokous parthenogenese bij sommige Trichogramma-wespen. We proberen een antwoord te vinden op het mechanisme van Wolbachia en geslachtsbepalingssystemen bij Trichogramma-wespen.We hebben de RNA-interferentiemethode ontwikkeld in Trichogramma-wespen. Deze methode is een essentiële genetische gereedschapskist voor h...

Video: Auteur in de schijnwerpers: onderzoek naar door wolbachia geïnduceerde thelytokous-parthenogenese en de ontwikkeling van genetische toolkits door middel van RNA-interferentie

Application of Two-spotted Spider Mite Tetranychus urticae for Plant-pest Interaction Studies

The two-spotted spider mite, Tetranychus urticae, is a ubiquitous polyphagous arthropod herbivore that feeds on a remarkably broad array of species, with more than 150 of economic value. It is a major pest of greenhouse crops, especially in Solanaceae and Cucurbitaceae (e.g., tomatoes, eggplants, peppers, cucumbers, zucchini) and greenhouse ornamentals (e.g., roses, chrysanthemum, carnations), annual field crops (such as maize, cotton, soybean, and sugar beet), and in perennial cultures (alfalfa, strawberries, grapes, citruses, and plums)1,2. In addition to the extreme polyphagy that makes it an important agricultural pest, T. urticae has a tendency to develop resistance to a wide array of insecticides and acaricides that are used for its control3-7.
T. urticae is an excellent experimental organism, as it has a rapid life cycle (7 days at 27 &#176;C) and can be easily maintained at high density in the laboratory. Methods to assay gene expression (including in situ hybridization and antibody staining) and to inactivate expression of spider mite endogenous genes using RNA interference have been developed8-10. Recently, the whole genome sequence of T. urticae has been reported, creating an opportunity to develop this pest herbivore as a model organism with equivalent genomic resources that already exist in some of its host plants (Arabidopsis thaliana and the&#160;tomato Solanum lycopersicum)11. Together, these&#160;model organisms could provide insights into molecular bases of plant-pest interactions.
Here, an efficient method for quick and easy collection of a large number of adult female mites, their application on an experimental plant host, and the assessment of the plant damage due to spider mite feeding are described. The presented protocol enables fast and efficient collection of hundreds of individuals at any developmental stage (eggs, larvae, nymphs, adult males, and females) that can be used for subsequent experimental application.

Application of Two-spotted Spider Mite Tetranychus urticae for Plant-pest Interaction Studies

Protocols for efficient preparation of homogenous samples of spider mites, infestation of experimental plants, and assessment of plant damage, as required for studies of plant-pest interaction were developed.

The two-spotted spider mite, Tetranychus urticae, is a ubiquitous polyphagous arthropod herbivore that feeds on a remarkably broad array of species, with ...

Environment

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. Infective eggs are given by oral gavage, hatch in the distal small intestine, invade the intestinal epithelial cells (IECs) 
that line the crypts of the cecum and proximal colon and upon maturation the worms release eggs into the environment1. This model is a powerful tool to 
examine factors that control CD4+ T helper (Th) cell activation as well as changes in the intestinal epithelium. The immune response that occurs in 
resistant inbred strains, such as C57BL/6 and BALB/c, is characterized by Th2 polarized cytokines (IL-4, IL-5 and IL-13) and expulsion of worms while Th1-associated 
cytokines (IL-12, IL-18, IFN-&gamma;) promote chronic infections in genetically susceptible AKR/J mice2-6. Th2 cytokines promote physiological changes in 
the intestinal microenvironment including rapid turnover of IECs, goblet cell differentiation, recruitment and changes in epithelial permeability and smooth muscle 
contraction, all of which have been implicated in worm expulsion7-15. Here we detail a protocol for propagating Trichuris muris eggs which can 
be used in subsequent experiments. We also provide a sample experimental harvest with suggestions for post-infection analysis. Overall, this protocol will provide 
researchers with the basic tools to perform a Trichuris muris mouse infection model which can be used to address questions pertaining to Th proclivity in 
the gastrointestinal tract as well as immune effector functions of IECs.

Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

Trichuris muris infection is an intestinal model of Th2 immunity where resistant mice generate a protective Th2 response and susceptible mice generate a pathological Th1 response.

Trichuris muris is a natural pathogen of mice and is biologically and antigenically similar to species of Trichuris that 
infect humans and livestock1. ...

Immunology and Infection

In Vitro Nuclear Assembly Using Fractionated Xenopus Egg Extracts

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm chromatin, cytosol, and light membrane fractions. Complete nuclei are formed, including nuclear membranes with pore complexes, and these reconstituted nuclei are capable of normal nuclear processes.

Nuclear membrane assembly is an essential step in the cell division cycle; this process can be replicated in the test tube by combining Xenopus sperm ...

Measuring Volatile and Non-volatile Antifungal Activity of Biocontrol Products

The protocol described is based on a plug-transfer technique that allows accurate determination of microorganism quantities and their developmental stages. A specified number of spores are spread on an agar plate. This agar plate is incubated for a defined period to allow the fungi to reach the expected developmental stage, except for spores where incubation is not required. Agar plugs covered by spores, hyphae, or mycelium are next withdrawn and transferred onto agar media containing the antifungal compound to be tested either placed at a distance from the fungi or in contact. This method is applicable to test both liquid extracts and solid samples (powders). It is particularly well suited for quantifying the relative contributions of volatile and non-volatile agents in bioactive mixtures and for determining their effects, specifically on spores, early hyphae, and mycelium.
The method is highly relevant for the characterization of the antifungal activity of biocontrol products, notably plant-derived products. Indeed, for plant treatment, the results can be used to guide the choice of mode of application and to establish the trigger thresholds.

We describe a modified agar-based method designed to quantify the antifungal effects of plant-derived products. Both volatile and non-volatile contributions to the antifungal activity can be assessed through this protocol. In addition, efficacy against fungi can be measured at key developmental stages in a single experimental setup.

The protocol described is based on a plug-transfer technique that allows accurate determination of microorganism quantities and their developmental ...

Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica

The firebrat Thermobia domestica is an ametabolous, wingless species that is suitable for studying the developmental mechanisms of insects that led to their successful evolutionary radiation on the earth. The application of genetic tools such as genome editing is the key to understanding genetic changes that are responsible for evolutionary transitions in an Evo-Devo approach. In this article, we describe our current protocol for generating and maintaining mutant strains of T. domestica. We report a dry injection method, as an alternative to the reported wet injection method, that allows us to obtain stably high survival rates in injected embryos. We also report an optimized environment setting to mate adults and obtain subsequent generations with high efficiency. Our method underlines the importance of taking each species&#8217; unique biology into account for the successful application of genome editing methods to non-traditional model organisms. We predict that these genome editing protocols will help in implementing T. domestica as a laboratory model and to further accelerate the development and application of useful genetic tools in this species.

Egg Microinjection and Efficient Mating for Genome Editing in the Firebrat Thermobia domestica

We provide a detailed protocol for rearing, microinjection of eggs and for efficient mating of the firebrat Thermobia domestica to generate and maintain mutant strains after genome editing.

The firebrat Thermobia domestica is an ametabolous, wingless species that is suitable for studying the developmental mechanisms of insects that led to ...

Developmental Biology

Artificial Host Parasitization by Trichogramma dendrolimi and Venom Extraction

Extracting Venom from the Parasitoid Wasp Trichogramma dendrolimi Using an Artificial Host

Parasitoid wasps are a diverse group of hymenopteran insects that serve as invaluable resources for pest biocontrol. To ensure successful parasitism, parasitoid wasps inject venom into their hosts to suppress their hosts&#39; immunity, modulate hosts&#39; development, metabolism, and even behavior. With over 600,000 estimated species, the diversity of parasitoid wasps surpasses that of other venomous animals, such as snakes, cone snails, and spiders. Parasitoid wasp venom is an underexplored source of bioactive molecules with potential applications in pest control and medicine. However, collecting parasitoid venom is challenging due to the inability to use direct or electrical stimulation and the difficulty in dissection because of their small size. Trichogramma is a genus of tiny (~0.5 mm) egg parasitoid wasps that are widely used for the biological control of lepidopteran pests in both agriculture and forests. Here, we report a method for extracting venom from T. dendrolimi using artificial hosts. These artificial hosts are created with polyethylene film and amino acid solutions and then inoculated with Trichogramma wasps for parasitism. The venom was subsequently collected and concentrated. This method enables the extraction of large amounts of Trichogramma venom while avoiding contamination from other tissues caused by dissection, a common issue in venom reservoir dissection protocols. This innovative approach facilitates the study of Trichogramma venom, paving the way for new research and potential applications.

Author Spotlight: Efficient Venom Extraction Method from Trichogramma Parasitoid Wasps 

Here, we present a protocol for extracting venom from Trichogramma dendrolimi using an artificial host created with polyethylene film and amino acid solution.

Parasitoid wasps are a diverse group of hymenopteran insects that serve as invaluable resources for pest biocontrol. To ensure successful parasitism, ...

Biochemistry

Fungal Clearance Efficiency of Macrophages

Viability Assay of Trichoderma stromaticum Conidia Inside Human Peripheral Blood Mononuclear-Derived Macrophages

Macrophages represent a crucial line of defense and are responsible for preventing the growth and colonization of pathogens in different tissues. Conidial phagocytosis is a key process that allows for the investigation of the cytoplasmic and molecular events involved in macrophage-pathogen interactions, as well as for the determination of the time of death of internalized conidia. The technique involving the phagocytosis of fungal conidia by macrophages is widely used for studies evaluating the modulation of the immune responses against fungi. The evasion of phagocytosis and escape of phagosomes are mechanisms of fungal virulence. Here, we report the methods that can be used for the analysis of the phagocytosis, clearance, and viability of T. stromaticum conidia, a fungus which is used as a biocontrol and biofertilizer agent and is capable of inducing human infections. The protocol consists of 1) Trichoderma culture, 2) washing to obtain conidia, 3) the isolation of peripheral blood mononuclear cells (PBMCs) using the polysucrose solution method and the differentiation of the PBMCs into macrophages, 4) an in vitro phagocytosis method using round glass coverslips and coloration, and 5) a clearance assay to assess the conidia viability after conidia phagocytosis. In summary, these techniques can be used to measure the&#160;fungal clearance efficiency of macrophages.

Author Spotlight: Unraveling the Interplay Between Trichoderma stromaticum and the Mammalian Immune System

The technique involving the phagocytosis of fungal conidia by macrophages is widely used for studies evaluating the modulation of the immune responses against fungi. The purpose of this manuscript is to present a method for evaluating the phagocytosis and clearance abilities of human peripheral blood mononuclear-derived macrophages stimulated with Trichoderma stromaticum conidia.

Macrophages represent a crucial line of defense and are responsible for preventing the growth and colonization of pathogens in different tissues. Conidial ...

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

The red flour beetle, Tribolium&#160;castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle&#8217;s body cavity.
In this report, we provide an overview of our larval RNAi technique in T. castaneum. The protocol includes (i) isolation of the proper stage of T. castaneum larvae for injection, (ii) preparation for the injection setting, and (iii) dsRNA injection. Larval RNAi is a simple, but powerful technique that provides us with quick access to loss-of-function phenotypes, including multiple gene knockdown phenotypes as well as a series of hypomorphic phenotypes. Since virtually all T. castaneum tissues are susceptible to extracellular dsRNA, the larval RNAi technique allows researchers to study a wide variety of tissues in diverse contexts, including the genetic basis of organismal responses to the outside environment. In addition, the simplicity of this technique stimulates more student involvement in research, making T. castaneum an ideal genetic system for use in a classroom setting.

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

RNA interference (RNAi)-based gene knockdown techniques are at the core of Tribolium research. Here, we provide an overview of our larval RNAi technique in Tribolium castaneum. Larval RNAi is a simple, but powerful technique that provides quick access to loss-of-function phenotypes, allowing researchers to study gene functions in diverse contexts.

The red flour beetle, Tribolium&#160;castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully ...

Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus

The pinewood nematode, Bursaphelenchus xylophilus, is one of the most destructive invasive species worldwide, causing the wilting and eventual death of pine trees. Despite the recognition of their economic and environmental significance, it has thus far been impossible to study the detailed gene functions of plant-parasitic nematodes (PPNs) using conventional forward genetics and transgenic methods. However, as a reverse genetics technology, RNA interference (RNAi) facilitates the study of the functional genes of nematodes, including B. xylophilus. 
This paper outlines a new protocol for RNAi of the ppm-1 gene in B. xylophilus, which has been reported to play crucial roles in the development and reproduction of other pathogenic nematodes. For RNAi, the T7 promoter was linked to the 5&#8242;-terminal of the target fragment by polymerase chain reaction (PCR), and double-stranded RNA (dsRNA) was synthesized by in vitro transcription. Subsequently, dsRNA delivery was accomplished by soaking the nematodes in a dsRNA solution mixed with synthetic neurostimulants. Synchronized juveniles of B. xylophilus (approximately 20,000 individuals) were washed and soaked in dsRNA (0.8 &#181;g/mL) in the soaking buffer for 24 h in the dark at 25 &#176;C.
The same quantity of nematodes was placed in a soaking buffer without dsRNA as a control. Meanwhile, another identical quantity of nematodes was placed in a soaking buffer with green fluorescent protein (gfp) gene dsRNA as a control. After soaking, the expression level of the target transcripts was determined using real-time quantitative PCR. The effects of RNAi were then confirmed using microscopic observation of the phenotypes and a comparison of the body size of the adults among the groups. The current protocol can help advance research to better understand the functions of the genes of B. xylophilus and other parasitic nematodes toward developing control strategies through genetic engineering.

Application of RNA Interference in the Pinewood Nematode, Bursaphelenchus xylophilus

Here, we introduce a detailed soaking method of RNA interference in Bursaphelenchus xylophilus to facilitate the study of gene functions.

The pinewood nematode, Bursaphelenchus xylophilus, is one of the most destructive invasive species worldwide, causing the wilting and eventual death of ...

Nanoparticle-Mediated dsRNA Feeding for Gene Silencing in Silkworm Larvae

Larval RNA Interference in Silkworm Bombyx mori through Chitosan/dsRNA Nanoparticle Delivery

The silkworm, Bombyx mori, is an important economic insect with thousands of years of history in China. Meanwhile, the silkworm is the model insect of Lepidoptera with a good accumulation of basic research. It is also the first insect in Lepidoptera with its complete genome sequenced and assembled, which provides a solid foundation for gene functional study. Although RNA interference (RNAi) is widely used in reverse gene functional study, it is refractory in silkworms and other Lepidopteran species. Previous successful RNAi-related research to deliver double-stranded RNA (dsRNA) was performed through injection only. Delivery of dsRNA through feeding is never reported. In this article, we describe step-by-step procedures to prepare the chitosan/dsRNA nanoparticles, which are fed to the silkworm larvae by ingestion. The protocol includes (i) selection of the proper stage of silkworm larvae, (ii) synthesis of dsRNA, (iii) preparation of the chitosan/dsRNA nanoparticles, and (iv) feeding the silkworm larvae with chitosan/dsRNA nanoparticles. Representative results, including gene transcript confirmation and phenotype observation, are presented. dsRNA feeding is a simple technique for RNAi in silkworm larvae. Since silkworm larvae are easy to rear and large enough to operate, it provides a good model to demonstrate larval RNAi in insects. In addition, the simplicity of this technique stimulates more student involvement in research, making silkworm larvae an ideal genetic system for use in a classroom setting.

Author Spotlight: Advancing Gene Silencing Research in Silkworms with dsRNA Delivery Through Feeding Chitosan Nanoparticles

Presented here is a protocol for chitosan/dsRNA nanoparticle delivery in silkworm Bombyx mori larvae to induce gene silencing through ingestion.

The silkworm, Bombyx mori, is an important economic insect with thousands of years of history in China. Meanwhile, the silkworm is the model insect of ...

RNA Interference in the Egg Parasitoid, Trichogramma dendrolimi Matsumura

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