We acknowledge financial support from the Natural Science Foundation of Hainan Province (Grant no. 323QN262), the National Natural Science Foundation of China (Grant no. 31701843 and 32172483), the Jiangsu Agriculture Science and Technology Innovation Fund (Grant No. CX(22)3012 and CX(21)3008), the &#34;Shuangchuang Doctor&#34; Foundation of Jiangsu Province (Grant No. 202030472), and the Nanjing Agricultural University startup fund (Grant No. 804018).

1. Insect rearing
<ol>
	<li>Feed Corcyra cephalonica on corn flour at a temperature of 26 &#177; 1&#176;C and a relative humidity of 40% &#177; 10%.</li>
	<li>Breed T. dendrolimi strain from the Jilin insectary indoors using the eggs of Corcyra cephalonica as hosts. Feed wasp adults 10% sucrose water in Drosophila tubes at a temperature of 26 &#177; 1 &#176;C, relative humidity of 70% &#177; 10%, light (L): dark (D) period of 14 h: 10 h.</li>
</ol>
2. Preparation of polyethylene plastic film egg cards
<ol>
	<li>Take a polyethylene plastic film with a length of 16 cm, a width of 12 cm, and a thickness of 20 &#181;m. Press out 30 semicircular protrusions with a diameter of 2-3 mm and a height of approximately 3 mm using a glass grinding rod according to the standard PCR plate layout of 96 holes. 
	NOTE: The process of pressing out 30 semicircular protrusions using a glass grinding rod needs to be done paying attention to the pressure because too hard a press will puncture the plastic and contaminate the extracted venomless grinding rod.</li>
	<li>Disinfect the pressed polyethylene plastic film by exposing both sides to ultraviolet (UV) light for 1 h.</li>
	<li>Add a small amount of 10% polyvinyl alcohol to the semicircular surface.</li>
</ol>
3. Trichogramma dendrolimi parasitism
<ol>
	<li>After CO2 anesthesia, place T. dendrolimi female wasps into a collection box, and the number of wasps was ~3000.</li>
	<li>Place the convex side of the film egg card toward the collection box and secure the edges with a rubber band.</li>
	<li>Add 4 &#181;L of amino acid solution (6 g/L leucine, 4 g/L phenylalanine, 4.25 g/L histidine) to each semicircular protrusion. Cover it with a flat polyethylene plastic film 16 cm long and 12 cm wide. Use a rubber band to tightly cover the collection box with two sheets of plastic.</li>
	<li>Let T. dendrolimi wasps parasitize freely for 4-8 h and provide 10% sucrose water through wetted cotton.</li>
</ol>
4. Collecting T. dendrolimi venom
<ol>
	<li>Obtain the parasitized amino acid solution from the inner protrusion of the artificial egg card and transfer it to the cap of 1.5 mL tubes.</li>
	<li>Cover the tube cap with a 10 &#181;m nylon net with a 25 mm diameter, fasten the nylon net and centrifuge tube tightly. Place the centrifuge tube upright for short centrifugation using a mini-centrifuge (1360 x g) for 10 s and collect the filtered solution (~100 &#181;L of T. dendrolimi venom).</li>
	<li>Measure the concentration of collected T. dendrolimi venom using a Bicinchoninic acid (BCA) assay kit (Table of Materials).</li>
	<li>Store the venom at -80 &#176;C for further analyses.</li>
</ol>
5. SDS-PAGE analyses
<ol>
	<li>Add 30 &#181;L of T. dendrolimi venom to 10 &#181;L of 4x sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer (Table of Materials) and heat at 95 &#176;C for 10 min.</li>
	<li>Perform SDS-PAGE gel run at 130 V for 120 min.</li>
	<li>Stain and decolorize the SDS-PAGE gel using the protein staining apparatus (Table of Materials).</li>
</ol>

Artificial Host Parasitization by &lt;em&gt;Trichogramma dendrolimi&lt;/em&gt; and Venom Extraction

<ol>
	<li>Pennacchio, F., Strand, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+developmental+strategies+in+parasitic+hymenoptera.">Evolution of developmental strategies in parasitic hymenoptera.</a> Annual Review of Entomology. 51, 233-258 (2006).</li><li>Yan, Z. C., Ye, X. H., Wang, B. B., Fang, Q., Ye, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+advances+on+composition,+function+and+evolution+of+venom+proteins+in+parasitoid+wasps.">Research advances on composition, function and evolution of venom proteins in parasitoid wasps.</a> Chinese Journal of Biological Control. 33 (1), 1-10 (2017).</li><li>Asgari, S., Rivers, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Venom+proteins+from+endoparasitoid+wasps+and+their+role+in+host-parasite+interactions.">Venom proteins from endoparasitoid wasps and their role in host-parasite interactions.</a> Annual Review of Entomology. 56, 313-335 (2011).</li><li>Moreau, S. J. M., Guillot, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+and+prospects+on+biosynthesis,+structures,+and+functions+of+venom+proteins+from+parasitic+wasps.">Advances and prospects on biosynthesis, structures, and functions of venom proteins from parasitic wasps.</a> Insect Biochemistry and Molecular Biology. 35 (11), 1209-1223 (2005).</li><li>Yan, Z. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+venom+serpin+splicing+isoform+of+the+endoparasitoid+wasp+Pteromalus+puparum+suppresses+host+prophenoloxidase+cascade+by+forming+complexes+with+host+hemolymph+proteinases.">A venom serpin splicing isoform of the endoparasitoid wasp Pteromalus puparum suppresses host prophenoloxidase cascade by forming complexes with host hemolymph proteinases.</a> Journal Biological Chemistry. 292 (3), 1038-1051 (2017).</li><li>Woelke, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Description+and+biology+of+two+new+egg+parasitoid+species+(Hymenoptera:+Trichogrammatidae)+reared+from+eggs+of+Heliconiini+butterflies+(Lepidoptera:+Nymphalidae:+Heliconiinae)+in+Panama.">Description and biology of two new egg parasitoid species (Hymenoptera: Trichogrammatidae) reared from eggs of Heliconiini butterflies (Lepidoptera: Nymphalidae: Heliconiinae) in Panama.</a> Journal of Natural History. 53 (11-12), 639-657 (2019).</li><li>Zang, L. S., Wang, S., Zhang, F., Desneux, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+control+with+Trichogramma+in+China:+History,+present+status,+and+perspectives.">Biological control with Trichogramma in China: History, present status, and perspectives.</a> Annual Review of Entomology. 66, 463-484 (2021).</li><li>Nettles, W. C. J., Morrison, R. K., Xie, Z. N., Ball, D., Shenkir, C. A., Vinson, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+action+of+potassium+chloride+and+magnesium+sulfate+on+parasitoid+wasp+oviposition.">Synergistic action of potassium chloride and magnesium sulfate on parasitoid wasp oviposition.</a> Science. 218, 4568 (1982).</li><li>Tilden, R. L., Ferkovich, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kairomonal+stimulation+of+oviposition+into+an+artificial+substrate+by+the+endoparasitoid+Microplitis+croceipes+(Hymenoptera)Braconidae).">Kairomonal stimulation of oviposition into an artificial substrate by the endoparasitoid Microplitis croceipes (Hymenoptera)Braconidae).</a> Annals of the Entomological Society of America. 81 (1), 152-156 (1988).</li><li>Xie, Z. N., Li, L., Xie, Y. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+culture+of+Habrobracon+hebetor.">In vitro culture of Habrobracon hebetor.</a> Chinese Journal of Biological Control. 5 (2), 49-51 (1989).</li><li>Han, S. T., Liu, W. H., Li, L. Y., Chen, Q. X., Zeng, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breeding+Trichogramma+ostriniae+with+artificial+eggs.">Breeding Trichogramma ostriniae with artificial eggs.</a> Journal of Environmental Entomology. 21 (1), 9-12 (1999).</li><li>Li, L. Y., Chen, Q. X., Liu, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oviposition+behavior+of+twelve+species+of+Trichogramma+and+its+influence+on+the+efficiency+of+rearing+them+in+vitro.">Oviposition behavior of twelve species of Trichogramma and its influence on the efficiency of rearing them in vitro.</a> Journal of Environmental Entomology. 11 (1), 31-35 (1989).</li><li>Xing, J. Q., Li, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rearing+of+an+egg+parasite+Anastatus+japonicus+Ashmead+in+vitro.">Rearing of an egg parasite Anastatus japonicus Ashmead in vitro.</a> Acta Entomologica Sinica. 33 (2), 166-173 (1990).</li><li>Moreau, S. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#34;It+stings+a+bit+but+it+cleans+well&#34;:+Venoms+of+Hymenoptera+and+their+antimicrobial+potential.">&#34;It stings a bit but it cleans well&#34;: Venoms of Hymenoptera and their antimicrobial potential.</a> Journal of Insect Physiology. 59 (2), 186-204 (2013).</li><li>Moreau, S. J. M., Asgari, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Venom+proteins+from+parasitoid+wasps+and+their+biological+function.">Venom proteins from parasitoid wasps and their biological function.</a> Toxins. 7 (7), 2385-2412 (2015).</li></ol>

The author has nothing to disclose and no competing financial interests.

Here, we present a method for extracting venom from T. dendrolimi using artificial hosts. The key points in the venom collection experiment are as follows. (1) During preparation, T. dendrolimi must be anesthetized rapidly with an appropriate concentration of CO2. If the CO2 concentration is too low, it will be insufficient to anesthetize the Trichogramma quickly. Conversely, if the concentration is too high, Trichogramma may die, reducing their ability to parasit...

Parasitoid wasps are parasitic hymenopteran insects that are important resources for biological control1. There is a wide variety of parasitoid wasps, with over 600,000 estimated species2. The diversity of parasitoid wasps far exceeds that of other venomous arthropods, such as snakes, cone snails, spiders, scorpions, and bees. Venom is an important parasitic factor in parasitoid wasps. For successful parasitism, venom is injected into the host, modulating the host&#39;s behavior, immunity, development, and metabolism3. Moreover, the venom of parasitoid wasps displays remarkable diversity in its molecular structures, targets, and functions, reflecting complex coevolution with their hosts. Thus, parasitoid venom is a valuable and underappreciated resource of active molecules for insecticidal or medical purposes4. Unlike the venom of snakes, cone snails, spiders, scorpions, and bees, parasitoid wasp venom cannot be collected by direct stimulation or electrical stimulation5. The current method of extraction of parasitoid wasp venom is to dissect the venom reservoir. However, parasitoid wasps are often small, and dissection of parasitoid wasps requires high technical skills. Therefore, if we can find a way to collect the venom of parasitoid wasps efficiently and conveniently, it will be of great help to research the venom of parasitoid wasps.
Trichogramma (Hymenoptera: Trichogrammatidae) is a genus of tiny (~0.5 mm long) parasitoid wasps6. These wasps are among the most widely used biocontrol agents, particularly targeting eggs of various lepidopteran pests in both agriculture and forests. For example, T. dendrolimi, one of the most widely used Trichogramma species in China, has been extensively applied to control a variety of agricultural and forestry pests, such as Dendrolimus superans, Ostrinia furnacalis, and Chilo suppressalis. Previous studies showed that Trichogramma wasps could inject their eggs into artificial hosts7. Artificial hosts can be created using materials such as wax8, agar9, Parafilm10, and plastic film11. The solution in artificial hosts that induces sufficient oviposition for Trichogramma can be simple, such as amino acids or inorganic salts12. Based on the characteristic that T. dendrolimi can parasitize artificial hosts, this study provides a new method for extracting venom from parasitoid wasps using artificial hosts. This approach aims to address the shortcomings of low yield, low purity, and susceptibility to contamination in current extraction techniques. By using this method, a large amount of high-purity venom from T. dendrolimi can be extracted, which meets the needs of scientific research and screening of bioactive molecules for insecticidal or medical purposes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10 &#956;m Nylon Net</td>
			<td>Millipore</td>
			<td>NY1002500</td>
			<td>For filtering the eggs</td>
		</tr>
		<tr>
			<td>10% Polyvinyl alcohol</td>
			<td>Aladdin</td>
			<td>P139533</td>
			<td>For attractting&#160; T. dendrolimi&#160; to lay eggs</td>
		</tr>
		<tr>
			<td>10% Sucrose water</td>
			<td>Sinopharm Chemical Reagent&#160;</td>
			<td>10021463</td>
			<td>Feed Trichogramma dendrolimi</td>
		</tr>
		<tr>
			<td>4x LDS loading buffer</td>
			<td>Ace Hardware</td>
			<td>B23010301</td>
			<td>SDS-PAGE</td>
		</tr>
		<tr>
			<td>Collection box</td>
			<td>Deli</td>
			<td>8555</td>
			<td>Container for T. dendrolimi parasitism</td>
		</tr>
		<tr>
			<td>Future PAGE&#160; 4&#8211;12% (12 wells)</td>
			<td>Ace Hardware</td>
			<td>J70236502X</td>
			<td>SDS-PAGE</td>
		</tr>
		<tr>
			<td>GenScript eStain L1 protein staining apparatus</td>
			<td>GenScript</td>
			<td>L00753</td>
			<td>SDS-PAGE</td>
		</tr>
		<tr>
			<td>Glass grinding rod&#160;&#160;</td>
			<td>Applygen</td>
			<td>tb6268</td>
			<td>Semicircular protrudations&#160;</td>
		</tr>
		<tr>
			<td>L- Leucine</td>
			<td>Solarbio</td>
			<td>L0011</td>
			<td>Artificial host components</td>
		</tr>
		<tr>
			<td>L-Histidine</td>
			<td>Aladdin</td>
			<td>A2219458</td>
			<td>Artificial host components</td>
		</tr>
		<tr>
			<td>L-Phenylalanine</td>
			<td>Solarbio</td>
			<td>P0010</td>
			<td>Artificial host components</td>
		</tr>
		<tr>
			<td>Mini-Centrifuges</td>
			<td>Scilogex</td>
			<td>D1008</td>
			<td>Centrifuge</td>
		</tr>
		<tr>
			<td>MOPS-SDS running buffer</td>
			<td>Ace Hardware</td>
			<td>B23021</td>
			<td>SDS-PAGE</td>
		</tr>
		<tr>
			<td>Omni-Easy Instant BCA protein assay kit</td>
			<td>Shanghai Yamay Biomedical Technology&#160;</td>
			<td>ZJ102</td>
			<td>For esimation of venom protein concentration</td>
		</tr>
		<tr>
			<td>PCR plate layout of 96 holes</td>
			<td>Thermo Fisher</td>
			<td>AB1400L</td>
			<td>Semicircular protrudations&#160;</td>
		</tr>
		<tr>
			<td>Polyethylene plastic film</td>
			<td>Suzhou Aopang Trading &#160;</td>
			<td>001c5427</td>
			<td>Artificial egg card</td>
		</tr>
		<tr>
			<td>Prestained color protein marker(10&#8211;180 kDa)</td>
			<td>YiFeiXue Biotech</td>
			<td>YWB007</td>
			<td>SDS-PAGE</td>
		</tr>
		<tr>
			<td>Rubber band</td>
			<td>Guangzhou qianrui biology science and technology</td>
			<td>009</td>
			<td>Tighten the plastic film and the collection box</td>
		</tr>
		<tr>
			<td>Silicone rubber septa mat, 96-well, round hole</td>
			<td>Sangon Biotech</td>
			<td>F504416-0001</td>
			<td>Semicircular protrudations&#160;</td>
		</tr>
	</tbody>
</table>

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 

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

The lack of efficient venom extraction methods often limits the research on the venom of parasitoid wasps, especially in tiny parasitoid wasps. This study provides an efficient method to extract the venom of Trichogramma with follow up post study of Trichogramma venom, such as the protein compensation and the venom function analysis. Previously, no methods of exchanging of parasitoid wasp venom requires the distinguishing of venom reserve.However, parasitoid wasps are tiny. Not only are the technical requirements of distinguishing venom was high, but continuation of other tissues during dissection is also common. Our normal method uses artificial host, thus avoiding such problems.In the future, we will focus on the agriculture and the medical applications of Trichogramma venom. We aim to utilize specific insecticidal venom genes for pest control. Meanwhile, we plan to investigate the untapped potential of Trichogramma venom in methodology, particularly its prospective utility in enhancing immunotherapy techniques, and in the discovery of novel antibiotic molecules.

The protein concentration of representative venom samples was measured using the protein assay kit, with the results presented in Table 1. The results showed that the concentration of venom protein collected by this method ranged from 0.35 &#181;g/&#181;L to 0.46 &#181;g/&#181;L, while the negative control of amino acid solution only had a protein concentration of 0.03 &#181;g/&#181;L to 0.05 &#181;g/&#181;L. The concentration of venom protein collected by this method is much higher than that of negative...

Watch this Scientific Journal Video about Efficient Venom Extraction Method from Trichogramma Parasitoid Wasps at JoVE.com

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, ...

extracting-venom-from-parasitoid-wasp-trichogramma-dendrolimi-using

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Biochemistry

Trichogramma dendrolimi Artificial Host Interaction and Venom Extraction

Trichogramma dendrolimi  Artificial Host Interaction and Venom Extraction  

To begin, take a polyethylene plastic film 16 centimeters long, 12 centimeters wide, and 20 micrometers thick. Use a glass grinding tool to press out 30 semi-circular protrusions in a standard 96-well PCR layout. Expose both sides of the pressed polyethylene plastic film to UV light for one hour to disinfect it.Add a small amount of 10%polyvinyl alcohol to the semi-circular surface. Next, place about 3, 000 anesthetized female trichogramma dendrolimi wasps into a collection box. Place the convex side of the film egg card towards the collection box and secure the edges with a rubber band.Now add four microliters of amino-acid solution to each protrusion. Then cover it with another piece of polyethylene plastic film. Use a rubber band to tightly cover the collection box with two sheets of plastic.Allow the wasps to parasitize freely for four to eight hours. Provide 10%sucrose water through wet cotton during parasitization. Next, obtain the parasitized amino-acid solution from the inner protrusion of the artificial egg card and transfer it to the cap of 1.45 milliliter tubes.Cover the tube cap with a 10-micrometer nylon net, having a diameter of 25 millimeters. Then fasten the nylon net in the tube lightly. Centrifuge the upper right tube at 1, 360 g for 10 seconds.Collect the filtered solution containing the venom. Store the venom at minus 80 degrees Celsius for further analyses. BCA analysis of the venom samples showed that the concentration of venom protein ranged from 0.35 to 0.46 micrograms per microliter.The negative control of amino-acid solution only had a protein concentration of 0.03 to 0.05 micrograms per microliter. SDS-PAGE analysis of the venom showed a protein range spanning from under 10 kilodaltons to over 130 kilodaltons in size.

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

Artificial Host Parasitization by Trichogramma dendrolimi and Venom Extraction

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